Handbook microbiological media parte3 by Marco Acuña

Heart Infusion Broth with Human Serum and Fresh Yeast Extract Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except antibiotic inhibitor solution and glucose solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile antibiotic inhibitor solution and glucose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Escherichia coli.

Heart Infusion Broth, HiVeg Composition per liter: Plant hydrolysate No. 1............................................................... 10.0g Plant infusion .............................................................................. 10.0g NaCl .............................................................................................. 5.0g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

815

Heart Infusion Broth with Horse Serum, Fresh Yeast Extract, and Penicillin Composition per 940.0mL: Heart infusion broth...............................................................720.0mL Horse serum, unheated...........................................................200.0mL Fresh yeast extract solution .....................................................10.0mL Penicillin solution ....................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Heart Infusion Broth: Composition per liter: Beef heart, infusion from.......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl.............................................................................................. 5.0g

Preparation of Heart Infusion Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant solution. Adjust pH to 6.6–6.8.

Use: For the isolation and cultivation of a wide variety of fastidious

Penicillin Solution: Composition per 10.0mL:

microorganisms.

Penicillin .............................................................................. 100,000U

Heart Infusion Broth with Horse Serum and Fresh Yeast Extract Composition per 930.0mL: Heart infusion broth ...............................................................720.0mL Horse serum, unheated...........................................................200.0mL Fresh yeast extract solution......................................................10.0mL pH 7.4 ± 0.2 at 25°C

Heart Infusion Broth: Composition per liter: Beef heart, infusion from .......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g

Preparation of Heart Infusion Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Preparation of Medium: To 720.0mL of sterile cooled heart infu-

Preparation of Penicillin Solution: Add penicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: To 720.0mL of sterile cooled heart infusion broth, aseptically add 200.0mL of horse serum, 10.0mL of fresh yeast extract solution, and 10.0mL of sterile penicillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Mycoplasma equigenitalium and Mycoplasma subdolum.

Heart Infusion Broth with Human Serum and Fresh Yeast Extract Composition per 930.0mL: Heart infusion broth...............................................................720.0mL Human serum, unheated ........................................................200.0mL Fresh yeast extract solution .....................................................10.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Heart Infusion Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

sion broth, aseptically add 200.0mL of horse serum and 10.0mL of fresh yeast extract solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Fresh Yeast Extract Solution: Composition per 100.0mL:

Use: For the cultivation and maintenance of Mycoplasma equigenita-

Preparation of Fresh Yeast Extract Solution: Add the live Bak-

lium and Mycoplasma subdolum.

er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90

Baker’s yeast, live, pressed, starch-free...................................... 25.0g

816

Heart Infusion Broth with Inactivated Horse Serum

min at 15 psi pressure–121°C. Allow to stand. Remove supernatant solution. Adjust pH to 6.6–6.8.

Preparation of Medium: To 720.0mL of sterile, cooled heart infusion broth, aseptically add 200.0mL of human serum and 10.0mL of fresh yeast extract solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Mycoplasma equigenitalium and Mycoplasma subdolum.

NaCl.............................................................................................. 5.0g Horse serum, inactivated .......................................................200.0mL Fresh yeast extract solution ...................................................100.0mL pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Heart Infusion Broth with Inactivated Horse Serum Composition per liter: Beef heart, infusion from .......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Horse serum, inactivated........................................................100.0mL pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Enterococcus faecium.

Heart Infusion Broth with Inactivated Horse Serum and Fresh Yeast Extract Composition per liter: Beef heart, infusion from .......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Horse serum, inactivated........................................................200.0mL Fresh yeast extract solution....................................................100.0mL pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Bak-

Preparation of Medium: Add components, except horse serum and fresh yeast extract solution, to distilled/deionized water and bring volume to 700.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse serum and fresh yeast extract solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Acholeplasma species, Mycoplasma species, and Streptobacillus species.

Heart Infusion Broth (pH 7.5) with Inactivated Human Serum and Yeast Extract (ATCC Medium 245) Composition per liter: Heart infusion broth with yeast extract..................................800.0mL Human serum, inactivated .....................................................200.0mL pH 7.5 ± 0.2 at 25°C

Heart Infusion Broth with Yeast Extract: Composition per liter: Beef heart, infusion from.......................................................... 500.0g Tryptose ...................................................................................... 10.0g Yeast extract (Oxoid) .................................................................... 6.3g NaCl.............................................................................................. 5.0g

Preparation of Heart Infusion Broth with Yeast Extract: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°.

er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant solution. Adjust pH to 6.6–6.8.

Source: Heart infusion broth without yeast extract is available as a

Preparation of Medium: Add components, except horse serum and

sion broth with yeast extract, aseptically add 200.0mL of heat inactivated human serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

fresh yeast extract solution, to distilled/deionized water and bring volume to 700.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse serum and fresh yeast extract solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Acholeplasma species, Mycoplasma species, and Streptobacillus species.

Heart Infusion Broth with Inactivated Horse Serum, Fresh Yeast Extract, and Sucrose Composition per liter: Beef heart, infusion from .......................................................... 500.0g Sucrose........................................................................................ 40.0g Tryptose ...................................................................................... 10.0g © 2010 by Taylor and Francis Group, LLC

premixed powder from BD Diagnostic Systems.

Preparation of Medium: To 800.0mL of sterile cooled heart infu-

Use: For the cultivation and maintenance of Corynebacterium pseudotuberculosis and Streptobacillus moniliformis.

Heart Infusion Broth with Porcine Serum and Fresh Yeast Extract Composition per liter: Beef heart, infusion from.......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl.............................................................................................. 5.0g Porcine serum, inactivated.....................................................200.0mL Fresh yeast extract (25% w/v solution) .................................100.0mL pH 7.5 ± 0.2 at 25°C

Hektoen Enteric Agar

Porcine Serum: Composition per 200.0mL: Porcine serum ........................................................................200.0mL

Preparation of Porcine Serum: Adjust the pH of 200.0mL of porcine serum to 4.4 with sterile 1N HCl. Do not overshoot pH below 4.2. Allow serum to stand at 4°C for 18–20 hr. Adjust pH to 7.0 with sterile NaOH. Aseptically centrifuge at 9000 rpm for 20 min. Discard sediment. Filter supernatant solution through a 0.85μm filter. Filter sterilize through a 0.22μm filter. Store at −70°C.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Preparation of Medium: Add components, except porcine serum and fresh yeast extract solution, to distilled/deionized water and bring volume to 700.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile porcine serum and fresh yeast extract solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Acholeplasma species.

Heart Infusion Broth with Sodium Chloride (HI with NaCl) (BAM M60) Composition per liter:

817

Heart Infusion Tyrosine Agar Composition per liter: Beef heart, infusion from.......................................................... 500.0g Agar ............................................................................................ 15.0g Tryptose ...................................................................................... 10.0g NaCl.............................................................................................. 5.0g L-Tyrosine ..................................................................................... 1.0g pH 7.4 ± 0.2 at 25°C

Use: For the cultivation and differentiation of Bordetella parapertussis based on browning of blood-free medium.

Hektoen Enteric Agar Composition per liter: Agar ............................................................................................ 13.5g Lactose........................................................................................ 12.0g Peptic digest of animal tissue ..................................................... 12.0g Sucrose........................................................................................ 12.0g Bile salts........................................................................................ 9.0g NaCl.............................................................................................. 5.0g Na2S2O3 ........................................................................................ 5.0g Yeast extract.................................................................................. 3.0g Salicin ........................................................................................... 2.0g Ferric ammonium citrate............................................................... 1.5g Acid Fuchsin................................................................................. 0.1g Bromthymol Blue ..................................................................... 0.064g pH 7.6 ± 0.2 at 25°C

NaCl ............................................................................................ 20.0g Tryptose ...................................................................................... 10.0g NaCl ............................................................................................ 20.0g Beef heart, infusion from 500.0g ..................................................1.0L pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Source: This medium without added NaCl is available as a premixed

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until components are dissolved. Do not autoclave. Pour into sterile Petri dishes. Allow agar to solidify with the Petri dish covers partially off.

powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation and cultivation of halophilic Vibrio spp.

Heart Infusion Medium with Fetal Bovine Serum Composition per liter: Beef heart, infusion from .......................................................... 500.0g Agar ............................................................................................ 15.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Fetal bovine serum.................................................................100.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components, except fetal bovine serum, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile fetal bovine serum. Aseptically distribute into sterile tubes or flasks.

agnostic Systems and Oxoid Unipath.

Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin.

Preparation of Medium: Add components to distilled/deionized

Use: For the isolation and cultivation of Gram-negative enteric microorganisms from a variety of clinical and nonclinical specimens based on lactose or sucrose fermentation and H2S production. For the isolation and differentiation of Salmonella and Shigella. Bacteria that ferment lactose or sucrose appear as yellow to orange colonies. Bacteria that produce H2S appear as colonies with black centers.

Hektoen Enteric Agar Composition per liter: Agar ............................................................................................ 15.0g Proteose peptone......................................................................... 12.0g Lactose........................................................................................ 12.0g Sucrose........................................................................................ 12.0g Bile salts........................................................................................ 9.0g NaCl.............................................................................................. 5.0g Na2S2O3 ........................................................................................ 5.0g Yeast extract.................................................................................. 3.0g Salicin ........................................................................................... 2.0g Ferric ammonium citrate............................................................... 1.5g

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Hektoen Enteric Agar, HiVeg

Acid Fuchsin ................................................................................. 0.1g Bromthymol Blue ..................................................................... 0.065g pH 7.5 ± 0.2 at 25°C

Antibiotic solution A ...............................................................10.0mL Antibiotic solution B................................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Antibiotic Solution A: Composition per 10.0mL:

Media.

Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until components are dissolved. Do not autoclave. Pour into sterile Petri dishes. Allow agar to solidify with the Petri dish covers partially off.

Vancomycin ............................................................................... 1.0mg Trimethoprim ............................................................................. 5.0mg Polymyxin B .............................................................................. 250 U

Preparation of Antibiotic Solution A: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Antibiotic Solution B: Composition per 10.0mL: Amphotericin B ......................................................................... 5.0mg Dimethylformamide...................................................................2.0mL

Preparation of Antibiotic Solution B: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except laked horse

Hektoen Enteric Agar, HiVeg Composition per liter: Plant peptone No. 3..................................................................... 19.0g Agar ............................................................................................ 15.0g Lactose ........................................................................................ 12.0g Sucrose........................................................................................ 12.0g NaCl .............................................................................................. 5.0g Na2S2O3 ........................................................................................ 5.0g Yeast extract.................................................................................. 3.0g Synthetic detergent No. I .............................................................. 2.0g Salicin ........................................................................................... 2.0g Ferric ammonium citrate............................................................... 1.5g Acid Fuchsin ................................................................................. 0.1g Bromthymol Blue ..................................................................... 0.065g pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until components are dissolved. Do not autoclave. Pour into sterile Petri dishes. Allow agar to solidify with the Petri dish covers partially off.

Helicobacter Medium Composition per 850.0mL: Agar ............................................................................................ 15.0g Beef extract ................................................................................. 10.0g Peptone........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Horse blood, laked ...................................................................50.0mL © 2010 by Taylor and Francis Group, LLC

blood, antibiotic solution A, and antibiotic solution B, to distilled/deionized water and bring volume to 930.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 50.0mL of sterile laked horse blood, 10.0mL of sterile antibiotic solution A, and 10.0mL of sterile antibiotic solution B. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Helicobacter muridarum and Helicobacter felis.

Helicobacter pylori Isolation Agar Composition per liter: Agar ............................................................................................ 15.0g Bitone.......................................................................................... 10.0g Pancreatic digest of casein............................................................ 5.0g NaCl.............................................................................................. 5.0g Peptic digest of animal tissue ....................................................... 5.0g Tryptic digest of beef heart ........................................................... 3.0g Cornstarch..................................................................................... 1.0g Horse blood, laked ...................................................................35.0mL Antibiotic inhibitor solution ....................................................10.0mL pH 7.3 ± 0.2 at 25°C

Antibiotic Inhibitor Solution: Composition per 10.0mL: Vancomycin ................................................................................ 0.01g Amphotericin B ......................................................................... 5.0mg Cefsulodin.................................................................................. 5.0mg Trimethoprim lactate.................................................................. 5.0mg

Preparation of Antibiotic Inhibitor Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except horse blood and antibiotic inhibitor solution, to distilled/deionized water and bring volume to 955.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse blood and sterile antibiotic inhibitor solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Helicobacter pylori from clinical specimens.

Heliorestis Medium

Helicobacter pylori Selective Medium Composition per 1080.0mL: Special peptone ........................................................................... 23.0g Agar ............................................................................................ 10.0g NaCl .............................................................................................. 5.0g Starch ............................................................................................ 1.0g Horse blood, laked ...................................................................70.0mL Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Horse Blood, Laked: Composition per 100.0mL: Horse blood, fresh..................................................................100.0mL

Preparation of Horse Blood, Laked: Add blood to a sterile polypropylene bottle. Freeze overnight at −20°C. Thaw at 8°C. Refreeze at −20°C. Thaw again at 8°C.

Selective Supplement Solution: Composition per 10.0mL: Vancomycin ............................................................................. 10.0mg Trimethoprim ............................................................................. 5.0mg Cefsulodin .................................................................................. 5.0mg Amphotericin B.......................................................................... 5.0mg

Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except selective supplement solution and laked horse blood, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptially add 10.0mL selective supplement solution and 70.0mL sterile laked horse blood. Mix thoroughly. Pour into sterile Petri dishes. Use: For the isolation of Helicobacter pylori from clinical specimens. H. pylori forms discrete, translucent, and non-coalescent colonies.

Heliobacillus mobilis Medium Composition per 966.0mL: Yeast extract................................................................................ 10.0g MgSO4 .......................................................................................... 0.1g EDTA ......................................................................................... 2.0mg Sodium pyruvate solution ......................................................100.0mL Trace elements solution B........................................................10.0mL K2HPO4 solution ........................................................................5.0mL Trace elements solution A..........................................................1.0mL pH 7.1 ± 0.2 at 25°C

Sodium Pyruvate Solution: Composition per 100.0mL: Sodium pyruvate ........................................................................... 1.1g

Preparation of Sodium Pyruvate Solution: Add sodium pyruvate to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution B: Composition per 100.0mL: CaCl2·2H2O................................................................................... 0.3g Ferric ammonium citrate............................................................... 0.2g © 2010 by Taylor and Francis Group, LLC

819

Preparation of Trace Elements Solution B: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. K2HPO4 Solution: Composition per 100.0mL: K2HPO4......................................................................................... 4.0g

Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution A: Composition per 100.0mL: H3BO3 ......................................................................................... 2.86g MnCl2·4H2O ............................................................................... 1.81g Na2MoO4·2H2O .......................................................................... 0.39g ZnSO4·7H2O ............................................................................. 0.222g CuSO4·5H2O............................................................................. 0.079g Co(NO3)2·6H2O ....................................................................... 49.4mg

Preparation of Trace Elements Solution A: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except sodium pyruvate solution, trace elements solution B, K2HPO4 solution, and trace elements solution A, to distilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Adjust pH to 7.1. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 100.0mL of sterile sodium pyruvate solution, 10.0mL of sterile trace elements solution B, 5.0mL of sterile K2HPO4 solution, and 1.0mL of sterile trace elements solution A. Mix thoroughly. Aseptically distibute into sterile tubes or flasks. Use: For the cultivation and maintenance of Heliobacillus mobilis.

Heliobacterium chlorum Medium Composition per liter: Yeast extract................................................................................ 10.0g K2HPO4........................................................................................ .1.0g MgSO4·7H2O ................................................................................ 1.0g Sodium ascorbate.......................................................................... 0.5g pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except sodium ascorbate, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Sparge with 100% N2 and continue boiling for 3–4 min. Add sodium ascorbate and continue to sparge with 100% N2. Adjust pH to 6.8. Under 100% N2, immediately dispense 45.0mL of medium into 50.0mL screw-capped tubes fitted with rubber septa. Tighten screw caps. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Heliobacillus mobilis and Heliobacterium chlorum.

Heliorestis Medium (DSMZ Medium 886) Composition per liter: Na-acetate ..................................................................................... 1.0g MgCl2·6H2O ................................................................................. 0.6g Yeast extract.................................................................................. 0.5g KH2PO4......................................................................................... 0.5g NaCl.............................................................................................. 0.5g Resazurin ...................................................................................... 0.2g

820

Hemmes Medium Base

CaCl2 ............................................................................................. 0.1g Vitamin B12 ...............................................................................20.0µg Biotin ........................................................................................20.0µg Solution A ................................................................................50.0mL Trace elements solution SL-6 ....................................................1.0mL pH 9.0–9.5 at 25°C

Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Solution A : Composition per 50.0mL:

Use: For the detection of Salmonella spp. and Shigella spp. based upon 7 different metabolic reactions.

Na2CO3 ......................................................................................... 2.5g NaHCO3 ........................................................................................ 2.5g NH4Cl ........................................................................................... 0.5g Na2S·9H2O .................................................................................... 0.4g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O.................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except solution A, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 50.0mL solution A. Mix thoroughly. Adjust pH to 9.0–9.5. Aseptically distribute to sterile tubes or flasks.

Use: For the cultivation of Heliorestis baculata. Hemin Medium for Mycobacterium See: Middlebrook 7H10 Agar with Middlebrook OADC Enrichment and Hemin

Hemmes Medium Base Composition per liter: Casein enzymatic hydrolysate .................................................... 10.0g Lactose ........................................................................................ 10.0g Sucrose........................................................................................ 10.0g Agar .............................................................................................. 5.5g NaCl .............................................................................................. 4.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 0.3g Na2S2O3·5H2O .............................................................................. 0.1g FeSO4·7H2O................................................................................ 0.04g Phenol Red ................................................................................ 0.015g Urea solution..............................................................................5.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Urea Solution: Composition per 10.0mL: Urea............................................................................................... 4.0g

Hemo ID Quad Plate with Growth Factors (Haemophilus Identification Quadrant Plate with Growth Factors) Composition per plate: Quadrant I ..................................................................................5.0mL Quadrant II.................................................................................5.0mL Quadrant III ...............................................................................5.0mL Quadrant IV ...............................................................................5.0mL Quadrant I: Composition per 5.0mL: Hemin ........................................................................................ 0.1mg Brain heart infusion agar ...........................................................5.0mL

Quadrant II: Composition per 5.0mL: Brain heart infusion agar ...........................................................5.0mL Supplement solution ................................................................0.05mL

Quadrant III: Composition per 5.0mL: Hemin ........................................................................................ 0.1mg Brain heart infusion agar ...........................................................5.0mL Supplement solution ................................................................0.05mL

Quadrant IV: Composition per 5.0mL: Hemin ........................................................................................ 0.1mg Brain heart infusion agar ...........................................................5.0mL Horse blood..............................................................................0.25mL Supplement solution ................................................................0.05mL

Source: The supplement solution (IsoVitaleX® enrichment) is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems.

Preparation of Quadrant Media: Sterilize Brain Heart Infusion Agar by autocalving for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Add additional components as filter sterilized solutions. Mix and distribute as 5.0mL aliquots into quadrants. Use: For the differentiation and presumptive identification of Haemophilus species. The Hemo ID Quad Plate is a four-sectored plate, each with a different medium.

Hemorrhagic Coli Agar (HC Agar) Composition per liter: Sorbitol ....................................................................................... 20.0g Pancreatic digest of casein.......................................................... 20.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 5.0g Bile salts No. 3............................................................................ 1.12g Bromcresol Purple .................................................................... 0.015g pH 7.2 ± 0.2 at 25°C

Herpetosiphon giganteus Medium Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the isolation and cultivation of enterohemorraghic Escherichia coli from food.

Hemorrhagic Coli Agar with MUG (HC Agar with MUG) (BAM M62) Composition per liter: Sorbitol........................................................................................ 20.0g Pancreatic digest of casein .......................................................... 20.0g Agar ............................................................................................ 15.0g NaCl .............................................................................................. 5.0g Bile salts No. 3............................................................................ 1.12g Bromcresol Purple .................................................................... 0.015g MUG reagent ................................................................................ 0.1g pH 7.2 ± 0.2 at 25°C

Source: MUG reagent is available from Hach Company, Loveland, Colorado.

Use: For the isolation and cultivation of enterohemorraghic Escherichia coli from food. For hemorrhagic colitis E. coli strains.

Heparin Medium Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein ............................................................ 3.5g NaCl .............................................................................................. 1.0g Pancreatic digest of soybean meal ................................................ 0.6g Glucose ......................................................................................... 0.5g K2HPO4 ......................................................................................... 0.5g Heparin solution.......................................................................10.0mL pH 6.5 ± 0.2 at 25°C

Heparin Solution: Composition per 10.0mL: Heparin........................................................................................ 0.02g

Preparation of Heparin Solution: Add heparin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except heparin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile heparin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Flavobacterium leparinum.

Herbaspirillum Agar

821

K2HPO4......................................................................................... 0.1g NaCl.............................................................................................. 0.1g Yeast extract................................................................................ 0.05g CaCl2 ........................................................................................... 0.02g FeCl2·6H2O................................................................................. 0.01g NaMoO4·2H2O........................................................................... 2.0mg Solution A................................................................................50.0mL pH 7.0 ± 0.2 at 25°C

Solution A: Composition per 50.0mL Sodium malate .............................................................................. 5.0g

Preparation of Solution A: Add sodium malate to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Adjust pH to 7.0. Filter sterilize. Preparation of Medium: Add components, except solution A, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 50.0mL of sterile solution A. Mix thoroughly. Aseptically pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Herbaspirillum seropedicae.

Herellea Agar Composition per liter: Agar ............................................................................................ 16.0g Pancreatic digest of casein.......................................................... 15.0g Lactose........................................................................................ 10.0g Maltose ....................................................................................... 10.0g Enzymatic digest of soybean meal ............................................... 5.0g NaCl.............................................................................................. 5.0g Bile salts...................................................................................... 1.25g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the isolation, cultivation, and differentiation of Gram-negative nonfermentative and fermentative bacteria. It is especially recommended for the differentiation of Acinetobacter (Herellea) species from Neisseria gonorrhoeae in urethral or vaginal specimens. Fermentative bacteria appear as yellow colonies surrounded by yellow zones. Nonfermentative bacteria, such as Acinetobacter species, appear as pale lavender colonies.

Herpetosiphon giganteus Medium Composition per liter: Pancreatic digest of casein............................................................ 3.0g Yeast extract.................................................................................. 1.0g CaC12·2H2O.................................................................................. 0.5g pH 7.2 ± 0.2 at 25°C

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Agar ............................................................................................ 15.0g KH2PO4 ......................................................................................... 4.0g MgSO4·7H2O ................................................................................ 0.2g

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

822

Hershey’s Tris-Buffered Salts Medium

Use: For the cultivation of Herpetosiphon giganteus.

Hershey’s Tris-Buffered Salts Medium Composition per liter: Tris(hydroxymethyl)aminomethane buffer (0.1M solution) ............................................ 12.1g NaCl .............................................................................................. 5.4g KCl................................................................................................ 3.0g NH4Cl ........................................................................................... 1.1g MgCl2 ........................................................................................ 0.095g KH2PO4 ..................................................................................... 0.087g Na2SO4 ...................................................................................... 0.023g CaCl2 ......................................................................................... 0.011g FeCl3 ........................................................................................ 0.16mg Glucose solution ....................................................................100.0mL pH 7.4 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: Glucose ......................................................................................... 2.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile glucose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of a variety of heterotrophic microorganisms.

HESNW Medium Composition per 1011.0mL: Natural seawater ...........................................................................1.0L Enrichment solution .................................................................10.0mL Vitamin solution.........................................................................1.0mL

Enrichment Solution: Composition per liter: NaNO3....................................................................................... 4.667g Na2SiO3·9H2O........................................................................... 3.000g Sodium glycerophosphate......................................................... 0.667g EDTA·2H2O .............................................................................. 0.553g H3BO3 ....................................................................................... 0.380g Fe(NH4)2(SO4)2·6H2O .............................................................. 0.234g MnSO4·4H2O ............................................................................ 0.054g FeCl3·6H2O ............................................................................... 0.016g ZnSO4·7H2O .............................................................................. 7.3mg CoSO4·7H2O .............................................................................. 1.6mg

Preparation of Enrichment Solution: Add Na2SiO3·9H2O to dis-

tilled/deionized water. Mix thoroughly. Neutralize Na2SiO3·9H2O with 1N HCl. Add 500.0mL of distilled/deionized water. Mix thoroughly. Add remaining components and bring volume to 1.0L with distilled/ deionized water. Mix thoroughly. Filter sterilize.

Vitamin Solution: Composition per liter: Thiamine ....................................................................................... 0.1g Vitamin B12 ................................................................................ 2.0mg Biotin ......................................................................................... 1.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Allow natural seawater to age for 2 months. Filter sterilize. Aseptically add 10.0mL of sterile enrichment solution and 1.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Amphiprora hyalina, Chlamydomonas hedleyi, Chlamydomonas provasolii, Chlorella saccharophila, Chroomonas salina, Pavlova lutheri, and Trichosphaerium species.

HESP1/SR1/TMC4/LUP Medium (DSMZ Medium 860) Composition per 1090.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.2g CaCl2·2H2O .................................................................................. 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................60.0mL Substrate solution.....................................................................20.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.5mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

HESP1/SR1/TMC4/LUP Medium

823

Substrate Solution: Composition per 10.0mL:

Na2S·9H2O Solution: Composition per 10.0mL:

Fructose......................................................................................... 2.0g

Na2S·9H2O.................................................................................... 0.3g

Preparation of Substrate Solution: Add fructose to distilled/de-

ionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except NaHCO3 solution, Na2S·9H2O solution, and substrate solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Boil for 10 min. Cool to room temperature while sparging with 80% N2 + 20% CO2. Adjust pH to 6.0. Dispense under 80% N2 + 20% CO2 into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 0.6mL sterile NaHCO3 solution per 10.0mL medium, 0.1mL Na2S·9H2O solution per 10.0mL medium, and 0.2mL substrate solution per 10.0mL medium. Final pH is 7.0.

Use: For the cultivation of Clostridium xylanovorans DSM 12503.

HESP1/SR1/TMC4/LUP Medium (DSMZ Medium 860) Composition per 1090.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.2g CaCl2·2H2O................................................................................... 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................60.0mL Substrate solution.....................................................................20.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.5mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. © 2010 by Taylor and Francis Group, LLC

Substrate Solution: Composition per 10.0mL: Na-syringate.................................................................................. 0.6g

Preparation of Substrate Solution: Add Na-syringate to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize.

Use: For the cultivation of Sporobacterium olearium (Clostridium sp.) DSM 12504.

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized

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HESP1/SR1/TMC4/LUP Medium

water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Substrate Solution: Composition per 10.0mL: Casamino acids ............................................................................. 0.5g

Preparation of Substrate Solution: Add casamino acids to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except NaHCO3 solution, Na2S·9H2O solution, and substrate solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Boil for 10 min. Cool to room temperature while sparging with 80% N2 + 20% CO2. Adjust pH to 6.0. Dispense under 80% N2 + 20% CO2 into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 0.6mL sterile NaHCO3 solution per 10.0mL medium, 0.1mL Na2S·9H2O solution per 10.0mL medium, and 0.2mL substrate solution per 10.0mL medium. Final pH is 7.0.

Use: For the cultivation of Clostridium peptidivorans DSM 12505.

HESP1/SR1/TMC4/LUP Medium (DSMZ Medium 860) Composition per 1090.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.2g CaCl2·2H2O................................................................................... 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................60.0mL Substrate solution.....................................................................20.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.5mL pH 7.0 ± 0.2 at 25°C

MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically. Substrate Solution: Composition per 10.0mL: Na-lactate.................................................................................... 1.25g Na2S2O3·5H2O ............................................................................ 0.05g

Preparation of Substrate Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize.

Use: For the cultivation of Desulfovibrio mexicanus DSM 13116.

HESP1/SR1/TMC4/LUP Medium

KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................60.0mL Substrate solution.....................................................................20.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.5mL pH 7.4 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Substrate Solution: Composition per 10.0mL: Na2S2O3·5H2O ............................................................................ 1.25g

Preparation of Substrate Solution: Add Na2S2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except NaHCO3 solution, Na2S·9H2O solution, and substrate solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Boil for 10 min. Cool to room temperature while sparging with 80% N2 + 20% CO2. Adjust pH to 6.0. Dispense under 80% N2 + 20% CO2 into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 0.6mL sterile NaHCO3 solution per 10.0mL medium, 0.1mL Na2S·9H2O solution per 10.0mL medium, and 0.2mL substrate solution per 10.0mL medium. Final pH is 7.4.

825

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically. Substrate Solution: Composition per 10.0mL: Glucose ......................................................................................... 1.0g

Preparation of Substrate Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Filter sterilize.

826

Heterotrophic Agar H3P

while sparging with 80% N2 + 20% CO2. Adjust pH to 6.0. Dispense under 80% N2 + 20% CO2 into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 0.6mL sterile NaHCO3 solution per 10.0mL medium, 0.1mL Na2S·9H2O solution per 10.0mL medium, and 0.2mL substrate solution per 10.0mL medium. Final pH is 7.4.

Use: For the cultivation of Sporanaerobacter acetigenes DSM 13106.

Heterotrophic Agar H3P (DSMZ Medium 428) Composition per 1010.0mL: Solution B ..............................................................................855.0mL Solution A ................................................................................50.0mL Solution E ................................................................................50.0mL Solution D ................................................................................30.0mL Solution C ................................................................................20.0mL Standard vitamin solution ..........................................................5.0mL

Solution A: Composition per 50.0mL: Na2HPO4·2H2O............................................................................. 2.9g KH2PO4 ......................................................................................... 2.3g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Solution B: Composition per 855.0mL: Agar ............................................................................................ 15.0g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g CaCl2·2H2O............................................................................... 0.010g MnCl2·4H2O.............................................................................. 0.005g NaVO2·H2O .............................................................................. 0.005g Trace elements solution SL-6 ....................................................5.0mL

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 855.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Solution D: Composition per 30.0mL: Yeast extract.................................................................................. 1.0g Na-acetate ..................................................................................... 1.0g Na2-succinate................................................................................ 1.0g DL-Malate...................................................................................... 1.0g

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Adjust to pH 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Solution E: Composition per 50.0mL: Na-lactate...................................................................................... 1.0g Na-pyruvate .................................................................................. 1.0g D-Mannitol .................................................................................... 1.0g D-Glucose...................................................................................... 2.0g

Preparation of Solution E: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Adjust to pH 7.0. Filter sterilize.

Standard Vitamin Solution: Composition per 100.0mL: Thiamine-HCl·2H2O................................................................ 50.0mg Nicotinic acid........................................................................... 50.0mg Pyridoxine-HCl........................................................................ 50.0mg Ca-pantothenate ....................................................................... 50.0mg Riboflavin ................................................................................ 10.0mg Vitamin B12 .............................................................................. 1.0mg Folic acid ................................................................................... 0.2mg Biotin ......................................................................................... 0.1mg

Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically mix solutions A, B, C, D, E, and standard vitamin solution. Pour into sterile Petri dishes or aseptically distribute to flasks or tubes.

Use: For the cultivation and maintenance of Bacillus spp., Pseudomonas spp., Xanthomonas campestris pvar. campestris, Xanthobacter sp., Aquaspirillum arcticum, Herbaspirillum seropedicae, Sphingobium chlorophenolicum=Sphingomonas chlorophenolica, and Azoarcus sp. Also H3P is a heterotrophic medium for growth, purity checking, and isolation of a broad spectrum of aerobic bacteria.

Heterotrophic Agar H3P Composition per 1010.0mL:

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Solution B ..............................................................................855.0mL Solution A................................................................................50.0mL Solution E ................................................................................50.0mL Solution C ................................................................................20.0mL Solution D................................................................................30.0mL Standard vitamin solution ..........................................................5.0mL pH 6.8 ± 0.2 at 25°C

Solution C: Composition per 20.0mL:

Solution A: Composition per 50.0mL:

Ferric ammonium citrate............................................................. 0.05g Distilled water..........................................................................20.0mL

Na2HPO4·2H2O............................................................................ .2.9g KH2PO4......................................................................................... 2.3g

Preparation of Solution C: Add ferric ammonium citrate to dis-

Preparation of Solution A: Add components to distilled/deionized

tilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Preparation of Trace Elements Solution SL-6: Add components

Heterotrophic Broth H3P

Solution B: Composition per 855.0mL: Agar ............................................................................................ 15.0g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g CaCl2·2H2O................................................................................. 0.01g MnCl2·4H2O............................................................................... 5.0mg NaVO3·H2O ............................................................................... 5.0mg Trace elements solution SL-6 ....................................................5.0mL

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 855.0mL. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

Preparation of Solution C: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Solution D: Composition per 30.0mL: Disodium succinate....................................................................... 1.0g DL-Malate...................................................................................... 1.0g Sodium acetate .............................................................................. 1.0g Yeast extract.................................................................................. 1.0g

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Solution E: Composition per 50.0mL: D-Glucose...................................................................................... 2.0g D-Mannitol .................................................................................... 1.0g

Sodium lactate............................................................................... 1.0g Sodium pyruvate ........................................................................... 1.0g

Preparation of Solution E: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C. Standard Vitamin Solution: Composition per 100.0mL: Calcium DL-pantothenate......................................................... 50.0mg Nicotinic acid ........................................................................... 50.0mg Pyridoxine HCl ........................................................................ 50.0mg Thiamine .................................................................................. 50.0mg Riboflavin ................................................................................ 10.0mg B12 .............................................................................................. 1.0mg Folic acid.................................................................................... 0.2mg Biotin ......................................................................................... 0.1mg © 2010 by Taylor and Francis Group, LLC

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Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C. Preparation of Medium: Aseptically combine 50.0mL of sterile solution A, 855.0mL of sterile solution B, 20.0mL of sterile solution C, 30.0mL of sterile solution D, 50.0mL of sterile solution E, and 5.0mL of sterile standard vitamin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the heterotrophic growth, isolation, and purity checking of a wide variety of aerobic bacteria, including Ancylobacter species, Aquaspirillum species, Azospirillum species, Beijerinckia species, Derxia gummosa, Herbaspirillum seropedicae, Rhodococcus species, Xanthobacter species, and Xanthomonas campestris.

Heterotrophic Broth H3P Composition per 1010.0mL: Solution B ..............................................................................855.0mL Solution A................................................................................50.0mL Solution E ................................................................................50.0mL Solution C ................................................................................20.0mL Solution D................................................................................30.0mL Standard vitamin solution ..........................................................5.0mL pH 6.8 ± 0.2 at 25°C

Solution A: Composition per 50.0mL: Na2HPO4·2H2O............................................................................ .2.9g KH2PO4......................................................................................... 2.3g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Solution B: Composition per 855.0mL: NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g CaCl2·2H2O ................................................................................ 0.01g MnCl2·4H2O .............................................................................. 5.0mg NaVO3·H2O ............................................................................... 5.0mg Trace elements solution SL-6 ....................................................5.0mL

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Solution B: Add components to distilled/deionized water and bring volume to 855.0mL. Autoclave for 15 min at 15 psi pressure–121°C.

Solution C: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

828

Heterotrophic Hyperthermophilic Archaea Medium

Preparation of Solution C: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Solution E: Composition per 50.0mL: D-Glucose...................................................................................... 2.0g D-Mannitol .................................................................................... 1.0g

Sodium lactate............................................................................... 1.0g Sodium pyruvate ........................................................................... 1.0g

Preparation of Solution E: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C.

Standard Vitamin Solution: Composition per 100.0mL: Calcium DL-pantothenate......................................................... 50.0mg Nicotinic acid ........................................................................... 50.0mg Pyridoxine HCl ........................................................................ 50.0mg Thiamine .................................................................................. 50.0mg Riboflavin ................................................................................ 10.0mg B12 .............................................................................................. 1.0mg Folic acid.................................................................................... 0.2mg Biotin ......................................................................................... 0.1mg

Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C.

Preparation of Medium: Aseptically combine 50.0mL of sterile solution A, 855.0mL of sterile solution B, 20.0mL of sterile solution C, 30.0mL of sterile solution D, 50.0mL of sterile solution E, and 5.0mL of sterile standard vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes.

Heterotrophic Hyperthermophilic Archaea Medium Composition per liter: Pancreatic digest of casein ............................................................ 3.0g Glucose ......................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Artificial seawater..................................................................990.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Artificial Seawater: Composition per liter: NaCl ............................................................................................ 20.0g MgSO4·7H2O ................................................................................ 6.0g © 2010 by Taylor and Francis Group, LLC

MgCl2·6H2O ................................................................................. 3.0g (NH4)2SO4 .................................................................................... 1.0g NaHCO3 ........................................................................................ 0.2g CaCl2·2H2O .................................................................................. 0.3g KCl................................................................................................ 0.5g KH2PO4...................................................................................... .0.42g NaBr............................................................................................ 0.05g SrCl2·6H2O ................................................................................. 0.02g Fe(NH4) citrate ........................................................................... 0.01g Wolfe’s mineral solution............................................................5.0mL Vitamin solution.........................................................................5.0mL

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoCl2·6H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Vitamin Solution: Composition per liter: Niacin....................................................................................... 10.0mg Pantothenate............................................................................. 10.0mg Lipoic acid ............................................................................... 10.0mg p-Aminobenzoic acid............................................................... 10.0mg Thiamine (B1) .......................................................................... 10.0mg Riboflavin (B2) ........................................................................ 10.0mg Pyridoxine (B6) ........................................................................ 10.0mg Cobalamin (B12)....................................................................... 10.0mg Biotin ......................................................................................... 4.0mg Folic acid ................................................................................... 4.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.8g

Heterotrophic Medium for Hydrogen-Oxidizing Bacteria

thoroughly. Adjust medium pH to 7.2 by adding sterile anaerobic HCl. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of hyperthermophilic archaea.

Heterotrophic Medium H3P (DSMZ Medium 428) Composition per 1010.0mL: Solution B ..............................................................................855.0mL Solution A ................................................................................50.0mL Solution E ................................................................................50.0mL Solution D ................................................................................30.0mL Solution C ................................................................................20.0mL Standard vitamin solution ..........................................................5.0mL Solution A: Composition per 50.0mL: Na2HPO4·2H2O............................................................................. 2.9g KH2PO4 ......................................................................................... 2.3g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution B: Composition per 855.0mL: NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g CaCl2·2H2O............................................................................... 0.010g MnCl2·4H2O.............................................................................. 0.005g NaVO2·H2O .............................................................................. 0.005g Trace elements solution SL-6 ....................................................5.0mL

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 855.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Solution C: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

Preparation of Solution C: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution D: Composition per 30.0mL: Yeast extract.................................................................................. 1.0g Na-acetate ..................................................................................... 1.0g Na2-succinate ................................................................................ 1.0g DL-Malate...................................................................................... 1.0g © 2010 by Taylor and Francis Group, LLC

829

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Adjust to pH 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution E: Composition per 50.0mL: D-Glucose...................................................................................... 2.0g Na-lactate...................................................................................... 1.0g Na-pyruvate .................................................................................. 1.0g D-Mannitol .................................................................................... 1.0g

Preparation of Solution E: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Adjust to pH 7.0. Filter sterilize.

Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically mix solutions A, B, C, D, E, and standard vitamin solution. Aseptically distribute to flasks or tubes.

Heterotrophic Medium for Hydrogenomonas Composition per liter: Agar ............................................................................................ 15.0g Tryptose ........................................................................................ 5.0g Cornstarch..................................................................................... 2.0g Sodium succinate·6H2O................................................................ 2.0g Sodium glutamate ......................................................................... 1.0g Yeast extract.................................................................................. 1.0g Sodium citrate·2H2O..................................................................... 0.5g Sodium acetate·3H2O.................................................................... 0.3g KH2PO4......................................................................................... 0.2g MgSO4 .......................................................................................... 0.1g pH 6.8–7.2 at 25°C

Use: For the heterotrophic cultivation of Hydrogenomonas species.

Heterotrophic Medium for Hydrogen-Oxidizing Bacteria Composition per 1010.0mL: Solution A..............................................................................900.0mL Solution C ..............................................................................100.0mL Solution B ................................................................................10.0mL

830

Heterotrophic Nitrobacter Medium

Solution A: Composition per 900.0mL: Noble agar................................................................................... 17.0g Na2HPO4·12H2O........................................................................... 9.0g KH2PO4 ......................................................................................... 1.5g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g Trace elements solution SL-6 ....................................................1.0mL

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O................................................................................. 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Solution B: Composition per 10.0mL:

MgSO4·7H2O ................................................................................ 0.5g CaCO3 ........................................................................................ 0.07g

Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution: Composition per liter: FeSO4·7H2O............................................................................. 97.3mg H3BO3 ...................................................................................... 49.4mg ZnSO4·7H2O ............................................................................ 43.1mg (NH4)6Mo7O24·4H2O ............................................................... 37.1mg MnSO4·2H2O ........................................................................... 33.8mg CuSO4·5H2O ............................................................................ 25.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Nitrospira moscoviensis, Nitrobacter hamburgensis, Nitrobacter vulgaris, and Nitrobacter winogradskyi.

Heterotrophic Nitrobacter Medium (LMG Medium 245) Composition per liter:

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Yeast extract.................................................................................. 1.5g Peptone ......................................................................................... 1.5g Na-pyruvate ................................................................................ 0.55g Stock solution ........................................................................100.0mL Trace elements solution .............................................................1.0mL pH 7.4 ± 0.2 at 25°C

Solution C: Composition per 100.0mL:

Stock Solution: Composition per liter:

CaCl2·2H2O................................................................................. 0.01g Ferric ammonium citrate............................................................ 5.0mg

Sodium 3-hydroxybutyrate ........................................................... 2.0g

Preparation of Solution C: Add sodium 3-hydroxybutyrate to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 45°–50°C.

Preparation of Medium: Aseptically combine 900.0mL of sterile solution A, 10.0mL of sterile solution B, and 100.0mL of sterile solution C. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the heterotrophic cultivation and maintenance of Xanthobacter agilis.

Heterotrophic Nitrobacter Medium (DSMZ Medium 756) Composition per liter:

NaCl.............................................................................................. 5.0g KH2PO4......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.5g CaCO3 ......................................................................................... 0.07g

Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution: Composition per liter: (NH4)Mo7O2.......................................................................... 437.1mg FeSO4·7H2O............................................................................. 97.3mg ZnSO4·7H2O ............................................................................ 43.1mg H3BO3 ...................................................................................... 39.4mg MnSO4·H2O ............................................................................. 33.8mg CuSO2·5H2O ............................................................................ 25.0mg

Yeast extract.................................................................................. 1.5g Peptone.......................................................................................... 1.5g Na-pyruvate ................................................................................ 0.55g Stock solution.........................................................................100.0mL Trace elements solution .............................................................1.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Trace Elements Solution: Add components to

Stock Solution: Composition per liter:

Use: For the cultivation of heterotrophic Nitrobactger spp.

distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4 with NaOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Heterotrophic Plate Count See: HPC Agar

Hickey Tresner Agar

Hexamita Medium Composition per liter: TYGM-9 medium ..................................................................250.0mL Sonneborn's Paramecium medium ........................................750.0mL

TYGM-9 Medium: Composition per liter: NaCl .............................................................................................. 7.5g K2HPO4 ......................................................................................... 2.8g Casein digest ................................................................................. 2.0g Gastric mucin ................................................................................ 2.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.4g Bovine serum, heat inactivated ................................................30.0mL Rice starch solution..................................................................30.0mL Tween™ solution .......................................................................0.5mL pH 7.4 ± 0.2 at 25°C

Tween™ Solution: Composition per 100.0mL:

831

Preparation of Solution 1: Add cerophyll to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Boil for 5 min. Filter through Whatman #1 filter paper. Add 0.5g of Na2HPO4. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Distribute 10.0mL volumes into tubes. Autoclave for 15 min at 15 psi pressure–121°C. Solution 2: Composition per liter: Agar ............................................................................................ 20.0g Yeast extract.................................................................................. 4.0g Glucose ....................................................................................... 0.16g

Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute 5.0mL into tubes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position. Preparation of Sonneborn's Paramecium Medium: Inoculate

Preparation of Tween™ Solution: Add Tween™ 80 to absolute

the surface of agar slants of solution 2 with a culture of Klebsiella pneumoniae. Incubate at 37°C for 24–48 hr. Scrape cells from the surface of the agar slants and add to 10.0mL of solution 1. Incubate at 30°C for 24 hr.

ethanol and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine 3.0mL of sterile

Rice Starch Solution: Composition per 100.0mL:

TYGM-9 medium with 9.0mL of Sonneborn's Paramecium medium in 16 × 125mm screw-capped test tubes.

Rice starch..................................................................................... 5.0g Phosphate-buffered saline solution ........................................100.0mL

Use: For the cultivation of Hexamita inflata, Hexamita pusilla, Masti-

Tween™ 80 ..............................................................................10.0mL

Phosphate-Buffered Saline Solution: Composition per liter: NaCl .............................................................................................. 9.0g Na2HPO4·7H2O......................................................................... 0.795g KH2PO4 ..................................................................................... 0.114g

Preparation of Phosphate-Buffered Saline Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C.

Preparation of Rice Starch Solution: Heat sterilize rice starch at 150°C for 2 hr. Aseptically add 100.0mL of sterile phosphate-buffered saline solution. Mix thoroughly. Use immediately.

Preparation of TYGM-9 Medium: Add components, except rice starch solution, Tween™ solution, and bovine serum, to distilled/deionized water and bring volume to 939.5mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 30.0mL of sterile bovine serum, 30.0mL of sterile rice starch solution, and 0.5mL of sterile Tween™ solution. Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks.

Sonneborn's Paramecium Medium: Composition per liter: Solution 1......................................................................................1.0L Klebsiella pneumoniae cultured on solution 2 ....................................................... variable

Solution 1: Composition per liter:

gamoeba invertens, and Trepomonas agilis.

HHD Medium Composition per liter: Agar ............................................................................................ 20.0g Pancreatic digest of casein.......................................................... 10.0g Casamino acids ............................................................................. 3.0g Fructose......................................................................................... 2.5g KH2PO4......................................................................................... 2.5g Papaic digest of soybean meal...................................................... 1.5g Tween™ 80................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Bromcresol Green solution ......................................................20.0mL pH 7.0 ± 0.2 at 25°C

Bromcresol Green Solution: Composition per 30.0mL: Bromcresol Green......................................................................... 0.1g NaOH (0.01N solution)............................................................30.0mL

Preparation of Bromcresol Green Solution: Add Bromcresol Green to 30.0mL of NaOH solution. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Salmonella species from foods.

Hickey Tresner Agar

Rye grass cerophyll....................................................................... 2.5g Na2HPO4 ....................................................................................... 0.5g

Composition per liter:

Source: Cerophyll can be obtained from Ward's Natural Science Establishment, Inc. Dairy Goat Nutrition distributes Grass Media Culture, which is equivalent. Cereal Leaf Product from Sigma Chemical is similar to cerophyll.

Agar ............................................................................................ 15.0g Dextrin ........................................................................................ 10.0g Pancreatic digest of casein............................................................ 2.0g Meat extract .................................................................................. 1.0g

832

HiCrome™ Aureus Agar Base with Egg Tellurite

Yeast extract.................................................................................. 1.0g CaCl2 .......................................................................................... 2.0mg pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Thermomonospora curvata.

Meat extract .................................................................................. 1.0g Phenol Red................................................................................ 0.025g Polymyxin B solution ................................................................1.0mL pH 7.1 ± 0.2 at 25°C

Source: This medium, without Polymyxin B solution, is available as a premixed powder from HiMedia.

Polymyxin B Solution: Composition per 1.0mL: Polymyxin B .............................................................................. 1.0mg

HiCrome™ Aureus Agar Base with Egg Tellurite (Aureus Agar Base, HiCrome™) (HiCrome™ Staphylococcus aureus Agar) (Staphylococcus aureus Agar, HiCrome) Composition per liter: Agar ............................................................................................ 20.0g Casein enzymic hydrolysate ....................................................... 12.0g Sodium pyruvate ......................................................................... 10.0g Beef extract ................................................................................... 6.0g LiCl ............................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Pancreatic digest of gelatin ........................................................... 3.0g Chromogenic mixture ................................................................... 2.1g Egg tellurite emulsion ..............................................................50.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Lithium chloride is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin, wash with plenty of water immediately.

Egg Yolk Tellurite Emulsion: Composition per 100.0mL: Sterile saline.............................................................................64.0mL Egg yolk ...................................................................................30.0mL Sterile potassium tellurite solution, 3.5% ..................................6.0mL

Source: This medium is available from Fluka, Sigma-Aldrich, and HiMedia.

Preparation of Medium: Add components, except egg yolk tellurite emulsion, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 50.0mL sterile egg yok tellurite emulsion. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and identification of staphylococci from environmental samples. For the isolation and enumeration of coagulase positive Staphylococcus aureus. Coagulase positive S. aureus gives brown-black colonies whereas S. epidermidis gives yellow, slightly brownish, colonies.

HiCrome™ Bacillus Agar wtih Polymyxin B (Bacillus cereus HiCrome™ Agar) Composition per liter: Agar ............................................................................................ 15.0g Peptic digest of animal tissue...................................................... 10.0g D-Mannitol .................................................................................. 10.0g NaCl ............................................................................................ 10.0g Chromogenic mixture ................................................................... 3.2g © 2010 by Taylor and Francis Group, LLC

Preparation of Polymyxin B Solution: Add Polymyxin B to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Polymyxin B solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until boiling. Boil until components are fully dissolved. Do not autoclave. Cool to 45°–50°C. Mix thoroughly. Aseptically add 1.0mL of Polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the rapid identification of Bacillus species from a mixed culture by chromogenic method. For the enumeration of Bacillus cereus and Bacillus thuringiensis when present in large numbers in certain foodstuffs.

HiCrome™ Candida Agar Composition per liter: Agar ............................................................................................ 15.0g Peptic digest of animal tissue ..................................................... 15.0g Chromogenic mixture ............................................................... 11.22g K2HPO4......................................................................................... 1.0g Chloramphenicol........................................................................... 0.5g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until boiling. Boil until components are fully dissolved. Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes.

Use: For the rapid isolation and identification of Candida species from mixed cultures.

HiCrome™ Candida Agar, HiVeg Composition per liter: Agar ............................................................................................ 15.0g Plant peptone .............................................................................. 15.0g Chromogenic mixture ................................................................. 11.2g K2HPO4......................................................................................... 1.0g Chloramphenicol........................................................................... 0.5g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the rapid isolation and identification of Candida species from mixed cultures.

HiCrome™ Coliform Agar

HiCrome™ Candida Differential Agar Base with Candida Selective Supplement

833

HiCrome™ Candida HiVeg Agar Base, Modified wtih Candida Selective Supplement

Composition per liter:

Agar ............................................................................................ 15.0g Peptone, special .......................................................................... 15.0g Yeast extract.................................................................................. 4.0g Chromogenic mixture ............................................................... 7.220g K2HPO4 ........................................................................................ 1.0g Chloramphenicol........................................................................... 0.5g Gentamicin solution .................................................................. 1.0mL pH 7.2 ± 0.2 at 25°C

Agar ............................................................................................ 18.0g Glucose ....................................................................................... 10.0g Plant peptone ................................................................................ 5.0g Malt extract................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Chromogenic mixture ................................................................... 3.0g Chloramphenicol......................................................................... 0.05g Gentamicn solution................................................................... 1.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Candida Selective Supplement: Composition per 10.0mL: Gentamicin.................................................................................... 0.1g

Preparation of Candida Selective Supplement: Add gentamicin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Candida selective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Do not autoclave. Do not overheat. Cool to 50°C. Aseptically add 10.0mL Candida selective supplement. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Use: For the rapid isolation and identification of Candida species from mixed cultures.

Source: This medium is available as a premixed powder from HiMedia.

Candida Selective Supplement: Composition per 10.0mL: Gentamicin.................................................................................... 0.1g

Preparation of Candida Selective Supplement: Add gentamicin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except Candida selective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Do not autoclave. Do not overheat. Cool to 50°C. Aseptically add 10.0mL Candida selective supplement. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Use: For the rapid isolation and identification of Candida species from mixed cultures.

HiCrome™ Candida Differential Agar Base, Modified with Candida Selective Supplement

HiCrome™ Coliform Agar (Coliform Agar, HiCrome™)

Composition per liter: Agar ............................................................................................ 18.0g Glucose ....................................................................................... 10.0g Peptic digest of animal tissue........................................................ 5.0g Malt extract ................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Chromogenic mixture ................................................................... 3.0g Chloramphenicol......................................................................... 0.05g Gentamicin solution .................................................................. 1.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Candida Selective Supplement: Composition per 10.0mL: Gentamicin.................................................................................... 0.1g

Preparation of Candida Selective Supplement: Add gentamicin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Composition per liter: Agar ............................................................................................ 12.0g Sodium chloride............................................................................ 5.0g Peptone, special ............................................................................ 3.0g K2HPO4......................................................................................... 3.0g KH2PO4......................................................................................... 1.7g Sodium pyruvate........................................................................... 1.0g Tryptophan.................................................................................... 1.0g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulphate ................................................................. 0.1g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes.

Use: For the simultaneous detection of Escherichia coli and total coliforms in water and food samples. Sodium lauryl sulphate inhibits Gram-positive organisms. The chromogenic mixture contains two chromogenic substrates as Salmon-GAL and X-glucuronide. The enzyme β-D-galactosidase produced by coliforms cleaves SalmonGAL, resulting in the salmon to red coloration of coliform colonies. The enzyme β-D-glucuronidase produced by E. coli cleaves Xglucuronide. Escherichia coli forms dark blue to violet coloued colonies due to cleavage of both Salmon-GAL and X-glucuronide. The

834

HiCrome™ Coliform Agar with Novobiocin

addition of tryptophan improves the indole reaction, thereby increasing detection reliability in combination with the two chromogens.

HiCrome™ Coliform Agar with Novobiocin (Coliform Agar with Novobiocin, HiCrome™) Composition per liter: Agar ............................................................................................ 12.0g Sodium chloride ............................................................................ 5.0g Peptone, special ............................................................................ 3.0g K2HPO4 ......................................................................................... 3.0g KH2PO4 ......................................................................................... 1.7g Sodium pyruvate ........................................................................... 1.0g Tryptophan .................................................................................... 1.0g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulphate ................................................................. 0.1g Novobiocin................................................................................. 5.0mg pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

HiCrome™ Coliform Agar with SLS and Novobiocin Composition per liter: Agar ............................................................................................ 12.0g NaCl.............................................................................................. 5.0g K2HPO4......................................................................................... 3.0g Peptone, special ............................................................................ 3.0g KH2PO4......................................................................................... 1.7g Sodium pyruvate........................................................................... 1.0g Tryptophan.................................................................................... 1.0g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulfate.................................................................... 0.1g Novobiocin ................................................................................ 5.0mg pH 7.1 ± 0.2 at 25°C

Source: This medium, wihout novobiocin, is available as a premixed powder from HiMedia.

Media.

Use: For the simultaneous detection of Escherichia coli and total coli-

Preparation of Medium: Add components to distilled/deionized

forms in water and food samples when a high number of Gram-positive accompanying bacteria are expected.

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes.

HiCrome™ Coliform HiVeg Agar wtih SLS

Use: For the simultaneous detection of Escherichia coli and total coli-

Composition per liter:

forms in water and food samples when high numbers of Gram-positive bacteria may be present. Novobiocin and sodium lauryl sulphate inhibit Gram-positive organisms. The chromogenic mixture contains two chromogenic substrates as Salmon-GAL and X-glucuronide. The enzyme β-D-galactosidase produced by coliforms cleaves SalmonGAL, resulting in the salmon to red coloration of coliform colonies. The enzyme β-D-glucuronidase produced by E. coli, cleaves Xglucuronide. Escherichia coli forms dark blue to violet colored colonies due to cleavage of both Salmon-GAL and X-glucuronide. The addition of tryptophan improves the indole reaction, thereby increasing detection reliability in combination with the two chromogens.

Agar ............................................................................................ 12.0g NaCl.............................................................................................. 5.0g K2HPO4......................................................................................... 3.0g Plant special peptone .................................................................... 3.0g KH2PO4......................................................................................... 1.7g Sodium pyruvate........................................................................... 1.0g Tryptophan.................................................................................... 1.0g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulfate.................................................................... 0.1g pH 7.1 ± 0.2 at 25°C

HiCrome™ Coliform Agar with SLS Composition per liter: Agar ............................................................................................ 12.0g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 3.0g Peptone, special ............................................................................ 3.0g KH2PO4 ......................................................................................... 1.7g Sodium pyruvate ........................................................................... 1.0g Tryptophan .................................................................................... 1.0g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulfate .................................................................... 0.1g pH 7.1± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the simultaneous detection of Escherichia coli and total coliforms in water and food samples.

HiCrome™ E. coli Agar Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Casein enzymic hydrolysate ....................................................... 14.0g Agar ............................................................................................ 12.0g Peptone, special ............................................................................ 5.0g NaCl.............................................................................................. 2.4g Bile salts mixture .......................................................................... 1.5g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ...................................................................................... 0.6g X-Glucuronide .......................................................................... 0.075g pH 7.1 ± 0.2 at 25°C

Use: For the simultaneous detection of Escherichia coli and total coli-

Source: This medium is available as a premixed powder from Hi-

forms in water and food samples.

Media.

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

HiCrome™ ECC Agar

835

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from Hi-

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Media.

Use: For the simultaneous detection of Escherichia coli in foods without further confirmation on membrane filter or by indole reagent.

HiCrome™ E. coli Agar Composition per liter: Casein enzymic hydrolysate ....................................................... 20.0g Agar ............................................................................................ 15.0g Bile salts mixture ........................................................................ 1.50g X-Glucuronide ........................................................................... 7.5mg pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the simultaneous detection of Escherichia coli in foods without further confirmation on membrane filter or by indole reagent.

HiCrome™ E. coli Agar A (E. coli Agar A, HiCrome™)

Use: For the detection and enumeration of Escherichia coli in foods without further confirmation on membrane filter or by indole reagent. The chromogenic agent X-glucuronide used in this medium helps to detect glucuronidase activity. E. coli cells absorb X-glucuronide and the intracellular glucuronidase splits the bond between the chromophore and the glucuronide. The released chromophore gives coloration to the colonies. Bile salts mixture inhibits Gram-positive organisms.

HiCrome™ E. coli Agar, HiVeg Composition per liter: Plant hydrolysate ........................................................................ 14.0g Agar ............................................................................................ 12.0g Plant special peptone .................................................................... 5.0g NaCl.............................................................................................. 2.4g Synthetic detergent ....................................................................... 1.5g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ...................................................................................... 0.6g X-Glucuronide .......................................................................... 0.075g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Composition per liter:

Media.

Casein enzymic hydrolysate ....................................................... 14.0g Agar ............................................................................................ 12.0g Peptone, special ............................................................................ 5.0g NaCl .............................................................................................. 2.4g Bile salts mixture .......................................................................... 1.5g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ....................................................................................... 0.6g X-Glucuronide .......................................................................... 0.075g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from HiMedia.

HiCrome™ E. coli Agar B (E. coli Agar B, HiCrome™) Composition per liter: Casein enzymic hydrolysate ....................................................... 20.0g Agar ............................................................................................ 15.0g Bile salts mixture .......................................................................... 1.5g X-Glucuronide .......................................................................... 0.075g pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the simultaneous detection of Escherichia coli in foods without further confirmation on membrane filter or by indole reagent.

HiCrome™ EC O157:H7 Agar Composition per liter: Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ......................................................... 8.0g Sorbitol ......................................................................................... 7.0g Bile salts mixture .......................................................................... 1.5g Chromogenic mixture ................................................................. 0.25g Sodium lauryl sulfate.................................................................... 0.1g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the isolation and differentiation of Escherichia coli O157:H7 from food and recommended for selective isolation and easy detection of Escherichia coli O157:H7.

HiCrome™ ECC Agar (ECC Agar, HiCrome™) Composition per liter: Chromogenic mixture ................................................................. 20.3g Agar ............................................................................................ 15.0g

836

HiCrome™ ECC HiVeg Agar

Peptone, special ............................................................................ 5.0g NaCl .............................................................................................. 5.0g Na2HPO4 ....................................................................................... 3.5g Yeast extract.................................................................................. 3.0g Lactose .......................................................................................... 2.5g KH2PO4 ......................................................................................... 1.5g Neutral Red ................................................................................. 0.03g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the presumptive identification of Escherichia coli and other coliforms in food and environmental samples. A differential medium for presumptive identification of E. coli and other coliforms in food samples. The chromogenic mixture contains two chromogens as

X-glucuronide and Salmon-GAL. X-glucuronide is cleaved by the enzyme β-glucuronidase produced by E. coli. Salmon-GAL is cleaved by the enzyme galactosidase produced by the majority of coliforms, including E. coli.

HiCrome™ ECC HiVeg Agar Composition per liter: Chromogenic mixture ................................................................. 20.3g Agar ............................................................................................ 15.0g Plant special peptone .................................................................... 5.0g NaCl .............................................................................................. 5.0g Na2HPO4 ....................................................................................... 3.5g Yeast extract.................................................................................. 3.0g Lactose .......................................................................................... 2.5g KH2PO4 ......................................................................................... 1.5g Neutral Red ................................................................................. 0.03g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until components are completely dissolved (approximately 35 min). Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes. Medium may show haziness.

Use: For detection of Escherichia coli and coliforms in water and food samples.Tergitol inhibits Gram-positive as well as some Gram-negative bacteria other than coliforms. The chromogenic mixture contains two chromogenic substrates as Salmon-GAL and X-glucuronide. The enzyme b-D-galactosidase produced by coliforms cleaves SalmonGAL, resulting in the salmon to red coloration of coliform colonies. The enzyme β-D-glucuronidase produced by E. coli, cleaves Xglucuronide. E. coli forms dark blue to violet colored colonies due to cleavage of both Salmon-GAL and X-glucuronide. The addition of tryptophan improves the indole reaction.

HiCrome™ ECC Selective Agar (ECC Selective Agar, HiCrome™) Composition per liter: Agar ............................................................................................ 10.0g Peptone, special ............................................................................ 6.0g Casein enzymic hydrolysate ......................................................... 3.3g NaCl.............................................................................................. 2.0g Sodium pyruvate........................................................................... 1.0g Tryptophan.................................................................................... 1.0g Sorbitol ......................................................................................... 1.0g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ....................................................................................... 0.6g Tergitol 7® .................................................................................. 0.15g Chromogenic mixture ................................................................. 0.43g Cefsulodin.................................................................................. 5.0mg pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Media.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until components are completely dissolved (approximately 35 min.). Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes. Medium may show haziness.

Use: For the presumptive identification of Escherichia coli and other

Use: For detection of Escherichia coli and coliforms in water and food

coliforms in food and environmental samples.

samples using pour plate or streak plate methods.Tergitol inhibits Gram-positive as well as some Gram-negative bacteria other than coliforms. The cefsulodin inhibits Pseudomonas and Aeromonas species. The chromogenic mixture contains two chromogenic substrates as Salmon-GAL and X-glucuronide. The enzyme β-D-galactosidase produced by coliforms cleaves Salmon-GAL, resulting in the salmon to red coloration of coliform colonies. The enzyme β-D-glucuronidase produced by E. coli, cleaves X-glucuronide. E. coli forms dark blue to violet colored colonies due to cleavage of both Salmon-GAL and Xglucuronide. The addition of tryptophan improves the indole reaction.

HiCrome™ ECC Selective Agar (ECC Selective Agar, HiCrome™) Composition per liter: Agar ............................................................................................ 10.0g Peptone, special ............................................................................ 6.0g Casein enzymic hydrolysate ......................................................... 3.3g NaCl .............................................................................................. 2.0g Sodium pyruvate ........................................................................... 1.0g Tryptophan .................................................................................... 1.0g Sorbitol.......................................................................................... 1.0g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ....................................................................................... 0.6g Tergitol 7®................................................................................... 0.15g Chromogenic mixture ................................................................. 0.43g pH 6.8 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

HiCrome™ ECD HiVeg Agar with MUG

837

NaCl .............................................................................................. 2.0g Sodium pyruvate ........................................................................... 1.0g Tryptophan .................................................................................... 1.0g Sorbitol.......................................................................................... 1.0g Na2HPO4 ....................................................................................... 1.0g NaH2PO4 ....................................................................................... 0.6g Tergitol 7® .................................................................................. 0.15g Chromogenic mixture ................................................................. 0.43g Cefsulodin ................................................................................ 10.0mg pH 6.8 ± 0.2 at 25°C

Plant hydrolysate .......................................................................... 3.3g NaCl.............................................................................................. 2.0g Sodium dihydrogen phosphate ..................................................... 0.6g Na2HPO4 ....................................................................................... 1.0g Sodium pyruvate........................................................................... 1.0g Sorbitol ......................................................................................... 1.0g Tryptophan.................................................................................... 1.0g Chromogenic mixture ................................................................. 0.43g Tergitol 7..................................................................................... 0.15g pH 7.0± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Media.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until components are completely dissolved (approximately 35 min). Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes. Medium may show haziness.

water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation in boiling water bath or flowing steam. Boil until components are fully dissolved (approximately 35 min). Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes. Medium may show haziness. To inhibit Pseudomonas and Aeromonas species 5 mg cefsulodin may be added for surface and pour plate methods or 10 mg cefsulodin may be aseptically added to the medium for membrane filter technique.

Use: For detection of Escherichia coli and coliforms in water and food samples using the membrane filter technique.Tergitol inhibits Grampositive as well as some Gram-negative bacteria other than coliforms. The cefsulodin inhibits Pseudomonas and Aeromonas species. The chromogenic mixture contains two chromogenic substrates as SalmonGAL and X-glucuronide. The enzyme β-D-galactosidase produced by coliforms cleaves Salmon-GAL, resulting in the salmon to red coloration of coliform colonies. The enzyme β-D-glucuronidase produced by E. coli cleaves X-glucuronide. E. coli forms dark blue to violet colored colonies due to cleavage of both Salmon-GAL and Xglucuronide. The addition of tryptophan improves the indole reaction.

HiCrome™ ECC Selective Agar Base Composition per liter: Agar ............................................................................................ 10.0g Peptone, special ............................................................................ 6.0g Casein enzymic hydrolysate ......................................................... 3.3g NaCl .............................................................................................. 2.0g Na2HPO4 ....................................................................................... 1.0g Sodium pyruvate ........................................................................... 1.0g Sorbitol.......................................................................................... 1.0g Tryptophan .................................................................................... 1.0g Sodium dihydrogen phosphate...................................................... 0.6g Chromogenic mixture ................................................................. 0.43g Tergitol 7 ..................................................................................... 0.15g pH 7.0± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation in boiling water bath or flowing steam. Boil until components are fully dissolved (approximately 35 min). Do not autoclave. Cool to 45°–50°C. Pour into sterile Petri dishes. Medium may show haziness. To inhibit Pseudomonas and Aeromonas species 5 mg cefsulodin may be added for surface and pour plate methods or 10 mg cefsulodin may be aseptically added to the medium for the membrane filter technique.

Use: For the detection of Escherichia coli and coliforms in water and food samples.

HiCrome™ ECC Selective Agar Base, HiVeg Composition per liter: Agar ............................................................................................ 10.0g Plant special peptone .................................................................... 6.0g © 2010 by Taylor and Francis Group, LLC

Use: For the detection of Escherichia coli and coliforms in water and food samples.

HiCrome™ ECD Agar with MUG Composition per liter: Casein enzymic hydrolysate ....................................................... 20.0g Agar ............................................................................................ 15.0g Lactose.......................................................................................... 5.0g NaCl.............................................................................................. 5.0g K2HPO4......................................................................................... 4.0g Bile salts mixture .......................................................................... 1.5g KH2PO4......................................................................................... 1.5g L-Tryptophan ................................................................................ 1.0g Chromogenic substrate ................................................................. 0.1g Fluorogenic substrate.................................................................. 0.07g pH 7.0 ± 0.2 at 37°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For detection of Escherichia coli in a variety of specimens. Fluorescence in the UV and a positive indole test demonstrate the presence of E. coli in the colonies.

HiCrome™ ECD HiVeg Agar with MUG Composition per liter: Plant hydrolysate ........................................................................ 20.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 5.0g Lactose.......................................................................................... 5.0g K2HPO4......................................................................................... 4.0g KH2PO4......................................................................................... 1.5g Synthetic detergent ....................................................................... 1.5g Tryptophan.................................................................................... 1.0g Chromogenic substrate ................................................................. 0.1g Fluorogenic substrate.................................................................. 0.07g pH 7.0 ± 0.2 at 37°C

838

HiCrome™ Enrichment Broth Base for EC O157:H7

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components to distilled/deionized

Media.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For detection of Escherichia coli in a variety of specimens, including foods and water. Fluorescence in the UV and a positive indole test demonstrate the presence of E. coli in the colonies.

HiCrome™ Enrichment Broth Base for EC O157:H7 Composition per liter: Casein enzymic hydrolysate ....................................................... 10.0g Sorbitol........................................................................................ 10.0g Bile salts mixture .......................................................................... 1.5g Chromogenic mixture ................................................................... 1.3g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the enrichment culture of Escherichia coli O157:H7 in foods and environmental samples

HiCrome™ Enterobacter sakazakii Agar Composition per liter: Casein enzymic hydrolysate ....................................................... 15.0g Agar ............................................................................................ 15.0g Chromogenic mixture ............................................................... 10.17g Papaic digest of soybean meal ...................................................... 5.0g NaCl .............................................................................................. 5.0g Na2S2O3 ........................................................................................ 1.0g Sodium deoxycholate.................................................................... 0.5g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the isolation and identification of Enterobacter sakazakii from food and environmental samples. Meets the formulation recommended by ISO Committee for the isolation and identification of Enterobacter sakazakii from milk and milk products.

HiCrome™ Enterococci Broth (Enterococci HiCrome™ Broth) Composition per liter: Peptone, special .......................................................................... 10.0g NaCl.............................................................................................. 5.0g Polysorbate 80 .............................................................................. 2.0g NaH2PO4 ..................................................................................... 1.25g NaN3 ............................................................................................. 0.3g Chromogenic mixture ................................................................. 0.04g pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Sodium azide is toxic. It also has a tendency to form explosive metal azides with plumbing materials. It is advisable to use enough water to flush off the disposables. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the rapid and easy identification and differentiation of enterococci. It contains a chromogenic substrate which aids in the detection of enterococci, especially in water samples.

HiCrome™ Enterococci HiVeg Broth Composition per liter: Plant special peptone .................................................................. 10.0g NaCl.............................................................................................. 5.0g Polysorbate 80 .............................................................................. 2.0g Na2HPO4 ..................................................................................... 1.25g NaN3 ............................................................................................. 0.3g Chromogenic mixture ................................................................. 0.04g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the isolation and identification of Enterobacter sakazakii from food and environmental samples.

Caution: Sodium azide is toxic. It also has a tendency to form explosive metal azides with plumbing materials. It is advisable to use enough water to flush off the disposables.

HiCrome™ Enterobacter sakazakii Agar, Modified

Preparation of Medium: Add components to distilled/deionized

Composition per liter: Agar ............................................................................................ 15.0g Casein enzymatic hydrolysate ...................................................... 7.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Sodium deoxycholate.................................................................... 0.6g Chromogenic substrate ............................................................... 0.15g Crystal Violet .............................................................................. 0.02g pH 7.2 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the identification and differentiation of Enterococci from water samples.

HiCrome™ Enterococcus faecium Agar Base Composition per liter: Peptone, special .......................................................................... 23.0g Agar ............................................................................................ 15.0g Arabinose.................................................................................... 10.0g

HiCrome™ MacConkey-Sorbitol Agar

NaCl .............................................................................................. 5.0g Cornstarch ..................................................................................... 1.0g Chromogenic substrate ................................................................. 0.1g Phenol Red .................................................................................... 0.1g pH 7.2 ± 0.2 at 25°C

Use: For the selective isolation and easy detection of Klebsiella species from water and sewage.

HiCrome™ Listeria Agar Base, Modified, with Moxalactam (Listeria HiCrome Agar Base, Modified)

Source: This medium is available as a premixed powder from HiMedia.

Use: For the chromogenic identification of Enterococcus faecium from water and sewage samples.

HiCrome™ Improved Salmonella Agar Composition per liter: Agar ............................................................................................ 12.0g Peptone, special ............................................................................ 8.0g Chromogenic mixture ................................................................. 3.25g Yeast extract.................................................................................. 2.0g Sodium deoxycholate.................................................................... 1.0g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

839

Composition per liter: Peptone, special .......................................................................... 23.0g Agar ............................................................................................ 13.0g Rhamnose ................................................................................... 10.0g Chromogenic mixture ................................................................. 5.13g LiCl ............................................................................................... 5.0g Meat extract .................................................................................. 5.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 1.0g Phenol Red.................................................................................. 0.12g Moxolactam solution ...............................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Lithium chloride is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin, wash with plenty of water immediately.

Moxalactam Solution (Listeria Selective Supplement): Composition per 10.0mL: Moxolactam .................................................................................. 0.2g

Preparation of Moxalactam Solution (Listeria Selective Supplement): Add moxalactam to distilled/deionized water and

Use: For the improved selective and differential medium for Salmo-

bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

nella species.

Preparation of Medium: Add components, except moxalactam so-

HiCrome™ Klebsiella Selective Agar Base with Carbenicillin Composition per liter: Agar ............................................................................................ 15.0g Peptone, special .......................................................................... 12.0g Yeast extract.................................................................................. 7.0g NaCl .............................................................................................. 5.0g Bile salts mixture .......................................................................... 1.5g Chromogenic mixture ................................................................... 0.2g Sodium lauryl sulfate .................................................................... 0.1g Klebsiella selective supplement.................................................2.0mL pH 7.1 ± 0.2 at 25°C

Source: This medium, without Klebsiella selective supplement, is available as a premixed powder from HiMedia.

Klebsiella Selective Supplement: Composition per 2.0mL: Carbenicillin............................................................................. 50.0mg

Preparation of Klebsiella Selective Supplement: Add components to distilled/deionized water and bring volume to 2.0mL. Mix thoroughly. Filter sterilize.

lution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Add 10.0mL moxalactam solution (Listeria selective supplement). Mix well. Pour into sterile Petri dishes.

Use: For the rapid and direct identification of Listeria species, specifically Listeria monocytogenes. A selective and differential agar medium recommended for rapid and direct identification of Listeria species, specifically Listeria monocytogenes.

HiCrome™ MacConkey-Sorbitol Agar (MacConkey-Sorbitol Agar, HiCrome) Composition per liter: Casein enzymic hydrolysate ....................................................... 17.0g Agar ............................................................................................ 13.5g Sorbitol ....................................................................................... 10.0g NaCl.............................................................................................. 5.0g Proteose peptone........................................................................... 3.0g Bile salts mixture .......................................................................... 1.5g 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide sodium salt.............................................................................. 0.1g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................ 0.001g pH 7.1 ± 0.3 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

840

HiCrome™ MacConkey-Sorbitol Agar with Tellurite-Cefixime Supplement

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Mix to completely dissolve components. Do not autoclave. Cool to 50°C. Pour into sterile Petri dishes.

Use: For the direct isolation and differentiation of E. coli O157:H7 strains from foodstuffs. Recommended for selective isolation of Escherichia coli O157:H7 from food and animal feeds. The medium contains sorbitol instead of lactose. Enteropathogenic strains of Escherichia coli O157:H7 ferment lactose but do not ferment sorbitol and hence produce colorless colonies. Sorbitol fermenting strains of Escherichia coli produce pink-red colonies. The red color is due to production of acid from sorbitol, absorption of Neutral Red and a subsequent color change of the dye when the pH of the medium falls below 6.8. The chromogenic indicator is added to detect the presence of an enzyme β-D-glucuronidase. Strains of Escherichia coli possessing β-Dglucuronidase appear as blue colored colonies on the medium. Enteropathogenic strains of Escherichia coli O157 do not possess β-Dglucuronidase activity and thus produce colorless colonies. Escherichia coli fermenting sorbitol and possessing β-D-glucuronidase activity produce purple colored colonies. Most of the Gram-positive organisms are inhibited by Crystal Violet and bile salts.

HiCrome™ MacConkey-Sorbitol Agar with Tellurite-Cefixime Supplement (MacConkey-Sorbitol Agar with Tellurite-Cefixime Supplement) Composition per 1004.0mL: Casein enzymic hydrolysate ....................................................... 17.0g Agar ............................................................................................ 13.5g Sorbitol........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Proteose peptone ........................................................................... 3.0g Bile salts mixture .......................................................................... 1.5g 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide sodium salt.............................................................................. 0.1g Neutral Red ................................................................................. 0.03g Crystal Violet ............................................................................ 0.001g Tellurite-cefixime supplement ...................................................4.0mL pH 7.1 ± 0.3 at 25°C

Source: This medium, without tellurite-cefixime supplement, is available as a premixed powder from HiMedia.

Tellurite-Cefixime Supplement: Composition per 4.0mL: K2TeO3 ....................................................................................... 5.0mg Cefixime..................................................................................... 0.1mg

Preparation of Tellurite-Cefixime Supplement: Add components to 4.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Caution: Potassium tellurite is toxic. Source: This medium is available from Fluka, Sigma-Aldrich. Tellurite-cefixime supplement is available from Oxoid Unipath.

Preparation of Medium: Add components, except tellurite-cefixime supplement, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Mix to completely dissolve components. Do not autoclave. Cool to 50°C. Add 4.0mL sterile tellurite-cefixime supplement. Mix thoroughly. Pour into sterile Petri dishes. © 2010 by Taylor and Francis Group, LLC

Use: For the direct isolation and differentiation of E. coli O157:H7strains from foodstuffs. Recommended for selective isolation of Escherichia coli O157:H7 from food and animal feeding stuffs. The medium contains sorbitol instead of lactose. Enteropathogenic strains of Escherichia coli O157:H7 ferment lactose but do not ferment sorbitol and hence produce colorless colonies. Sorbitol fermenting strains of Escherichia coli produce pink-red colonies. The red color is due to production of acid from sorbitol, absorption of Neutral Red and a subsequent color change of the dye when the pH of the medium falls below 6.8. The chromogenic indicator is added to detect the presence of an enzyme β-D-glucuronidase. Strains of Escherichia coli possessing β-Dglucuronidase appear as blue colored colonies on the medium. Enteropathogenic strains of Escherichia coli O157 do not possess β-Dglucuronidase activity and thus produce colorless colonies. Escherichia coli fermenting sorbitol and possessing β-D-glucuronidase activity produce purple colored colonies. Most of the Gram-positive organisms are inhibited by Crystal Violet and bile salts. Addition of telluritecefixime supplement makes the medium selective. Potassium tellurite selects the serogroups O157 from other E. coli serogroups and inhibits Aeromonas species and Providencia species. Cefixime inhibits Proteus species.

HiCrome™ M-CP Agar Base (M-CP HiCrome™ Agar Base) (Membrane Clostridium perfringens HiCrome™ Agar Base) Composition per liter: Tryptose ...................................................................................... 30.0g Yeast extract................................................................................ 20.0g Agar ............................................................................................ 15.0g Sucrose.......................................................................................... 5.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g FeCl3·6H2O ................................................................................. 0.09g Indoxyl-β-D-glucoside ................................................................ 0.06g Bromcresol Purple ...................................................................... 0.04g pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the detection of Clostridium perfringens. Recommended by the Directive of the Council of the European Union 98/83/EC for isolation and enumeration of C. perfringens from water samples using the membrane filtration technique.

HiCrome™ MeReSa Agar with Methicillin Composition per liter: NaCl........................................................................................... 40.0g Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ...................................................... 13.0 g Chromogenic mixture .................................................................. 5.3 g Sodium pyruvate.......................................................................... 5.0 g Beef extract.................................................................................. 2.5 g Yeast extract................................................................................. 2.5 g MRSA selective supplement....................................................10.0mL pH 7.0 ± 0.2 at 25°C

HiCrome™ MS.O157 Agar

841

Source: This medium, without MRSA supplement, is available as a

Source: This medium is available as a premixed powder from Hi-

premixed powder from HiMedia.

Media.

MRSA Selective Supplement: Composition per 10.0mL:

Preparation of Medium: Add components to distilled/deionized

Methicillin.................................................................................. 4.0mg

Preparation of MRSA Selective Supplement: Add methicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the differentiation and enumeration of Escherichia coli and other coliforms by the membrane filtration method.

Preparation of Medium: Add components, except MRSA supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL MRSA supplement. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation and cultivation of methicillin-resistant Staphylococcus aureus (MRSA).

HiCrome™ MeReSa Agar with Oxacillin Composition per liter: NaCl ........................................................................................... 40.0g Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ...................................................... 13.0 g Chromogenic mixture .................................................................. 5.3 g Sodium pyruvate .......................................................................... 5.0 g Beef extract .................................................................................. 2.5 g Yeast extract................................................................................. 2.5 g MRSA selective supplement....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without MRSA supplement, is available as a premixed powder from HiMedia.

MRSA Selective Supplement: Composition per 10.0mL: Oxacillin..................................................................................... 2.0mg

Preparation of MRSA Selective Supplement: Add oxacillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the isolation and cultivation of methicillin-resistant Staphylococcus aureus (MRSA).

HiCrome™ M-Lauryl Sulfate Agar Composition per liter: Peptic digest of animal tissue...................................................... 40.0g Lactose ........................................................................................ 30.0g Agar ............................................................................................ 10.0g Yeast extract.................................................................................. 6.0g Sodium lauryl sulfate .................................................................... 1.0g Sodium pyruvate ........................................................................... 0.5g Chromogen.................................................................................... 0.2g Phenol Red .................................................................................... 0.2g pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

HiCrome™ MM Agar (HiCrome™ Miller and Mallinson Agar) (MM Agar HiCrome™) Composition per liter: Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g Peptic digest of animal tissue ..................................................... 10.0g Chromogenic mixture ................................................................... 6.6g D-Cellobiose.................................................................................. 3.0g Beef extract................................................................................... 2.0g D-Trehalose ................................................................................. 1.33g D-Mannitol .................................................................................... 1.2g pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the identification and differentiation of Salmonella and nonSalmonella like Citrobacter from water samples.

HiCrome™ MM HiVeg Agar Composition per liter: Agar ............................................................................................ 15.0g Plant peptone .............................................................................. 10.0g Lactose........................................................................................ 10.0g Chromogenic mixture ................................................................... 6.6g D-Cellobiose.................................................................................. 3.0g Plant extract .................................................................................. 2.0g D-Trehalose ................................................................................. 1.33g D-Mannitol .................................................................................... 1.2g pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the identification and differentiation of Salmonella and nonSalmonella like Citrobacter from water samples.

HiCrome™ MS.O157 Agar (MS.O157 Agar HiCrome™) Composition per liter: Agar ............................................................................................ 12.0g Peptone, special .......................................................................... 10.0g Sorbitol ......................................................................................... 4.0g

842

HiCrome™ MS.O157 Agar with Tellurite

Bile salt mixture............................................................................ 1.0g Chromogenic mixture ............................................................... 0.731g pH 6.8 ± 0.2 at 25°C

Sodium lauryl sulfate.................................................................... 0.2g Sodium deoxycholate.................................................................... 0.1g pH 7.3 ± 0.2 at 25°C

Source: This medium is available from Fluka, Sigma-Aldrich.

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components to distilled/deionized

Media.

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Do not autoclave. Cool to 50°C. Pour into sterile Petri dishes.

Use: For the simultaneous detection of Escherichia coli, Escherichia coli O157:H7 and coliforms in water and food samples. Escherichia coli O157:H7 gives colorless colonies because of non-fermentation of sorbitol and absence of β-glucuronidase activity, whereas other strains of Escherichia coli having β-glucuronidase activity and fermenting sorbitol appear as steel blue colored colonies. Some non Escherichia coli O157:H7 may have some colony color.

HiCrome™ MS.O157 Agar with Tellurite (MS.O157 Agar with Tellurite, HiCrome™) Composition 1000.25mL: Agar ............................................................................................ 12.0g Peptone, special .......................................................................... 10.0g Sorbitol.......................................................................................... 4.0g Bile salt mixture............................................................................ 1.0g Chromogenic mixture ............................................................... 0.731g Tellurite solution ......................................................................0.25mL pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Tellurite Solution: Composition per 10.0mL: K2TeO3 .......................................................................................... 0.1g

Preparation of Tellurite Solution: Add K2TeO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Caution: Potassium tellurite is toxic. Preparation of Medium: Add components, exept tellurite solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Do not autoclave. Cool to 45°C. Aseptically add 0.25mL sterile tellurite solution. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the simultaneous detection of Escherichia coli, Escherichia coli O157:H7, and coliforms in water and food samples. Escherichia coli O157:H7 gives colorless colonies because of non-fermentation of sorbitol and absence of β-glucuronidase activity, whereas other strains of Escherichia coli having β-glucuronidase activity and fermenting sorbitol appear as steel blue colored colonies. Addition of tellurite makes the medium much more specific and selective.

HiCrome™ M-TEC Agar Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 7.5g Proteose peptone ........................................................................... 5.0g K2HPO4 ......................................................................................... 3.3g Yeast extract.................................................................................. 3.0g KH2PO4 ......................................................................................... 1.0g Chromogen.................................................................................... 0.5g © 2010 by Taylor and Francis Group, LLC

Use: For the differentiation and enumeration of thermotolerant E. coli from water by the membrane filtration method.

HiCrome Nickels and Leesment Medium Composition per liter: Casein enzymatic hydrolysate .................................................... 18.0g Agar ............................................................................................ 15.0g Calcium lactate pentahydrate........................................................ 8.0g Tricalcium dicitrate tetrahydrate................................................. 6.65g Yeast extract.................................................................................. 4.5g Glucose ......................................................................................... 4.5g Lactose.......................................................................................... 4.5g NaCl.............................................................................................. 3.6g Gelatin......................................................................................... 2.25g Trisodium citrate dihydrate........................................................... 1.8g Carboxymethyl cellulose .............................................................. 0.4g Chromogenic substrate (X-gal)..................................................... 0.2g Selective supplement solution .................................................10.0mL pH 6.65 ± 0.05 at 25°C

Source: This medium is available from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Vancomycin .................................................................................. 0.2g

Preparation of Selective Supplement Solution: Add vancomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the enumeration of citrate-fermenting lactic acid bacteria from milk.

HiCrome™ OGYE Agar Base (OGYE Agar Base HiCrome) (Oxytetracycline Glucose Yeast Extract Agar HiCrome) Composition per liter: Glucose ...................................................................................... 20.0g Agar ............................................................................................ 12.0g Yeast extract ................................................................................. 4.0g Chromogenic mixture .................................................................. 1.1g Oxytetracycline selective supplement .....................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

HiCrome™ Salmonella Agar

Oxytetracycline Selective Supplement: Composition per 10.0mL:

843

Use: For the identification and differentiation of Salmonella species from the members of Enterobacteriaceae, especially Proteus species.

Oxytetracycline ............................................................................. 0.1g

HiCrome™ Rapid Coliform Broth (Coliform Rapid HiCrome™ Broth) (Rapid Coliform HiCrome™ Broth)

Preparation of Oxytetracycline Selective Supplement: Add 0.1g oxytetracycline to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except oxytetracycline selective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL sterile oxytetracycline selective supplement. Mix thoroughly. Pour into sterile Petri dishes. Use: For the rapid isolation of yeasts and molds from milk and milk products. Oxytetracycline makes the medium more selective by inhibiting the growth of lactobacilli. Aspergillus niger appears as light blue colored colonies with black spores due to the presence of chromogenic mixture; Candida albicans shows green colored colonies and Saccharomyces cerevisiae gives colorless colonies.

Composition per liter: Peptone, special ............................................................................ 5.0g NaCl.............................................................................................. 5.0g Na2HPO4 ....................................................................................... 2.7g KH2PO4......................................................................................... 2.0g Sorbitol ......................................................................................... 1.0g Sodium lauryl sulfate.................................................................... 0.1g IPTG ............................................................................................. 0.1g Chromogenic substrate ............................................................... 0.08g Fluorogenic substrate.................................................................. 0.05g pH 6.8 ± 0.3 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

HiCrome™ RajHans Medium (Salmonella Agar) Composition per liter: Agar ............................................................................................ 13.5g Casein enzymic hydrolysate ......................................................... 8.0g Chromogenic mixture ................................................................... 7.3g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Peptone.......................................................................................... 4.0g Lactose .......................................................................................... 3.0g Sodium deoxycholate.................................................................... 1.0g Neutral Red ................................................................................. 0.02g pH 7.3 ± 0.2 at 25°C

Use: For the identification and differentiation of Salmonella species from the members of Enterobacteriaceae, especially Proteus species.

HiCrome™ RajHans Medium, Modified (Salmonella Agar, Modified) Composition per liter: Agar ............................................................................................ 12.0g Casein enzymic hydrolysate ......................................................... 8.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Chromogenic mixture ................................................................. 4.32g Peptic digest of animal tissue........................................................ 4.0g Lactose .......................................................................................... 3.0g Sodium deoxycholate.................................................................... 1.0g Neutral Red ................................................................................. 0.02g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Do not autoclave. Do not overheat. Cool to 50°C. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes. © 2010 by Taylor and Francis Group, LLC

Use: For the detection and confirmation of Escherichia coli and coliforms on the basis of enzyme substrate reaction from water samples, using a combination of chromogenic and fluorogenic substrate.

HiCrome™ Rapid Enterococci Agar (Enterococci Rapid HiCrome™ Agar) (Rapid Enterococci HiCrome™ Agar) Composition per liter: Agar ............................................................................................ 15.0g Peptone special ........................................................................... 10.0g NaCl.............................................................................................. 5.0g Polysorbate 80 .............................................................................. 2.0g Na2HPO4 ..................................................................................... 1.25g NaN3 ............................................................................................. 0.3g Chromogenic mixture ................................................................. 0.06g pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Sodium azide is toxic. It also has a tendency to form explosive metal azides with plumbing materials. It is advisable to use enough water to flush off the disposables.

Use: For the rapid and easy identification and differentiation of enterococci. It contains a chromogenic substrate, which aids in the detection of enterococci, especially from water samples.

HiCrome™ Salmonella Agar Composition per liter: Agar ............................................................................................ 13.0g Peptic digest of animal tissue ....................................................... 6.0g Chromogenic mixture ................................................................... 5.4g

844

HiCrome™ Salmonella Agar

Yeast extract.................................................................................. 2.5g Bile salts mixture .......................................................................... 1.0g pH 7.3 ± 0.2 at 25°C

stirring and bring to boiling. Mix to completely dissolve components. Do not autoclave. Mix and boil in 5 min sequences for 35–40 min. Cool to 50°C. Shake gently for 30–35 min. Pour into sterile Petri dishes.

Source: This medium is available as a premixed powder from Hi-

Use: A selective medium used for the detectgion of Salmonella species. This medium exploits a novel phenotypic characteristic of Salmonella spp.: the formation of acid from propylene glycol. This characteristic may be used in combination with a chromogenic indicator of βgalactosidase to differentiate Salmonella spp. from Proteus spp. and the other members of the Enterobacteriaceae. Deoxycholate is included in the plate medium as an inhibitor of Gram-positive organisms. Nontyphi Salmonella spp. yield distinct, bright red colonies on this medium, allowing facilitated identification and unambiguous differentiation from Proteus spp. Coliforms produce blue-green to blue-violet colonies. Other Enterobacteriaceae and Gram-negative bacteria such as Proteus, Shigella, Pseudomonas, Salmonella typhi, and S. paratyphi A form colorless or yellow colonies.

Media.

Use: For the simultaneous detection of Escherichia coli and Salmonella from food and water. For the identification, differentiation, and confirmation of enteric bacteria from specimens such as urine, water, or food which may contain large numbers of Proteus species as well as potentially pathogenic Gram-positive organisms.

HiCrome™ Salmonella Agar (Salmonella Agar, HiCrome™)

HiCrome™ UTI Agar, HiVeg

Composition per liter:

Agar ............................................................................................ 13.0g Peptic digest of animal tissue........................................................ 6.0g Chromogenic mixture ................................................................... 5.4g Yeast extract.................................................................................. 2.5g Chromogenic mix ......................................................................... 1.5g Bile salt mixture............................................................................ 1.0g pH 7.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components to distilled/deionized

Media.

Use: A selective chromogenic medium used for the isolation and differentiation of Salmonella species from coliforms in food and water. E. coli and Salmonella are easily distinguishable due to the colony characteristics. Salmonella give light purple colonies with a halo. E. coli have a characteristic blue color. Other organisms give colorless colonies. The characteristic light purple and blue color is due to the chromogenic mixture. Chromogenic medium for detecting and identifying Enterobacteria, Proteus species, and other Gram-positive organisms.

HiCrome™ Salmonella Chromogen Agar (Salmonella Chromogen Agar, HiCrome™) (Rambach Equivalent Agar) Composition per liter: Agar ............................................................................................ 15.0g Peptic digest of animal tissue........................................................ 5.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 2.0g Meat extract .................................................................................. 1.0g Na-deoxycholate ........................................................................... 1.0g Chromogenic mixture ................................................................. 1 vial pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Source: This medium is available as a premixed powder from HiMedia. water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the differentiation and enumeration of thermotolerant E. coli from water by the membrane filtration method. For the identification, differentiation, and confirmation of enteric bacteria from specimens such as urine, water, or food which may contain large numbers of Proteus species as well as potentially pathogenic Gram-positive organisms.

HiCrome™ UTI Agar, Modified Composition per liter: Peptic digest of animal tissue ..................................................... 18.0g Agar ............................................................................................ 15.0g Chromogenic mixture ............................................................... 12.44g Beef extract................................................................................... 6.0g Casein enzymic hydrolysate ......................................................... 4.0g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Hippea Medium

HiCrome™ UTI Agar, Modified (UTI Agar, Modified HiCrome™) Composition per liter: Peptic digest of animal tissue...................................................... 18.0g Agar ............................................................................................ 15.0g Chromogenic mixture ............................................................... 12.44g Beef extract ................................................................................... 4.0g Casein enzymatic hydrolysate ...................................................... 4.0g pH 7.2 ± 0.2 at 25°C

845

High Salt Nutrient Agar Composition per liter: NaCl............................................................................................ 30.0g Agar ............................................................................................ 15.0g Peptic digest of animal tissue ....................................................... 5.0g Meat extract .................................................................................. 5.0g pH 8.5 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized

Media.

Preparation of Medium: Add components to distilled/deionized

Use: For the isolation and cultivation of salt-tolerant Vibrio species.

Source: This medium is available as a premixed powder from Hi-

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Mix to completely dissolve components. Do not autoclave. Cool to 50°C. Pour into sterile Petri dishes.

Use: A chromogenic medium used for detecting and identifying Enterobacteria, Proteus species, and other bacteria involved in urinary tract infections.

HiFluoro™ Pseudomonas Agar Base Composition per liter: Pancreatic digest of gelatin ......................................................... 18.0g Agar ............................................................................................ 15.0g K2SO4 .......................................................................................... 10.0g Fluorogenic mixture.................................................................... 2.05g MnCl2 ............................................................................................ 1.4g Cetrimide ...................................................................................... 0.3g Glycerol ......................................................................................10.ml pH 7.2 ± 0.2 at 25°C

Source: This medium, wihout glycerol, is available as a premixed powder from HiMedia.

Use: For the selective isolation of Pseudomonas aeruginosa from clinical and nonclinical specimens by the fluorogenic method.

High Plate Count Agar Composition per liter: Agar ............................................................................................ 15.0g Peptic digest of animal tissue........................................................ 3.0g Casein, soluble .............................................................................. 0.5g K2HPO4 ......................................................................................... 0.2g MgSO4·7H2O .............................................................................. 0.05g FeCl3·4H2O ................................................................................ 1.0mg pH 7.2 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

High Salt Peptone Yeast Extract Agar Composition per liter: NaCl............................................................................................ 30.0g Agar ............................................................................................ 15.0g Peptic digest of animal tissue ..................................................... 10.0g Yeast extract.................................................................................. 6.0g Meat extract .................................................................................. 2.0g Glucose ......................................................................................... 2.0g L-Cysteine·HCl·H2O ..................................................................... 0.3g pH 7.5 ± 0.2 at 25°C

Use: For the confirmation of Vibrio species.

Hippea Medium (DSMZ Medium 854) Composition per 1010.0mL: NaCl............................................................................................ 25.0g Sulfur, powdered......................................................................... 10.0g Na-acetate ..................................................................................... 5.0g MOPS [3-(N-morpholino) propane sulfonic acid] .......................................................................... 3.0g Na2S·9H2O.................................................................................... 0.5g NH4Cl ......................................................................................... 0.33g CaCl2·2H2O ................................................................................ 0.33g MgCl2·6H2O ............................................................................... 0.33g KCl.............................................................................................. 0.33g KH2PO4....................................................................................... 0.33g Yeast extract.................................................................................. 0.1g Resazurin ................................................................................... 0.5mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 6.1 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g

846

Hippurate Hydrolysis Broth

CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Add components, except vitamin solution, sulfur, and Na2S·9H2O, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0. Sparge the medium with 80% N2 + 20% CO2 gas mixture for 30 min. Add Na2S·9H2O. Mix thoroughly. Readjust the pH to 6.0–6.2. Dispense medium under 80% N2 + 20% CO2 gas mixture into anaerobe tubes or bottles containing 100.0mg sulfur powder per 10mL medium. Autoclave 20 min at 110°C. Prior to use inject 0.1mL sterile vitamin solution per 10.0mL medium.

Use: For the cultivation of Hippea maritima. Hippurate Broth See: Sodium Hippurate Broth

Hippurate Hydrolysis Broth Composition per liter: Heart infusion powder ................................................................ 10.0g Peptic digest of animal tissue...................................................... 10.0g Sodium hippurate ........................................................................ 10.0g NaCl .............................................................................................. 5.0g pH 7.4 ± 0.2 at 25°C

Use: For the detection of hippurate-hydrolyzing bacteria.

Hirschia Medium Composition per liter: Pancreatic digest of casein ............................................................ 5.0g HEPES .......................................................................................... 4.0g © 2010 by Taylor and Francis Group, LLC

Yeast extract.................................................................................. 2.0g Artificial seawater..................................................................250.0mL Glucose solution ....................................................................100.0mL pH 7.4 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 0.25g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Artificial Seawater: Composition per liter: NaCl............................................................................................ 27.5g MgCl2·6H2O ............................................................................... 5.38g MgSO4·7H2O .............................................................................. 6.78g KCl.............................................................................................. 0.72g NaHCO3 ....................................................................................... 0.2g CaCL2·2H2O ................................................................................. 1.4g

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Aseptically add 100.0mL of sterile glucose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Hirschia baltica.

Hi-Sensitivity Test Agar Composition per liter: Casein enzymic hydrolysate ....................................................... 11.0g Agar .............................................................................................. 8.0g NaCl.............................................................................................. 3.0g Peptic digest of animal tissue ....................................................... 3.0g Glucose ......................................................................................... 2.0g Na2HPO4 ....................................................................................... 2.0g Sodium acetate.............................................................................. 1.0g Starch, soluble............................................................................... 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-Cystine hydrochloride ............................................................. 0.02g L-Tryptophan .............................................................................. 0.02g Adenine....................................................................................... 0.01g Guanine....................................................................................... 0.01g Uracil .......................................................................................... 0.01g Xanthine...................................................................................... 0.01g Calcium pantothenate ................................................................ 3.0mg Biotin ......................................................................................... 3.0mg Nicotinamide.............................................................................. 3.0mg Pyridoxine hydrochloride .......................................................... 3.0mg Manganese chloride ................................................................... 2.0mg ZnSO4 ........................................................................................ 1.0mg CoSO4 ........................................................................................ 1.0mg CuSO4 ........................................................................................ 1.0mg Cyanocobalamin ........................................................................ 1.0mg FeSO4 ......................................................................................... 1.0mg Menadione ................................................................................. 1.0mg Thiamine hydrochloride........................................................... 0.04mg pH 7.2 ± 0.2 at 25°C

Hi-Sensitivity Test HiVeg Broth Source: This medium is available as a premixed powder from HiMedia.

Use: For antimicrobial susceptibility tests.

Hi-Sensitivity Test Broth Composition per liter: Casein enzymic hydrolysate ....................................................... 11.0g NaCl .............................................................................................. 3.0g Peptic digest of animal tissue........................................................ 3.0g Glucose ......................................................................................... 2.0g Na2HPO4 ....................................................................................... 2.0g Sodium acetate .............................................................................. 1.0g Starch, soluble............................................................................... 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-Cystine hydrochloride.............................................................. 0.02g L-Tryptophan............................................................................... 0.02g Adenine ....................................................................................... 0.01g Guanine ....................................................................................... 0.01g Uracil .......................................................................................... 0.01g Xanthine...................................................................................... 0.01g Calcium pantothenate ................................................................ 3.0mg Biotin ......................................................................................... 3.0mg Nicotinamide.............................................................................. 3.0mg Pyridoxine hydrochloride .......................................................... 3.0mg Manganese chloride ................................................................... 2.0mg ZnSO4 ........................................................................................ 1.0mg CoSO4 ........................................................................................ 1.0mg CuSO4 ........................................................................................ 1.0mg Cyanocobalamin ........................................................................ 1.0mg FeSO4 ......................................................................................... 1.0mg Menadione ................................................................................. 1.0mg Thiamine hydrochloride........................................................... 0.04mg pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For antimicrobial susceptibility testing.

Hi-Sensitivity Test HiVeg Agar Composition per liter: Plant hydrolysate......................................................................... 11.0g Agar .............................................................................................. 8.0g NaCl .............................................................................................. 3.0g Plant peptone................................................................................. 3.0g Glucose ......................................................................................... 2.0g Na2HPO4 ....................................................................................... 2.0g Sodium acetate .............................................................................. 1.0g Starch, soluble............................................................................... 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-Cystine hydrochloride.............................................................. 0.02g © 2010 by Taylor and Francis Group, LLC

847

L-Tryptophan .............................................................................. 0.02g Adenine....................................................................................... 0.01g Guanine....................................................................................... 0.01g Uracil .......................................................................................... 0.01g Xanthine...................................................................................... 0.01g Calcium pantothenate ................................................................ 3.0mg Biotin ......................................................................................... 3.0mg Nicotinamide.............................................................................. 3.0mg Pyridoxine hydrochloride .......................................................... 3.0mg Manganese chloride ................................................................... 2.0mg ZnSO4 ........................................................................................ 1.0mg CoSO4 ........................................................................................ 1.0mg CuSO4 ........................................................................................ 1.0mg Cyanocobalamin ........................................................................ 1.0mg FeSO4 ......................................................................................... 1.0mg Menadione ................................................................................. 1.0mg Thiamine hydrochloride........................................................... 0.04mg pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For antimicrobial susceptibility tests.

Hi-Sensitivity Test HiVeg Broth Composition per liter: Plant hydrolysate ........................................................................ 11.0g NaCl.............................................................................................. 3.0g Plant peptone ................................................................................ 3.0g Glucose ......................................................................................... 2.0g Na2HPO4 ....................................................................................... 2.0g Sodium acetate.............................................................................. 1.0g Starch, soluble............................................................................... 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-Cystine hydrochloride ............................................................. 0.02g L-Tryptophan .............................................................................. 0.02g Adenine....................................................................................... 0.01g Guanine....................................................................................... 0.01g Uracil .......................................................................................... 0.01g Xanthine...................................................................................... 0.01g Calcium pantothenate ................................................................ 3.0mg Biotin ......................................................................................... 3.0mg Nicotinamide.............................................................................. 3.0mg Pyridoxine hydrochloride .......................................................... 3.0mg Manganese chloride ................................................................... 2.0mg ZnSO4 ........................................................................................ 1.0mg CoSO4 ........................................................................................ 1.0mg CuSO4 ........................................................................................ 1.0mg Cyanocobalamin ........................................................................ 1.0mg FeSO4 ......................................................................................... 1.0mg Menadione ................................................................................. 1.0mg Thiamine hydrochloride........................................................... 0.04mg pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring

848

Hisitest Agar

to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For antimicrobial susceptibility testing.

Hisitest Agar Composition per liter: Casein enzymic hydrolysate ....................................................... 11.0g Agar .............................................................................................. 8.0g Buffer salt...................................................................................... 3.3g Peptic digest of animal tissue........................................................ 3.0g NaCl .............................................................................................. 3.0g Glucose ......................................................................................... 2.0g Starch ............................................................................................ 1.0g Nucleoside basis ......................................................................... 0.02g Thiamine .................................................................................. 0.02mg pH 7.2 ± 0.2 at 25°C

Solution 6...................................................................................0.1mL Solution 7...................................................................................0.1mL pH 6.5 ± 0.2 at 25°C

Solution 1: Composition per liter: KH2PO4......................................................................................... 8.0g (NH4)2SO4 .................................................................................... 8.0g MgSO4·7H2O .............................................................................. 0.86g CaCl2, anhydrous ........................................................................ 0.08g ZnSO4·7H2O ............................................................................... 0.05g

Media.

FeSO4·7H2O.................................................................................. 5.7g MnCl2·6H2O ................................................................................. 0.8g NaMoO4·2H2O............................................................................ 0.15g HCl, concentrated ......................................................................1.0mL

Preparation of Medium: Add components to distilled/deionized

Preparation of Solution 2: Add 1.0mL of concentrated HCl to

Source: This medium is available as a premixed powder from Hi-

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For determination of antibiotic susceptibility of fastidious microorganisms.

Histidans Agar Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 10.0g Yeast extract................................................................................ 10.0g Na2HPO4 ..................................................................................... 0.95g KH2PO4 ....................................................................................... 0.91g MgSO4·7H2O ................................................................................ 0.5g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Streptomyces species.

Histoplasma capsulatum Agar Composition per liter: Agar ............................................................................................ 12.5g Glucose ....................................................................................... 10.0g Citric acid.................................................................................... 10.0g Potato starch.................................................................................. 2.0g α-Ketoglutaric acid ....................................................................... 1.0g L-Cystine·HCl·H2O ....................................................................... 1.0g Glutathione, reduced ..................................................................... 0.5g L-Asparagine ................................................................................. 0.1g L-Tryptophan ............................................................................... 0.02g Solution 1 ...............................................................................250.0mL Solution 3 .................................................................................40.0mL Solution 2 .................................................................................10.0mL Solution 4 .................................................................................10.0mL Solution 8 .................................................................................10.0mL Solution 5 ...................................................................................1.0mL © 2010 by Taylor and Francis Group, LLC

100.0mL of distilled water in a 1.0L volumetric flask. Dissolve each component completely in the sequence given. Bring volume to 1.0L with distilled/deionized water. Store at 5°C. Discard if red color or red precipitate appears.

Solution 3: Composition per 100.0mL: Casein, acid-hydrolyzed, vitamin-free........................................ 10.0g

Preparation of Solution 3: Add casein to distilled/deionized water and bring volume to 100.0mL.

Solution 4: Composition per liter: Calcium pantothenate ................................................................... 0.2g Inositol .......................................................................................... 0.2g Riboflavin ..................................................................................... 0.2g Thiamine·HCl ............................................................................... 0.2g Nicotinamide................................................................................. 0.1g Biotin .......................................................................................... 0.01g

Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Store at −20°C. Solution 5: Composition per 100.0mL: Hemin ........................................................................................... 0.2g NH4OH, concentrated ................................................................0.3mL

Preparation of Solution 5: Add hemin to approximately 30.0mL of distilled/deionized water. Add NH4OH. Mix thoroughly until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5° C. Solution 6: Composition per 10.0mL: DL-Thioctic

acid .......................................................................... 0.01g Ethanol (95% solution) ............................................................10.0mL

Preparation of Solution 6: Add DL-thioctic acid to 10.0mL of ethanol. Mix thoroughly. Store at −20°C.

Solution 7: Composition per 10.0mL: Coenzyme A ............................................................................... 0.01g Na2S·5H2O (0.05% solution) .....................................................0.2mL

Histoplasma capsulatum Agar Preparation of Solution 7: Prepare Na2S·5H2O solution in freshly boiled distilled/deionized water. Add coenzyme A to 9.8mL of distilled/deionized water. Mix thoroughly. Add freshly prepared Na2S·5H2O solution. Mix thoroughly. Store the solution at −20°C. Solution 8: Composition per 100.0mL: Oleic acid ...................................................................................... 0.1g

Preparation of Solution 8: Add oleic acid to 50.0mL of distilled/

849

Preparation of Solution 2: Add the 1.0mL of concentrated HCl to 100.0mL of distilled water in a 1.0L volumetric flask. Dissolve each component completely in the sequence given. Bring volume to 1.0L with distilled/deionized water. Store at 5°C. Discard if red color or red precipitate appears.

Solution 3: Composition per 100.0mL: Casein, acid-hydrolyzed, vitamin-free........................................ 10.0g

deionized water. Adjust pH to 9.0 with NaOH. Gently heat until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5°C.

Preparation of Solution 3: Add casein to distilled/deionized water

Preparation of Medium: Add components—except agar, potato

Solution 4: Composition per liter:

starch, and solution 8—to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 6.5 with 20% KOH solution. Filter sterilize. In a separate flask, add potato starch to 50.0mL of distilled/deionized water. Add the starch solution to 450.0mL of boiling distilled/deionized water. Add 10.0mL of solution 8 and the agar. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 70°C. Aseptically combine the two sterile solutions. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Histoplasma capsulatum in the yeast phase. For the cultivation of Histoplasma duboisii, Blastomyces dermatitidis, and Sprotrichum schenckii.

Histoplasma capsulatum Agar Composition per liter: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Potato starch.................................................................................. 2.0g α-Ketoglutaric acid ....................................................................... 1.0g L-Cystine·HCl·H2O ....................................................................... 1.0g Glutathione, reduced ..................................................................... 0.5g L-Asparagine ................................................................................. 0.1g L-Tryptophan ............................................................................... 0.02g Solution 1 ...............................................................................250.0mL Solution 3.................................................................................40.0mL Solution 2.................................................................................10.0mL Solution 4.................................................................................10.0mL Solution 8.................................................................................10.0mL Solution 5...................................................................................1.0mL Solution 6...................................................................................0.1mL Solution 7...................................................................................0.1mL pH 6.5 ± 0.2 at 25°C

Solution 1: Composition per liter: KH2PO4 ......................................................................................... 8.0g (NH4)2SO4 ..................................................................................... 8.0g MgSO4·7H2O .............................................................................. 0.86g CaCl2, anhydrous ........................................................................ 0.08g

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Store at 5°C.

Solution 2: Composition per liter: FeSO4·7H2O.................................................................................. 5.7g MnCl2·6H2O.................................................................................. 0.8g NaMoO4·2H2O............................................................................ 0.15g HCl, concentrated ......................................................................1.0mL © 2010 by Taylor and Francis Group, LLC

and bring volume to 100.0mL. Do not use enzymatically digested casein.

Calcium pantothenate ................................................................... 0.2g Inositol .......................................................................................... 0.2g Riboflavin ..................................................................................... 0.2g Thiamine·HCl ............................................................................... 0.2g Nicotinamide................................................................................. 0.1g Biotin .......................................................................................... 0.01g

Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Store at −20°C. Solution 5: Composition per 100.0mL: Hemin ........................................................................................... 0.2g NH4OH, concentrated................................................................0.3mL

acid .......................................................................... 0.01g Ethanol (95% solution) ............................................................10.0mL

Preparation of Solution 6: Add

DL-thioctic acid to 10.0mL of ethanol. Mix thoroughly. Store solution at −20°C.

Preparation of Solution 7: Prepare Na2S·5H2O solution in freshly boiled distilled/deionized water. Add coenzyme A to 9.8mL of distilled/deionized water. Mix thoroughly. Add freshly prepared Na2S·5H2O solution. Mix thoroughly. Store the solution at −20°C. Solution 8: Composition per 100.0mL: Oleic acid ...................................................................................... 0.1g

Preparation of Solution 8: Add oleic acid to 50.0mL of distilled/ deionized water. Adjust pH to 9.0 with NaOH. Gently heat until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5°C.

Preparation of Medium: Add components—except agar, potato starch, and solution 8—to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 6.5 with 20% KOH solution. Filter sterilize. In a separate flask, add potato starch to 50.0mL of distilled/deionized water. Add the starch solution to 450.0mL of boiling distilled/deionized water. Add 10.0mL of solution 8 and the agar. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool

850

Histoplasma capsulatum Broth

to 70°C. Aseptically combine the two sterile solutions. Pour into sterile Petri dishes or distribute into sterile tubes.

Nicotinamide................................................................................. 0.1g Biotin .......................................................................................... 0.01g

Use: For the cultivation and maintenance of Histoplasma capsulatum in the mycelial phase.

Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Store at −20°C.

Histoplasma capsulatum Broth Composition per liter: Glucose ....................................................................................... 10.0g Citric acid.................................................................................... 10.0g α-Ketoglutaric acid ....................................................................... 1.0g L-Cystine·HCl·H2O ....................................................................... 1.0g Potato starch.................................................................................. 0.5g Glutathione, reduced ..................................................................... 0.5g L-Asparagine ................................................................................. 0.1g L-Tryptophan ............................................................................... 0.02g Solution 1 ...............................................................................250.0mL Solution 3 .................................................................................40.0mL Solution 2 .................................................................................10.0mL Solution 4 .................................................................................10.0mL Solution 5 ...................................................................................1.0mL Solution 8 ...................................................................................1.0mL Solution 6 ...................................................................................0.1mL Solution 7 ...................................................................................0.1mL pH 6.5 ± 0.2 at 25°C

Solution 1: Composition per liter: KH2PO4 ......................................................................................... 8.0g (NH4)2SO4 ..................................................................................... 8.0g MgSO4·7H2O .............................................................................. 0.86g CaCl2, anhydrous ........................................................................ 0.08g ZnSO4·7H2O ............................................................................... 0.05g

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Store at 5°C. Solution 2: Composition per liter: FeSO4·7H2O.................................................................................. 5.7g MnCl2·6H2O.................................................................................. 0.8g NaMoO4·2H2O............................................................................ 0.15g HCl, concentrated ......................................................................1.0mL

Preparation of Solution 2: Add 1.0mL of concentrated HCl to 100.0mL of distilled water in a 1.0L volumetric flask. Dissolve each component completely in the sequence given. Bring volume to 1.0L with distilled/deionized water. Store at 5°C. Discard if red color or red precipitate appears. Solution 3: Composition per 100.0mL: Casein, acid-hydrolyzed, vitamin-free........................................ 10.0g

Preparation of Solution 3: Add casein to distilled/deionized water and bring volume to 100.0mL. Do not use enzymatically digested casein.

Solution 5: Composition per 100.0mL: Hemin ........................................................................................... 0.2g NH4OH, concentrated ................................................................0.3mL

acid .......................................................................... 0.01g Ethanol (95% solution) ............................................................10.0mL

Preparation of Solution 6: Add DL-thioctic acid to 10.0mL of ethanol. Mix thoroughly. Store solution at −20°C.

Preparation of Solution 8: Add oleic acid to 50.0mL of distilled/deionized water. Adjust pH to 9.0 with NaOH. Gently heat until dissolved. Bring volume to 100.0mL with distilled/deionized water. Store at 5°C.

Preparation of Medium: Add components—except potato starch and solution 8—to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 6.5 with 20% KOH solution. Filter sterilize. In a separate flask, add potato starch to 50.0mL of distilled/deionized water. Add the starch solution to 450.0mL of boiling distilled/deionized water. Add 1.0mL of solution 8. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 70°C. Aseptically combine the two sterile solutions. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Histoplasma capsulatum in the yeast phase. For the cultivation of Histoplasma duboisii, Blastomyces dermatitidis, and Sprotrichum schenckii.

HiVeg Hydrolysate Agar with 2.5% Agar Composition per liter: Agar ............................................................................................ 25.0g Plant hydrolysate .......................................................................... 5.0g Plant peptone ................................................................................ 5.0g NaCl.............................................................................................. 5.0g Na2HPO4 ...................................................................................... 2.5g Plant infusion ................................................................................ 1.5g Yeast autolysate ............................................................................ 1.5g Glycerol ...................................................................................22.0mL pH 7.8 ± 0.2 at 25°C

HNS Agar

851

Source: This medium, without glycerol, is available as a premixed powder from HiMedia.

Columbia Blood Top Agar: Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Agar ............................................................................................ 13.5g Pancreatic digest of casein.......................................................... 12.0g NaCl.............................................................................................. 5.0g Peptic digest of animal tissue ....................................................... 5.0g Beef extract................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Cornstarch..................................................................................... 1.0g Horse blood, defibrinated ........................................................50.0mL

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Vibrio cholerae. For the production of cholera vaccine.

HiVeg Magnesium Broth Composition per liter: Plant hydrolysate......................................................................... 10.0g NaCl .............................................................................................. 5.0g MgSO4 ........................................................................................ 0.94g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of recombinant strains of Escherichia coli.

HiVeg Peptone Water Composition per liter: Plant peptone............................................................................... 10.0g NaCl .............................................................................................. 5.0g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

Preparation of Columbia Blood Top Agar: Add components, except horse blood, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse blood. Mix thoroughly.

Preparation of Medium: Pour cooled, sterile Columbia agar base into sterile Petri dishes in 10.0mL volumes. Allow agar to solidify. Pour 5.0mL of cooled, sterile Columbia blood top agar over Columbia agar base that has solidified but is still warm. Use: For the cultivation of Listeria monocytogenes.

HM Medium Composition per liter: NaCl............................................................................................ 81.0g Yeast extract................................................................................ 10.0g MgSO4 .......................................................................................... 9.6g MgCl2............................................................................................ 7.0g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.36g NaHCO3 ................................................................................... 60.0mg NaBr......................................................................................... 26.0mg pH 7.1 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized wa-

Use: For the cultivation of various bacteria.

Use: For the cultivation of Salinicoccus roseus and Salinicoccus hispani-

ter and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. cus.

HL Agar Composition per plate: Columbia agar base..................................................................10.0mL Columbia blood top agar............................................................5.0mL pH 7.3 ± 0.2 at 25°C

Columbia Agar Base: Composition per liter: Agar ............................................................................................ 13.5g Pancreatic digest of casein .......................................................... 12.0g NaCl .............................................................................................. 5.0g Peptic digest of animal tissue........................................................ 5.0g Beef extract ................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Cornstarch ..................................................................................... 1.0g

Preparation of Columbia Agar Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. © 2010 by Taylor and Francis Group, LLC

HNS Agar (ATCC Medium 923) Composition per liter: Agar ............................................................................................ 15.0g NaCl.............................................................................................. 9.6g Heart infusion broth...............................................................990.0mL Horse serum .............................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Heart Infusion Broth: Composition per 900.0mL: Beef heart, infusion from.......................................................... 500.0g Tryptose ...................................................................................... 10.0g NaCl.............................................................................................. 5.0g

Preparation of Heart Infusion Broth: Add agar and NaCl to 990.0mL heart infusion broth. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL sterile horse serum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

852

HNW Medium

Use: For the cultivation and maintenance of Corynebacterium species.

HNW Medium (DSMZ Medium 997) Composition per liter: DMJ synthetic seawater ................................................................1.0L Vitamin solution.......................................................................10.0mL NaHCO3solution ......................................................................10.0mL NaNO3solution.........................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Tungstate solution ....................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Tungstate Solution: Composition per 10.0mL: Na2WO4·2H2O ........................................................................... 0.1mg

Preparation of Tungstate Solution: Add Na2WO4·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% H2. Filter sterilize. NaNO3 Solution: Composition per 10.0mL: NaNO3........................................................................................... 1.0g

Preparation of NaNO3 Solution: Add NaNO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

MgSO4·7H2O ................................................................................ 3.4g KCl.............................................................................................. 0.33g NH4Cl ........................................................................................ 0.25g K2HPO4 ...................................................................................... 0.14g CaCl2·2H2O ................................................................................ 0.14g Fe(NH4)2(SO4)2·6H2O ............................................................... 0.01g NiCl2·6H2O ................................................................................ 0.5mg Na2SeO3·5H2O........................................................................... 0.5mg Trace elements solution SL-10 ................................................10.0mL

Trace Elements Solution SL-10: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution SL-10: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/ deionized water to 1.0L. Mix thoroughly. Adjust pH to 7.0.

Preparation of DMJ Synthetic Seawater: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Aseptically add 10.0mL each of vitamin solution, NaHCO3solution, NaNO3solution, Na2S·9H2O solution, and tungstate solution to 1.0L sterile DMJ synthetic seawater. Mix thoroughly. Distribute into tubes. Tightly seal the tubes with butyl rubber stoppers under a gas phase of 80% H2 + 20% CO2 (300 kPa).

Use: For the cultivation of Persephonella hydrogeniphila and Hydrogenivirga caldilitoris.

Hofer’s Alkaline Medium Composition per liter: Agar ............................................................................................ 15.0g Mannitol...................................................................................... 10.0g Yeast extract.................................................................................. 1.0g K2HPO4......................................................................................... 0.5g MgSO4.......................................................................................... 0.2g NaCl.............................................................................................. 0.1g Thymol Blue ............................................................................. 0.016g pH 11.0 ± 0.2 at 25°C

Use: For the selective isolation of Agrobacterium spp. from soil.

Horikoshi-1 Medium with 10% Sodium Chloride

853

Hohn’s Medium, Modified See: Steenken and Smith Agar

KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g pH 9.0 ± 0.2 at 25°C

HO-LE Trace Elements Solution

Preparation of Medium: Add components to distilled/deionized

Composition per liter: H3BO3 ......................................................................................... 2.85g MnCl2·4H2O.................................................................................. 1.8g Sodium tartrate............................................................................ 1.77g FeSO4 .......................................................................................... 1.36g CoCl2·6H2O ................................................................................ 0.04g CuCl2·2H2O .............................................................................. 0.026g Na2MoO4·2H2O ........................................................................ 0.025g ZnCl2 ......................................................................................... 0.021g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For use as an enrichment to other media that require trace minerals.

Hominis Agar See: H Agar Hominis Broth See: H Broth

Horie Arabinose Ethyl Violet Broth (HAEB) Composition per liter: NaCl ............................................................................................ 30.0g Peptone.......................................................................................... 5.0g Beef extract ................................................................................... 3.0g Bromthymol Blue ....................................................................... 0.03g Ethyl Violet ................................................................................ 1.0mg Arabinose solution .................................................................100.0mL pH 9.0 ± 0.2 at 25°C

Arabinose Solution: Composition per 100.0mL: Arabinose ...................................................................................... 5.0g

Preparation of Arabinose Solution: Add arabinose to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except arabinose solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 9.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile arabinose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Vibrio species from foods.

Horikoshi Alkaline Medium (DSMZ Medium 940) Composition per liter: Agar ............................................................................................ 15.0g D-glucose..................................................................................... 10.0g Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Na2CO3 ......................................................................................... 5.0g © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Pannonibacter phragmitetus.

Horikoshi-1 Medium (DSMZ Medium 1081) Composition per liter: Agar ............................................................................................ 15.0g Glucose ...................................................................................... 10.0g Polypeptone .................................................................................. 5.0g Yeast extract ................................................................................. 5.0g KH2PO4 ........................................................................................ 1.0g K2HPO4 ........................................................................................ 1.0g MgSO4·7H2O ................................................................................ 0.2g NaCO3solution.......................................................................100.0mL pH 10.0 ± 0.2 at 25°C

NaCO3 Solution: Composition per 100.0mL: NaCO3 ......................................................................................... 10.0g

Preparation of NaCO3 Solution: Add NaCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except NaCO3 solu-

tion, to double distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Adjust pH to 10.0. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 100.0mL NaCO3 solution. Adjust pH to 10.0. Pour into Petri dishes or aseptically distribute into tubes.

Use: For the cultivation of “Streptomyces sannurensis” and Salinicoccus alkaliphilus.

Horikoshi-1 Medium with 10% Sodium Chloride (DSMZ Medium 1081a) Composition per liter: NaCl.......................................................................................... 100.0g Agar ............................................................................................ 15.0g Glucose ...................................................................................... 10.0g Polypeptone .................................................................................. 5.0g Yeast extract ................................................................................. 5.0g KH2PO4 ........................................................................................ 1.0g K2HPO4 ........................................................................................ 1.0g MgSO4·7H2O ................................................................................ 0.2g NaCO3solution.......................................................................100.0mL pH 10.0 ± 0.2 at 25°C

NaCO3 Solution: Composition per 100.0mL: NaCO3 ......................................................................................... 10.0g

Preparation of NaCO3 Solution: Add NaCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

854

Horse Blood Agar

Preparation of Medium: Add components, except NaCO3 solu-

Use: For the cultivation of Salinicoccus alkaliphilus.

Horse Blood Agar Composition per liter: Beef heart, infusion from .......................................................... 500.0g Agar ............................................................................................ 15.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Horse blood, defibrinated ........................................................50.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse blood, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse blood. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Yersinia pseudotuberculosis.

Horse Serum Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of gelatin ........................................................... 5.0g Beef extract ................................................................................... 3.0g Horse serum ...........................................................................200.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse serum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Pseudomonas aeruginosa and Streptobacillus moniliformis.

Hottinger Broth Composition per liter: Fish peptone................................................................................ 20.0g Yeast extract.................................................................................. 2.0g Tryptophan.................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For cultivation of less fastidious microorganisms and determination of indole as per USSR State Pharmacopoeia.

Howardella Medium (DSMZ Medium 1085) Composition per liter: Casitone ..................................................................................... 20.0g Yeast extract ................................................................................. 5.0g Na2HPO4 ...................................................................................... 5.0g MgCl2·6H2O ................................................................................. 1.1g Urea .............................................................................................. 1.0g Na-thioglycolate ........................................................................ 0.75g Resazurin .................................................................................. 0.5mg Urea solution............................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Urea Solution: Composition per 10.0mL: Urea .............................................................................................. 1.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Add components, except thioglycolate and urea solution, to double distilled/deionized water and bring volume to 990.0mL. Cool to room temperature while sparging with 80% N2 + 20% CO2. Add thioglycolate. Mix thoroughly. Distribute into tubes or bottles under an atmosphere of 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL urea solution. Adjust pH to 7.4. Use: For the cultivation of Howardella spp.

Horse Serum Broth Composition per liter: Pancreatic digest of gelatin ........................................................... 5.0g Beef extract ................................................................................... 3.0g Horse serum ...........................................................................200.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Pseudomonas aeruginosa and Streptobacillus moniliformis. © 2010 by Taylor and Francis Group, LLC

Hoyer’s Medium Composition per liter: (NH4)2SO4 .................................................................................... 1.0g KH2PO4......................................................................................... 0.9g MgSO4·7H2O .............................................................................. 0.25g K2HPO4......................................................................................... 0.1g FeCl3·6H2O ................................................................................. 0.02g Ethanol solution .....................................................................200.0mL

Ethanol Solution: Composition per 200.0mL: Ethanol.....................................................................................30.0mL

Preparation of Ethanol Solution: Add ethanol to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Filter sterilize.

Hoyle Medium Base Preparation of Medium: Add components, except ethanol solution, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Distribute into tubes in 4.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 1.0mL of sterile ethanol solution to each tube. Mix thoroughly.

Use: For the cultivation of Acetobacter species.

Hoyle Medium Composition per 1060.0mL: Agar ............................................................................................ 15.0g Lab Lemco powder ..................................................................... 10.0g Peptone........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Horse blood, laked ...................................................................50.0mL Tellurite solution ......................................................................10.0mL pH 7.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Horse Blood, Laked: Composition per 50.0mL: Horse blood, fresh....................................................................50.0mL

Preparation of Horse Blood, Laked: Add blood to a sterile poly-

Preparation of Horse Blood, Laked: Add blood to a sterile polypropylene bottle. Freeze overnight at −20°C. Thaw at 8°C. Refreeze at −20°C. Thaw again at 8°C.

Tellurite Solution: Composition per 100.0mL: K2TeO3 .......................................................................................... 3.5g

Preparation of Tellurite Solution: Add K2TeO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Caution: Potassium tellurite is toxic. Preparation of Medium: Add components, except laked horse blood and tellurite solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 50.0mL sterile laked horse blood and 10.0mL sterile tellurite solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and differentiation of Corynebacterium diphtheriae strains. This medium permits very rapid growth of all types of Corynebacterium diphtheriae, so that diagnosis is possible after 18 hours’ incubation.

propylene bottle. Freeze overnight at −20°C. Thaw at 8°C. Refreeze at −20°C. Thaw again at 8°C.

Tellurite Solution: Composition per 100.0mL: K2TeO3 .......................................................................................... 3.5g

Preparation of Tellurite Solution: Add K2TeO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Caution: Potassium tellurite is toxic. Preparation of Medium: Add components, except laked horse blood and tellurite solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 50.0mL sterile laked horse blood and 10.0mL sterile tellurite solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Hoyle HiVeg Medium Base Composition per liter: Agar ............................................................................................ 15.0g Plant extract ................................................................................ 10.0g Plant peptone............................................................................... 10.0g NaCl .............................................................................................. 5.0g Horse blood, laked ...................................................................50.0mL Tellurite solution ......................................................................10.0mL pH 7.8 ± 0.2 at 25°C

Source: This medium, without tellurite solution and laked blood, is available as a premixed powder from HiMedia.

855

Hoyle Medium Base Composition per liter: Agar ............................................................................................ 15.0g Beef extract................................................................................. 10.0g Peptone ....................................................................................... 10.0g NaCl.............................................................................................. 5.0g Blood, laked.............................................................................50.0mL Tellurite solution ......................................................................10.0mL pH 7.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Tellurite Solution: Composition per 100.0mL: K2TeO3 .......................................................................................... 3.5g

Caution: Potassium tellurite is toxic. Preparation of Tellurite Solution: Add K2TeO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Horse Blood, Laked: Composition per 50.0mL: Horse blood, fresh....................................................................50.0mL

Preparation of Horse Blood, Laked: Add blood to a sterile polypropylene bottle. Freeze overnight at −20°C. Thaw at 8°C. Refreeze at −20°C. Thaw again at 8°C.

Preparation of Medium: Add components, except laked blood, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile laked blood. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Horse Blood, Laked: Composition per 50.0mL:

Use: For the isolation and differentiation of Corynebacterium diphthe-

Horse blood, fresh....................................................................50.0mL

riae.

856

HP 6 Agar

Preparation of Medium: Add components, except glucose solu-

Agar ............................................................................................ 15.0g Sodium glutaminate .................................................................... 10.0g MgSO4·7H2O ................................................................................ 1.0g Yeast extract.................................................................................. 1.0g Cyanocobalamin ........................................................................ 0.5mg Glucose solution ....................................................................100.0mL pH 7.2 ± 0.2 at 25°C

Use: For the isolation and cultivation of Cytophaga, Herpetosiphon, Saprospira, and Flexithrix species.

Composition per liter:

Glucose Solution: Composition per 100.0mL: D-Glucose ...................................................................................... 5.0g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Cytophaga species, Herpetosiphon species, Saprospira species, and Flexithrix species.

HP 6 Agar Base Composition per liter: Plant hydrolysate......................................................................... 15.0g Glucose ......................................................................................... 5.5g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 2.5g Agar .............................................................................................. 1.0g Sodium hydrosulphite ................................................................... 0.5g Resazurin ................................................................................... 1.0mg pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Cytophaga species, Herpetosiphon species, Saprospira species, and Flexithrix species.

HP 6 Agar Base Composition per liter: Agar ............................................................................................ 15.0g Sodium glutaminate .................................................................... 10.0g MgSO4·7H2O ................................................................................ 1.0g Yeast extract.................................................................................. 1.0g Cyanocobalamin ........................................................................ 0.5mg Glucose solution ....................................................................100.0mL

Source: This medium is available from HiMedia. Glucose Solution: Composition per 100.0mL:

tion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add glucose solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

HP 74 Broth Composition per liter: Sodium glutaminate .................................................................... 10.0g MgSO4·7H2O ................................................................................ 2.0g Yeast extract.................................................................................. 2.0g Glucose solution ....................................................................100.0mL Phosphate buffer solution ........................................................20.0mL pH 6.5 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: D-Glucose .................................................................................... 10.0g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Phosphate Buffer Solution: Composition per 100.0mL: K2HPO4....................................................................................... 6.81g

Preparation of Phosphate Buffer Solution: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 6.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Add components, except glucose solution and phosphate buffer solution, to distilled/deionized water and bring volume to 880.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 100.0mL of sterile glucose solution and 20.0mL of sterile phosphate buffer solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the isolation and cultivation of Cytophaga species, Herpetosiphon species, Saprospira species, and Flexithrix species.

HP 101 Halophile Medium Composition per liter: NaCl.......................................................................................... 100.0g Agar ............................................................................................ 20.0g Peptone ....................................................................................... 10.0g MgSO4·7H2O ................................................................................ 4.3g NaNO3 .......................................................................................... 2.0g Yeast extract.................................................................................. 1.0g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Pseudomonas species.

Glucose ......................................................................................... 5.0g

HP Medium

Preparation of Glucose Solution: Add glucose to distilled/deion-

Composition per liter:

ized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Pancreatic digest of soybean meal.............................................. 20.0g Beef extract................................................................................. 10.0g

HQGö1 Medium

Yeast extract.................................................................................. 6.0g Ammonium citrate ........................................................................ 5.0g Tween™ 80 ................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g MnSO4·4H2O .............................................................................. 0.05g FeSO4·7H2O................................................................................ 0.04g Glucose solution ......................................................................10.0mL Tetracycline solution................................................................10.0mL

Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 10.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Tetracycline Solution: Composition per 100.0mL: Tetracycline................................................................................. 10.0g

Preparation of Tetracycline Solution: Add tetracycline to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except glucose solution and tetracycline solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution and tetracycline solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and enumeration of Leuconostoc species. HPC Agar See: NWRI Agar

HPC Agar (Heterotrophic Plate Count Agar) (m-HPC Agar) Composition per liter: Gelatin......................................................................................... 25.0g Pancreatic digest of gelatin ......................................................... 20.0g Agar ............................................................................................ 15.0g Glycerol ...................................................................................10.0mL pH 7.1 ± 0.2 at 25°C

857

KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................10.0mL Butanediol solution..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Vitamin solution.......................................................................10.0mL Gentisic acid solution ................................................................1.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................. 0.36g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Butanediol Solution: Composition per 10.0mL:

Source: This medium is available from BD Diagnostic Systems.

2,3 butanediol ............................................................................... 0.9g

Preparation of Medium: Add components, except glycerol, to dis-

Preparation of Butanediol Solution: Add butanediol to distilled/

tilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Add glycerol. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Pour into sterile Petri dishes.

Gentisic Acid Solution: Composition per 100.0mL:

Use: For the the cultivation and enumeration of microorganisms from potable water sources, swimming pools, and other water specimens by the membrane filter method and heterotrophic plate count technique.

HQGö1 Medium (DSMZ Medium 298a) Composition per liter: NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g © 2010 by Taylor and Francis Group, LLC

deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Gentisic acid ............................................................................... 3.08g

Preparation of Gentisic Acid Solution: Add gentisic acid to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.0. Sparge with 100% N2. Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg

858

HR Antifungal Assay Medium Buffered with MOPS

NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except NaHCO3 solution, butanediol solution, Na2S·9H2O solution, vitamin solution, gentisic acid solution, and trace elements solution SL-10, to distilled/ deionized water and bring volume to 958.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL NaHCO3 solution, 10.0mL butanediol solution, 10.0mL Na2S·9H2O solution, 10.0mL vitamin solution, 1.0mL gentisic acid solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% N2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Syntrophus gentianae.

HR Antifungal Assay Medium Buffered with MOPS Composition per liter: MOPS (3-N-morpholinopropanesulfonic acid) buffer............................................... 34.53g Glucose ....................................................................................... 10.0g (NH4)2SO4 ..................................................................................... 2.5g KH2PO4 ......................................................................................... 1.0g NaHCO3 ........................................................................................ 1.0g Glutamine.................................................................................... 0.58g MgSO4·7H2O ................................................................................ 0.5g CaCl2·2H2O................................................................................... 0.1g NaCl .............................................................................................. 0.1g L-Lysine....................................................................................... 0.07g L-Isoleucine ................................................................................. 0.05g L-Leucine..................................................................................... 0.05g L-Threonine ................................................................................. 0.05g L-Valine ....................................................................................... 0.05g L-Arginine ................................................................................... 0.04g L-Histidine................................................................................... 0.02g L-Methionine ............................................................................... 0.01g L-Tryptophan .............................................................................. 8.2mg DL-Methionine............................................................................ 2.0mg DL-Tryptophan............................................................................ 2.0mg Inositol ....................................................................................... 2.0mg L-Histidine·HCl .......................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg Calcium pantothenate ................................................................ 0.4mg MnSO4·H2O ............................................................................... 0.4mg Niacin......................................................................................... 0.4mg Pyridoxine .................................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg ZnSO4·7H2O .............................................................................. 0.4mg p-Aminobenzoic acid ................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Riboflavin .................................................................................. 0.2mg Na2MoO3.................................................................................... 0.2mg © 2010 by Taylor and Francis Group, LLC

KI ............................................................................................... 0.1mg CuSO4·5H2O ............................................................................ 0.04mg Biotin .......................................................................................... 2.0μg Folic acid .................................................................................... 2.0μg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except NaHCO3 and

MOPS buffer, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Add NaHCO3 and MOPS buffer. Mix thoroughly. Adjust pH to 7.0. Bring volume to 1.0L with distilled/deionized water. Filter sterilize.

Use: For testing the effectiveness of antifungal agents against clinical fungal isolates using the broth dilution susceptibility testing method.

HS HiVeg Medium Plant hydrolysate ........................................................................ 15.0g Glucose ......................................................................................... 5.5g Yeast extract.................................................................................. 5.0g Agar .............................................................................................. 1.0g NaCl.............................................................................................. 2.5g Na2S2O4 ........................................................................................ 0.5g Resazurin ................................................................................. 0.001g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For cultivation of aerobic as well as anaerobic bacteria and sterility testing.

HS Medium Casein enzymic hydrolysate ....................................................... 15.0g Glucose ......................................................................................... 5.5g Yeast extract.................................................................................. 5.0g Agar ............................................................................................. 1.0g NaCl.............................................................................................. 2.5g Na2S2O4 ........................................................................................ 0.5g Resazurin ................................................................................. 0.001g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For cultivation of aerobic as well as anaerobic bacteria and sterility testing.

HTM See: Haemophilus Test Medium

Hugh Leifson Glucose Broth Composition per liter: NaCl............................................................................................ 30.0g Glucose ....................................................................................... 10.0g Agar .............................................................................................. 3.0g Peptone ......................................................................................... 2.0g

Hungate’s Habitat-Simulating Medium

Yeast extract.................................................................................. 0.5g Bromcresol Purple .................................................................... 0.015g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Adjust pH to 7.4. Distibute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria based on their ability to ferment glucose. Bacteria that ferment glucose turn the medium yellow.

Hugh Leifson Glucose HiVeg Medium Composition per liter: NaCl ............................................................................................ 30.0g Glucose ....................................................................................... 10.0g Agar .............................................................................................. 3.0g Plant peptone................................................................................. 2.0g Yeast extract.................................................................................. 0.5g Bromcresol Purple .................................................................... 0.015g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Adjust pH to 7.4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria based on their ability to ferment glucose. Bacteria that ferment glucose turn the medium yellow.

Hugh Leifson HiVeg Medium Composition per liter: Glucose ....................................................................................... 10.0g NaCl .............................................................................................. 5.0g Agar .............................................................................................. 2.0g Plant peptone................................................................................. 2.0g K2HPO4 ......................................................................................... 0.3g Bromthymol Blue ....................................................................... 0.05g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Adjust pH to 7.4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria based on their ability to ferment glucose. Bacteria that ferment glucose turn the medium yellow.

Hugh Leifson Oxidation-Fermentation Medium See: Oxidation-Fermentation Medium, Hugh-Leifson Human Blood Tween™ Bilayer Medium See: HBT Bilayer Medium

Hungate’s Habitat-Simulating Medium Composition per 1140.2mL: Rumen fluid ...........................................................................333.0mL Mineral solution A .................................................................167.0mL © 2010 by Taylor and Francis Group, LLC

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Mineral solution B .................................................................167.0mL NaHCO3 solution.....................................................................53.0mL L-Cysteine·HCl solution ..........................................................10.6mL Substrate solution.....................................................................10.6mL Resazurin solution .....................................................................1.0mL

Mineral Solution A: Composition per liter: NaCl.............................................................................................. 6.0g KH2PO4......................................................................................... 3.0g (NH4)2SO4 .................................................................................... 3.0g CaCl2 ............................................................................................. 0.6g MgSO4 .......................................................................................... 0.6g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Mineral Solution B: K2HPO4........................................................................................... 3.0

Preparation of Solution B: Add K2HPO4 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Resazurin Solution: Composition per 100.0mL: Resazurin ...................................................................................... 0.1g

Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 100.0L. Mix thoroughly. L-Cysteine·HCl

Solution: Composition per 100.0mL:

L-Cysteine·HCl ............................................................................. 3.0g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to O2-free distilled/deionized water and bring volume to 100.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 2 min. Cool to 25°C under 100% N2. Seal tube with a stopper that is wired in place. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to O2-free distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Gas with 100% CO2 for 15 min. Substrate Solution: Composition per 100.0mL: Sugar ........................................................................................... 10.0g

Preparation of Substrate Solution: Add sugar to O2-free dis-

tilled/deionized water. Mix thoroughly. Gas with 100% N2 for 15 min. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Medium: Add 167.0mL of solution A, 167.0mL of solution B, and 1.0mL of resazurin solution to distilled/deionized water and bring volume to 733.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until resazurin turns colorless, indicating reduction. Bring volume back to 733.0mL (some evaporation will have occurred) with O2-free distilled/deionized water. Cool to 45°–50°C under O2-free 100% CO2. Anaerobically add rumen fluid. Anaerobically distribute into tubes in 10.0mL volumes. Cap with butyl rubber stoppers. Place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Immediately prior to inoculation, aseptically and anaerobically add 0.1mL of sterile L-cysteine·HCl solution, 0.5mL of sterile NaHCO3 solution, and 0.1mL of substrate solution per 10.0mL of medium in each tube.

860

Hutner’s Medium for Euglena

Use: For the cultivation of Bacteroides species from rumens.

Hutner’s Medium for Euglena

boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Flavobacterium species.

Composition per liter: Agar, noble.................................................................................. 12.0g Pancreatic digest of peptone ......................................................... 0.6g Yeast extract.................................................................................. 0.4g KH2PO4 ....................................................................................... 0.02g Potassium citrate·H2O................................................................. 0.04g MgSO4·3H2O .............................................................................. 0.02g Thiamine .................................................................................... 0.4mg Vitamin B12 .................................................................................0.5μg

Use: For the cultivation of Astasia longa, Euglena gracilis, Polytoma species, Polytomella parva, and Polytomella caeca.

Hutner’s Medium for Euglena Composition per liter: Agar, noble.................................................................................. 12.0g Pancreatic digest of peptone ......................................................... 0.6g Liver concentrate .......................................................................... 0.2g Potassium citrate·H2O................................................................. 0.04g KH2PO4 ....................................................................................... 0.02g MgSO4·3H2O .............................................................................. 0.02g

Use: For the cultivation of Astasia longa, Euglena gracilis, Polytoma species, Polytomella parva, and Polytomella caeca.

HY Agar for Flavobacterium Composition per liter: Agar .............................................................................................. 8.0g Glutamic acid ................................................................................ 5.0g K2HPO4 ......................................................................................... 0.1g MgSO4·7H2O ................................................................................ 0.1g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Glutamic acid may be replaced by 1.0g of folic acid if desired. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Flavobacterium species.

HY Medium for Flavobacterium Composition per liter: Glutamic acid ................................................................................ 5.0g K2HPO4 ......................................................................................... 0.1g MgSO4·7H2O ................................................................................ 0.1g pH 7.3 ± 0.2 at 25°C

HYA Agar Composition per liter: Agar ............................................................................................ 15.0g Proteose peptone No. 3 ............................................................... 10.0g Beef extract................................................................................... 1.0g Lactose solution .......................................................................10.0mL Galactose solution....................................................................10.0mL Glucose solution ......................................................................10.0mL pH 6.8 ± 0.2 at 25°C

Lactose Solution: Composition per 10.0mL: Lactose.......................................................................................... 5.0g

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Galactose Solution: Composition per 10.0mL: Galactose....................................................................................... 2.5g

Preparation of Galactose Solution: Add galactose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Glucose Solution: Composition per 10.0mL: Glucose ......................................................................................... 2.5g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except lactose solution, galactose solution, and glucose solution—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile lactose solution, galactose solution, and glucose solution. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the cultivation of acidogenic microorganisms, especially Lactobacillus bulgaricus and Streptococcus thermophilus, from foods.

Hydrogen-Oxidizing Bacteria Medium Composition per 1020.0mL: Solution I ......................................................................................1.0L Solution II ................................................................................10.0mL Solution III...............................................................................10.0mL

Solution I: Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g KH2PO4......................................................................................... 1.5g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g Trace elements solution .............................................................1.0mL

Preparation of Solution I: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until dissolved. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Hydrogenobacter acidophilus Medium

861

Trace Elements Solution: Composition per liter:

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g NaMoO4·2H2O............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Na-EDTA ...................................................................................... 0.5g CoCl2 .......................................................................................... 0.15g MnCl2·4H2O ................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g ZnCl2 ............................................................................................. 0.1g AlCl3·6H2O................................................................................. 0.04g NaWoO4·2H2O............................................................................ 0.03g CuCl2·2H2O ................................................................................ 0.02g NiSO4·6H2O................................................................................ 0.02g H3BO3 ......................................................................................... 0.01g Na2SeO4 ...................................................................................... 0.01g NaMoO4·2H2O............................................................................ 0.01g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Solution II: Composition per 100.0mL: CaCl2·2H2O................................................................................... 0.1g Ferric ammonium citrate............................................................. 0.05g

Preparation of Solution II: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution III: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution III: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine 1.0L of cooled, sterile solution I, 10.0mL of cooled, sterile solution II, and 10.0mL of sterile solution III. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of hydrogen-oxidizing bacteria.

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust the pH to 3.0.

Preparation of Medium: Sparge 1.0L of distilled/deionized water with 100% CO2 to produce anaerobic water. Add components to 1.0L of the anaerobic water. Mix thoroughly. The pH should be 6.0. Sparge with 100% CO2 for 20 min. Dispense 5.0mL aliquots into sealable culture tubes. Place stopper on culture tube and crimp tube cap onto stopper. Autoclave for 20 min at 15 psi pressure–121°C. Add 1.0mL of O2 to each tube before inoculation. After inoculation pressurize the tubes with H2 (138 KP). Use: For the cultivation of Sulfurihydrogenibium azorense.

Hydrogenivirga okinawensis Medium (DSMZ Medium 1131) Composition per liter:

Hydrogen-oxidizing Medium (DSMZ Medium 1003) Composition per liter: MgSO4·7H2O ................................................................................ 7.0g NaS2O3 .......................................................................................... 2.0g MES ............................................................................................ 1.95g MgCl2·6H2O................................................................................ 0.78g KCl.............................................................................................. 0.48g CaCl2·2H2O................................................................................... 0.4g Trace elements solution ...........................................................10.0mL Solution A ..................................................................................2.0mL Solution B ..................................................................................1.5mL pH 6.0 ± 0.2 at 25°C

Solution A: Composition per liter: NH4Cl ...................................................................................... 100.0g MgCl2·6H2O.............................................................................. 100.0g CaCl2·2H2O................................................................................. 40.0g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 4.0 with HCl.

Solution B: Composition per liter: K2HPO4·3H2O........................................................................... 200.0g

Preparation of Solution B: Add K2HPO4·3H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Agar ............................................................................................ 20.0g Mannitol...................................................................................... 10.0g Yeast extract.................................................................................. 0.3g K2HPO4 ........................................................................................ 0.2g MgSO4·7H2O ................................................................................ 0.2g NaCl............................................................................................ 0.05g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Pour into Petri dishes or leave in tubes.

Use: For the cultivation of Hydrogenivirga okinawensis.

Hydrogenobacter acidophilus Medium (DSMZ Medium 743) Composition per liter: Sulfur ............................................................................................ 5.0g (NH4)2SO4 .................................................................................... 1.0g K2HPO4......................................................................................... 1.0g NaCl.............................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.3g FeSO4·7H2O............................................................................... 1.0mg CaCl2 ......................................................................................... 1.0mg NiSO4·6H2O............................................................................. 0.06mg Trace elements solution .............................................................0.5mL pH 3.0 ± 0.2 at 25°C

862

Hydrogenobacter halophilus Medium

Trace Elements Solution: Composition per liter:

NiSO4·7H2O............................................................................... 0.6mg Trace elements solution .............................................................2.0mL

ZnSO4·7H2O ............................................................................ 28.0mg MoO3.......................................................................................... 4.0mg H3BO3 ........................................................................................ 4.0mg MnSO4·5H2O ............................................................................. 4.0mg CoCl2·6H2O ............................................................................... 4.0mg CuSO4·5H2O .............................................................................. 2.0mg

Trace Elements Solution: Composition per liter:

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2.

Preparation of Medium: Autoclave sulfur for 15 min at 9 psi pressure–113°C. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Add 5.0g sterile sulfur. Mix thoroughly by swirling. Adjust pH to 3.0 with HCl. Aseptically distribute into sterile tubes or flasks.

ZnSO4·7H2O .............................................................................. 7.0mg MoO3 ......................................................................................... 1.0mg H3BO3 ........................................................................................ 1.0mg MnSO4·H2O ............................................................................... 1.0mg CoCl2·6H2O ............................................................................... 1.0mg CuSO4·5H2O .............................................................................. 0.5mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Hydrogenovibrio marinus.

Use: For the cultivation of Hydrogenobaculum acidophilum=Hydrogenobacter acidophilus.

Hydrogenobacter halophilus Medium (DSMZ Medium 744) Composition per liter: NaCl ............................................................................................ 29.3g K2HPO4 ......................................................................................... 2.5g (NH4)2SO4 ..................................................................................... 2.0g KH2PO4 ......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2 ........................................................................................ 10.0mg FeSO4·7H2O............................................................................. 10.0mg NiSO4·7H2O............................................................................... 0.6mg Trace elements solution .............................................................0.5mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: ZnSO4·7H2O ............................................................................ 28.0mg MoO3.......................................................................................... 4.0mg H3BO3 ........................................................................................ 4.0mg MnSO4·5H2O ............................................................................. 4.0mg CoCl2·6H2O ............................................................................... 4.0mg CuSO4·5H2O .............................................................................. 2.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Adjust pH to 6.9. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Hydrogenovibrio marinus.

Hydrogenobacter halophilus Medium Composition per liter: NaCl ............................................................................................ 29.3g K2HPO4 ......................................................................................... 2.5g (NH4)2SO4 ..................................................................................... 2.0g KH2PO4 ......................................................................................... 0.5g CaCl2·2H2O................................................................................. 0.25g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O............................................................................. 10.0mg © 2010 by Taylor and Francis Group, LLC

Hydrogenobacter thermophilus Medium Composition per liter: Na2HPO4 ....................................................................................... 4.5g KH2PO4......................................................................................... 1.5g NH4NO3 ........................................................................................ 1.0g NaCl.............................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2 ........................................................................................ 10.0mg FeSO4·7H2O............................................................................. 10.0mg NiSO4·7H2O............................................................................. 0.06mg Trace elements solution .............................................................2.0mL

Trace Elements Solution: Composition per liter: ZnSO4·7H2O .............................................................................. 7.0mg MoO3 ......................................................................................... 1.0mg H3BO3 ........................................................................................ 1.0mg MnSO4·H2O ............................................................................... 1.0mg CoCl2·6H2O ............................................................................... 1.0mg CuSO4·5H2O .............................................................................. 0.5mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Calderobacterium hydrogenophilum and Hydrogenobacter thermophilus.

Hydrogenothermus hirschii Medium (DSMZ Medium 783) Composition per liter: MgSO4·7H2O ................................................................................ 7.0g MgCl2·6H2O ................................................................................. 5.5g NaHCO3 ........................................................................................ 2.0g KCl............................................................................................. 0.65g CaCl2·2H2O .................................................................................. 0.5g Sulfur, powdered........................................................................... 0.5g NH4Cl ......................................................................................... 0.15g K2HPO4....................................................................................... 0.15g NaBr.............................................................................................. 0.1g

Hydroxybenzoate Broth

Trace elements solution ...........................................................10.0mL CaCO3 solution ..........................................................................5.0mL pH 7.0 ± 0.2 at 25°C

CaCO3 Solution: Composition per 10.0mL: CaCO3 ........................................................................................... 1.0g

Preparation of CaCO3 Solution: Add CaCO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Use: For the cultivation of Hydrogenophilus hirschii (Hydrogenothermophilus hirschii).

863

bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Solution B: Composition per 10.0mL: MgSO4·7H2O .............................................................................. 0.27g

Preparation of Solution B: Add MgSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution C: Composition per 1.0mL: Fe(NH4)2(SO4)2·6H2O ................................................................ 0.05g

Preparation of Solution C: Add component to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize. Prepare solution immediately before adding to solutions A and B.

Solution D: Composition per 500.0mL: Agar ............................................................................................ 14.0g

Preparation of Solution D: Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Medium: Aseptically combine cooled sterile solution A, cooled sterile solution B, and cooled sterile solution D. Immediately add 1.0mL of freshly prepared sterile solution C. Adjust pH to 7.0 with 6N NaOH. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Comamonas testosteroni.

Hydroxybenzoate Agar (p-Hydroxybenzoate Agar) Composition per liter: Agar ............................................................................................ 20.0g (NH4)2HPO4.................................................................................. 3.0g p-hydroxybenzoic acid.................................................................. 3.0g K2HPO4......................................................................................... 1.2g NaCl.............................................................................................. 0.5g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O.................................................................................. 0.1g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Hydroxybenzoate Agar Composition per 1001.0mL: Solution A ..............................................................................490.0mL Solution D ..............................................................................500.0mL Solution B ................................................................................10.0mL Solution C ..................................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Solution A: Composition per 490.0mL: 4-Hydroxybenzoic acid................................................................. 3.0g (NH4)2SO4 ..................................................................................... 3.0g NaCl .............................................................................................. 2.5g K2HPO4 ......................................................................................... 1.6g Yeast extract.................................................................................. 0.5g

Use: For the cultivation of p-hydroxybenzoate-utilizing bacteria.

Hydroxybenzoate Broth Composition per 1001.0mL: Solution A..............................................................................990.0mL Solution B ................................................................................10.0mL Solution C ..................................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Solution A: Composition per 990.0mL: 4-Hydroxybenzoic acid................................................................. 3.0g (NH4)2SO4 .................................................................................... 3.0g NaCl.............................................................................................. 2.5g

864

Hydroxybenzoate Medium

K2HPO4 ......................................................................................... 1.6g Yeast extract.................................................................................. 0.5g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution B: Composition per 10.0mL: MgSO4·7H2O .............................................................................. 0.27g

Preparation of Solution C: Add component to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize. Prepare solution immediately before adding to solutions A and B.

Preparation of Medium: Aseptically combine cooled sterile solution A and cooled sterile solution B. Immediately add 1.0mL of freshly prepared sterile solution C. Adjust pH to 7.0 with 6N NaOH. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Comamonas testosteroni.

Hydroxybenzoate Medium Composition per liter: Noble agar................................................................................... 20.0g (NH4)2HPO4 .................................................................................. 3.0g K2HPO4 ......................................................................................... 1.2g NaCl .............................................................................................. 0.5g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O.................................................................................. 0.1g p-Hydroxybenzoic acid solution ..............................................50.0mL pH 7.0 ± 0.2 at 25°C

p-Hydroxybenzoic Acid Solution: Composition per 50.0mL: p-Hydroxybenzoic acid................................................................. 3.0g

Preparation of p-Hydroxybenzoic Acid Solution: Add p-hydroxybenzoic acid to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except p-hydroxybenzoic acid solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0 with 5N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile p-hydroxybenzoic acid solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Pseudomonas putida.

Hydroxybenzoate Medium Composition per 1002.0mL: Solution A ..............................................................................920.0mL Solution B ................................................................................50.0mL Solution E (Vitamin solution) ..................................................10.0mL Solution F.................................................................................10.0mL Solution G ................................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

Solution C (Trace elements solution SL-10) .............................1.0mL Solution D..................................................................................1.0mL pH 7.2–7.5 at 25°C

Solution A: Composition per 920.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution B: Composition per 50.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of Solution B: Add NaHCO3 to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution C (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Solution C (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Gas under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution D: Composition per 10.0mL: NaOH......................................................................................... 5.0mg Na2WO4·2H2O .......................................................................... 40.0μg Na2SeO3·5H2O.......................................................................... 30.0μg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Hyperthermus butylicus Medium

Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution F: Composition per 10.0mL:

865

Buffer Solution: KH2PO4....................................................................................... 0.45g Na2HPO4·12 H2O........................................................................ 2.39g

Preparation of Buffer Solution: Add components to distilled/de-

Sodium dihydroxybenzoate .......................................................... 0.4g

ionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Solution F: Add sodium dihydroxybenzoate to dis-

Preparation of Medium: Add components to distilled/deionized

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2.

Solution G: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.36g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Aseptically and anaerobically combine 920.0mL of sterile solution A, 50.0mL of sterile solution B, 1.0mL of sterile solution C, 1.0mL of sterile solution D, 10.0mL of sterile solution E, 10.0mL of sterile solution F, and 10.0mL of sterile solution G. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Clostridium species.

Hydroxybenzoic Acid Medium Composition per liter: Agar ............................................................................................ 15.0g K2HPO4·3H2O............................................................................. 4.25g NH4Cl ........................................................................................... 2.0g 4-Hydroxybenzoic acid................................................................. 1.0g NaH2PO4·H2O............................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g Nitrilotriacetic acid ....................................................................... 0.1g FeSO4·7H2O.............................................................................. 0.012g MnSO4·H2O ............................................................................... 3.0mg ZnSO4·7H2O .............................................................................. 3.0mg CoSO4 ........................................................................................ 1.0mg pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add 4-hydroxybenzoic acid and nitrilotriacetic acid to approximately 600.0mL of distilled/deionized water. Adjust pH to 8.0 with concentrated NaOH. Add remaining components. Mix thoroughly. Readjust pH to 7.2. Bring volume to 1.0L with distilled/deionized water. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Bacillus species.

Hydroxybutyrate Medium (3HB Medium) (LMG Medium 186)

Use: For the cultivation and maintenance of Paucimonas lemoignei.

Hyperthermus butylicus Medium Composition per 1010.0mL: NaCl............................................................................................ 17.0g Pancreatic digest of casein............................................................ 6.0g Sulfur, powdered........................................................................... 6.0g MgSO4·7H2O ................................................................................ 3.5g MgCl2·6H2O ............................................................................... 2.75g NiCl2·6H2O................................................................................... 2.0g Yeast extract.................................................................................. 2.0g CaCl2·2H2O ................................................................................ 0.75g KH2PO4......................................................................................... 0.5g NH4Cl ........................................................................................... 0.5g KCl............................................................................................ 0.325g NaBr............................................................................................ 0.05g H3BO3 ....................................................................................... 0.015g (NH4)2SO4 ............................................................................... 10.0mg SrCl2·6H2O ................................................................................ 7.5mg Citric acid................................................................................... 5.0mg KI ............................................................................................... 2.5mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.0–6.5 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7 H2O............................................................................... 3.0g Nitrilotriacetic acid ...................................................................... 1.5 g CaCl2·2 H2O ................................................................................ .1.0g NaCl.............................................................................................. 1.0g MnSO4·2 H2O............................................................................... 0.5g CoSO4·7 H2O.............................................................................. 0.18g ZnSO4·7 H2O.............................................................................. 0.18g FeSO4·7 H2O ................................................................................ 0.1g NiCl2·6 H2O.............................................................................. 0.025g KAI(SO4)2·12 H2O..................................................................... 0.02g CuSO4·5 H2O.............................................................................. 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2 H2O......................................................................... 0.01g Na2SeO3·5 H2O.......................................................................... 0.3mg

Composition per liter:

Preparation of Trace Elements Solution: Add nitrilotriacetic

Agar ............................................................................................ 20.0g DL-3-hydroxybutyrate ................................................................... 3.0g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g Ferric ammonium citrate.......................................................... 50.0mg Yeast extract............................................................................. 50.0mg Buffer solution .......................................................................333.3mL pH 6.8 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL:

acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0 with KOH. Add distilled/deionized water to 1.0L.

Na2S·9H2O.................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

866

Hyphomicrobium Enrichment Medium

Gas under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per 100.0mL:

Preparation of Medium: Add components, except Na2S·9H2O solu-

CuCl2 .......................................................................................... 0.15g FeSO4·7H2O.................................................................................. 0.1g Na2MoO4·2H2O .......................................................................... 0.05g MnSO4·H2O .............................................................................. 0.035g

tion, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0–6.5 with 6N H2SO4. Sparge wih 100% N2. Sterilize by bringing to 90°C for 60 min on 3 consecutive days. Immediately prior to inoculation, add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Use: For the cultivation and maintenance of Hyperthermus butylicus.

Hyphomicrobium Enrichment Medium

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Methylamine·HCl Solution: Composition per 20.0mL:

Composition per 100.0mL:

Methylamine·HCl ....................................................................... 3.38g

KNO3 .......................................................................................... 0.04g Na2HPO4·7H2O........................................................................... 0.02g MgSO4·7H2O ........................................................................... 0.48mg FeCl3·7H2O .............................................................................. 0.02mg MnCl2·4H2O............................................................................. 0.01mg pH 7.2 ± 0.2 at 25°C

Preparation of Methylamine·HCl Solution: Add methyl-

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and enrichment of Hyphomicrobium species.

Hyphomicrobium Medium

amine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except methylamine·HCl solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile methylamine·HCl solution. Mix thoroughly. Adjust pH to 7.1, if necessary. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Hyphomicrobium aestuarii, Hyphomicrobium facilis, Hyphomicrobium hollandicum, Hyphomicrobium vulgare, and Hyphomicrobium zavarzinii.

Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ..................................................................................... 2.13g KH2PO4 ....................................................................................... 1.36g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O.............................................................................. 9.95mg FeSO4·7H2O............................................................................... 5.0mg MnSO4·4H2O ............................................................................. 2.5mg Na2MoO4·2H2O ......................................................................... 2.5mg Urea solution............................................................................30.0mL Methanol ....................................................................................4.0mL

Urea Solution: Composition per 100.0mL: Urea............................................................................................. 20.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Filter sterilize methanol. Add components, except urea solution and methanol, to distilled/deionized water and bring volume to 966.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile urea solution and sterile methanol. Mix thoroughly. Aseptically distribute into sterile tubes or bottles.

Use: For the cultivation of Hyphomicrobium species.

Hyphomicrobium Medium Composition per liter: Noble agar................................................................................... 18.0g Na2HPO4 ..................................................................................... 2.15g KH2PO4 ....................................................................................... 1.36g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Trace elements solution .............................................................5.0mL Methylamine·HCl solution.......................................................20.0mL pH 7.1 ± 0.1 at 25°C © 2010 by Taylor and Francis Group, LLC

Hyphomicrobium Medium 337a Composition per liter: KH2PO4......................................................................................... 1.3g Na2HPO4 ..................................................................................... 1.13g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ............................................................................. 3.09mg FeSO4·7H2O............................................................................... 2.0mg Na2MoO4·2H2O ......................................................................... 1.0mg MnSO4·4H2O ........................................................................... 0.88mg pH 7.2–7.5 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the enrichment and cultivation of Hyphomicrobium species.

Hyphomicrobium methylovorum Medium Composition per liter: Agar ............................................................................................ 15.0g (NH4)2HPO4.................................................................................. 3.0g NaCl.............................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O............................................................................. 10.0mg MnSO4·2H2O ............................................................................. 5.0mg Tap water ......................................................................................1.0L Methanol ..................................................................................10.0mL Vitamin mixture .........................................................................5.0mL

Vitamin Mixture: Composition per liter: Inositol ................................................................................... 200.0mg Choline................................................................................... 100.0mg Calcium DL-pantothenate......................................................... 40.0mg

Hypoxanthine Agar

Niacin....................................................................................... 40.0mg Pyridoxine·HCl ........................................................................ 40.0mg Riboflavin ................................................................................ 40.0mg p-Aminobenzoic acid............................................................... 20.0mg Thiamine·HCl .......................................................................... 20.0mg Biotin ......................................................................................... 0.2mg Folic acid.................................................................................... 0.2mg Cyanocobalamin .........................................................................2.0μg

Preparation of Vitamin Mixture: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Methanol: Filter sterilize 10.0mL of methanol. Preparation of Medium: Add components, except methanol and vitamin mixture, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile methanol and 5.0mL of sterile vitamin mixture. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Hyphomicrobium methylovorum.

Hyphomicrobium Strain X Agar Composition per liter: Agar ............................................................................................ 15.0g Methylamine·HCl ......................................................................... 3.4g K2HPO4 ....................................................................................... 1.55g (NH4)2SO4 ..................................................................................... 1.0g NaH2PO4·H2O............................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Trace elements solution .............................................................0.2mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter:

867

Trace Elements Solution: Composition per liter: Disodium EDTA ......................................................................... 50.0g ZnSO4·7H2O ............................................................................... 22.0g CaCl2·2H2O ................................................................................ 5.54g MnCl2·4H2O ............................................................................... 5.06g FeSO4·7H2O.................................................................................. 5.0g CoCl2·6H2O ................................................................................ 1.61g CuSO4·5H2O............................................................................... 1.57g (NH4)6Mo7O24·4H2O .................................................................... 1.1g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Adjust pH to 7.0 with KOH. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Hyphomicrobium species.

Hyphomonas Enrichment Medium Composition per liter: Peptone ....................................................................................... 0.05g Yeast extract................................................................................ 0.05g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation and cultivation of Hyphomonas species.

Hyphomonas Medium Composition per liter:

Disodium EDTA ......................................................................... 50.0g ZnSO4·7H2O ............................................................................... 22.0g CaCl2·2H2O................................................................................. 5.54g MnCl2·4H2O................................................................................ 5.06g FeSO4·7H2O.................................................................................. 5.0g CoCl2·6H2O ................................................................................ 1.61g CuSO4·5H2O ............................................................................... 1.57g (NH4)6Mo7O24·4H2O .................................................................... 1.1g

Pancreatic digest of casein............................................................ 2.0g MgCl2·2H2O ................................................................................. 2.0g Yeast extract.................................................................................. 1.0g pH 7.5 ± 0.2 at 25°C

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Adjust pH to 7.0 with KOH.

Use: For the cultivation and maintenence of Hyphomonas polymorpha.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Hyphomicrobium species.

Hyphomicrobium Strain X Broth Composition per liter: K2HPO4 ....................................................................................... 1.55g (NH4)2SO4 ..................................................................................... 1.0g Methylamine·HCl ......................................................................... 0.7g NaH2PO4·H2O............................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Trace elements solution .............................................................0.2mL pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5 using indicator paper. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Hypoxanthine Agar Composition per 1100.0mL: Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g Hypoxanthine solution.................................................................. 5.0g Beef extract................................................................................... 3.0g pH 7.0 ± 0.1 at 25°C

Hypoxanthine Solution: Composition per 100.0mL: Hypoxanthine................................................................................ 5.0g

Preparation of Hypoxanthine Solution: Add hypoxanthine to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except hypoxanthine solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile hypoxanthine solu-

868

Idiomarina Medium

tion. Mix thoroughly. Pour into sterile 15mm × 100mm Petri dishes in 25.0mL volumes.

Use: For the cultivation and differentiation of bacteria based on hypoxanthine hydrolysis. Bacteria that hydrolyze hypoxanthine, such as Streptomyces griseus, appear with a clear zone under and around the colonies. Nocardia asteroides does not hydrolyze hypoxanthine.

IBB Agar See: Inositol Brilliant Green Bile Salts Agar

Idiomarina Medium (DSMZ Medium 1016) Composition per liter: NaCl ...................................................................................30.0-60.0g Glucose ...................................................................................... 10.0g Proteose peptone .......................................................................... 5.0g Yeast extract ................................................................................. 3.0g Malt extract .................................................................................. 3.0g pH 7.0 ± 0.2 at 25°C

FeSO4·7H2O.................................................................................. 0.2g H3BO3 ......................................................................................... 0.03g CoCl2·6H2O ................................................................................ 0.02g ZnSO4·7H2O ............................................................................... 0.01g MnCl2·4H2O .............................................................................. 3.0mg Na2MoO4·2H2O ......................................................................... 3.0mg NiCl2·6H2O ................................................................................ 2.0mg CaCl2·2H2O ............................................................................... 1.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Combine components, except lactose solution and trace elements solution. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile lactose solution and 1.0mL of sterile trace elements solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Bacillus species.

Preparation of Medium: Add componentsto distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Idiomarina spp.

IE Medium Composition per 1011.0mL: Agar ............................................................................................ 15.0g Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g Basal salts solution........................................................................1.0L Lactose solution .......................................................................... 10.0g Trace elements solution .............................................................1.0mL

Basal Salts Solution: Composition per liter: MgSO4 .......................................................................................... 0.5g Phosphate solution ...................................................................20.0mL (NH4)SO4 (36% solution) ..........................................................5.0mL

Phosphate Solution: Composition per liter: K2HPO4 ....................................................................................... 95.0g NaH2PO4·2H2O........................................................................... 78.0g

Preparation of Phosphate Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Basal Salts Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8-7.0. Lactose Solution: Composition per 10.0mL: Lactose .......................................................................................... 2.5g

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Warm to 50°–55°C. Trace Elements Solution: Composition per liter: Disodium EDTA ........................................................................... 0.5g © 2010 by Taylor and Francis Group, LLC

IFO Agar Composition per liter: Agar ............................................................................................ 20.0g (NH4)2HPO4.................................................................................. 3.0g NaCl.............................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O............................................................................. 10.0mg MnSO4·6H2O ............................................................................. 5.0mg Riboflavin ................................................................................ 0.02mg Calcium pantothenate .............................................................. 0.02mg Pyridoxine·HCl ........................................................................ 0.02mg Nicotinic acid........................................................................... 0.02mg p-Aminobenzoic acid............................................................... 0.01mg Thiamine·HCl .......................................................................... 0.01mg Biotin .......................................................................................... 1.0μg Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except agar and methanol, to distilled/deionized water and bring volume to 490.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. In a separate flask, add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically combine the two sterile solutions. Aseptically add 10.0mL of filter-sterilized methanol. Mix thoroughly. Adjust pH to 7.0. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Hyphomicrobium methylovorum.

IFO Broth Composition per liter: (NH4)2HPO4.................................................................................. 3.0g NaCl.............................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O............................................................................. 10.0mg MnSO4·6H2O ............................................................................. 5.0mg Riboflavin ................................................................................. 20.0μg Calcium pantothenate ............................................................... 20.0μg Pyridoxine·HCl ......................................................................... 20.0μg Nicotinic acid............................................................................ 20.0μg

Ignisphaera Medium

p-Aminobenzoic acid................................................................10.0μg Thiamine·HCl ...........................................................................10.0μg Biotin ..........................................................................................1.0μg Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of filter-sterilized methanol. Mix thoroughly. Adjust pH to 7.0. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Hyphomicrobium methylovorum.

IFO Medium 802 Composition per liter: Polypeptone™............................................................................. 10.0g Yeast extract.................................................................................. 2.0g MgSO4·7H2O ................................................................................ 1.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Sphingomonas asaccharolytica, Sphingomonas pruni, Sphingomonas mali, and Sphingomonas rosa.

869

before inoculation. After inoculation pressurize the vessels with 80% H2 and 20% CO2 gas mixture to 2 bar overpressure.

Use: For the cultivation of Ignicoccus islandicus and Ignicoccus pacificus.

Ignisphaera Medium (DSMZ Medium 1043) Composition per liter: Trypticase peptone........................................................................ 2.0g Starch, soluble............................................................................... 2.0g (NH4)2SO4 .................................................................................... 1.3g MgSO4·7H2O .............................................................................. 0.28g KH2PO4 ...................................................................................... 0.28g Yeast extract.................................................................................. 0.1g L-Cysteine ..................................................................................... 0.3g CaCl2·2H2O ............................................................................. 74.0mg Resazurin ................................................................................... 0.5mg FeCl2·6H2O ................................................................................ 0.5mg Trace elements solution ...........................................................10.0mL FeCl2 solution ..........................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.5 ± 0.2 at 25°C

FeCl2 Solution: Composition per 10.0mL: FeCl2·6H2O ................................................................................ 0.5mg

Preparation of FeCl2 Solution: Add FeCl2·6H2O to distilled/de-

Ignicoccus Medium (DSMZ Medium 897) Composition per liter: NaCl .......................................................................................... 13.65g Sulfur, powdered........................................................................... 5.0g MgSO4·7H2O ................................................................................ 3.5g MgCl2·6H2O................................................................................ 2.75g Meat extract .................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g CaCl2·2H2O................................................................................. 0.38g KCl.............................................................................................. 0.33g (NH4)2SO4 ................................................................................... 0.25g NaBr............................................................................................ 0.05g H3BO3 ...................................................................................... 15.0mg SrCl3·6H2O............................................................................... 7.50mg KI ............................................................................................ 0.05mg Resazurin ................................................................................... 0.5mg Na2S·9H2O solution .................................................................10.0mL pH 5.5 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.2g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Preparation of Medium: Add components, except Na2S·9H2O so-

lution, to distilled/deionized water and bring volume to 990.0mL. Sparge medium with N2 gas for 30–60 min. Mix thoroughly. Add 10.0mL Na2S·9H2O solution. Mix thoroughly. Adjust pH to 5.5 with H2SO4. Distribute into tubes or bottles under 80% H2 and 20% CO2 gas mixture. Heat the vessels containing medium in boiling water for 1 hr © 2010 by Taylor and Francis Group, LLC

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

870

Ilyobacter Broth

min. Cool to room temperature while sparging with 100% N2. Add FeCl3 solution. Adjust pH to 6.3. Dispense under an atmosphere of 100% N2 into suitable culture vessels (e.g., aliquots of 20mL medium into 50mL serum bottles). Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Adjust pH to 6.5. Prior to inoculation aseptically and anoxically add sulfide solution.

Use: For the cultivation of Ignisphaera spp. IGP Medium See: Intracellular Growth Phase Medium

Ilyobacter Agar Composition per liter: NaCl ............................................................................................ 20.0g Agar ............................................................................................ 15.0g MgCl2·6H2O.................................................................................. 3.0g KCl................................................................................................ 0.5g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 1.0mg Sodium sulfide solution ...........................................................10.0mL Sodium L-tartrate solution........................................................10.0mL NaHCO3 solution .....................................................................10.0mL Trace elements solution SL-7 ....................................................1.0mL pH 7.2 ± 0.2 at 25°C

Sodium Sulfide Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 3.6g

Preparation of Sodium L-Tartrate Solution: Add sodium L-tartrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

Trace Elements Solution SL-7: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.19g MnCl2·4H2O.................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g H3BO3 ....................................................................................... 0.062g Na2MoO4·2H2O ........................................................................ 0.036g NiCl2·6H2O ............................................................................... 0.024g CuCl2·2H2O .............................................................................. 0.017g HCl (25% solution) ..................................................................10.0mL

ume to 1.0L. Add remaining components. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

Preparation of Medium: Add components—except agar, sodium sulfide solution, sodium L-tartrate solution, NaHCO3 solution, and trace elements solution SL-7—to distilled/deionized water and bring volume to 469.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Maintain under 80% N2 + 20% CO2. Aseptically add 10.0mL of sodium L-tartrate solution, 10.0mL of NaHCO3 solution, and 1.0mL of trace elements solution SL-7 under 80% N2 + 20% CO2. Mix thoroughly. In a separate flask, add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Combine sterile agar and sterile basal medium. Adjust pH to 7.2. Aseptically add 10.0mL of sodium sulfide solution. Pour into sterile Petri dishes or distribute into sterile tubes. Maintain under 80% N2 + 20% CO2.

Use: For the cultivation and maintenance of Ilyobacter tartaricus.

Ilyobacter Broth Composition per liter: NaCl............................................................................................ 20.0g MgCl2·6H2O ................................................................................. 3.0g KCl................................................................................................ 0.5g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 1.0mg Sodium sulfide solution ...........................................................10.0mL Sodium L-tartrate solution........................................................10.0mL NaHCO3 solution .....................................................................10.0mL Trace elements solution SL-7 ....................................................1.0mL pH 7.2 ± 0.2 at 25°C

Sodium Sulfide Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................... 3.6g

Preparation of Sodium L-Tartrate Solution: Add sodium L-tartrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2. Trace Elements Solution SL-7: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.19g MnCl2·4H2O ................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g

Ilyobacter polytropus Medium

H3BO3 ....................................................................................... 0.062g Na2MoO4·2H2O ........................................................................ 0.036g NiCl2·6H2O ............................................................................... 0.024g CuCl2·2H2O .............................................................................. 0.017g HCl (25% solution) ..................................................................10.0mL

871

Selenite-Tungstate Solution: Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5HO ............................................................................ 3.0mg

Preparation of Trace Elements Solution SL-7: Add the FeCl2·4H2O to the HCl. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2.

Preparation of Medium: Add components—except sodium sulfide

solution, modified SL-7 trace elements solution, and selenite-tungstate solution—to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Adjust pH to 5.5. Cool to 45°–50°C under 100% N2. Maintain under 100% N2. Aseptically add 1.0mL of sterile modified SL-7 trace elements solution and 1.0mL of sterile selenitetungstate solution under 100% N2. Mix thoroughly. Aseptically distribute into sterile tubes or flasks under 100% N2. At time of inoculation, add sodium sulfide solution to a final concentration of 1.0%. Maintain under 100% N2.

solution, sodium L-tartrate solution, NaHCO3 solution, and trace elements solution SL-7—to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Maintain under 80% N2 + 20% CO2. Aseptically add 10.0mL of sodium L-tartrate solution, 10.0mL of NaHCO3 solution, and 1.0mL of trace elements solution SL-7 under 80% N2 + 20% CO2. Mix thoroughly. Aseptically distribute into sterile tubes or flasks under 80% N2 + 20% CO2. Adjust pH to 7.2. At time of inoculation add sodium sulfide solution to a final concentration of 0.1%.

Use: For the cultivation and maintenance of Ilyobacter tartaricus.

Ilyobacter Medium Composition per liter: Crotonic acid................................................................................. 1.7g NaCl .............................................................................................. 1.0g Yeast extract.................................................................................. 1.0g Na2HPO4·12H2O........................................................................... 0.7g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ........................................................................................... 0.3g Na2SO4 .......................................................................................... 0.1g Sodium sulfide solution ...........................................................10.0mL CaCl2·2H2O (1.0% )...................................................................1.0mL FeCl3 (0.5% ) .............................................................................1.0mL Modified SL-7 trace elements solution......................................1.0mL Resazurin (0.1% ) ......................................................................1.0mL Selenite-tungstate solution.........................................................1.0mL pH 6.8–7.2 at 25°C

Sodium Sulfide Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 3.6g

Preparation of Sodium Sulfide Solution: Add Na2S·9H2O to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C under N2. Maintain under 100% N2.

Modified SL-7 Trace Elements Solution: Composition per liter: CoCl2·6H2O .................................................................................. 0.2g MnCl2·4H2O.................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g H3BO3 ......................................................................................... 0.06g Na2MoO4·2H2O .......................................................................... 0.04g CuCl2·2H2O ................................................................................ 0.02g NiCl2·6H2O ................................................................................. 0.02g HCl (1N) ....................................................................................3.0mL

Preparation of Modified SL-7 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Maintain under 80% N2 + 20% CO2. © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components—except sodium sulfide

Use: For the cultivation and maintenance of Ilyobacter delafieldii.

Ilyobacter polytropus Medium Composition per 1011.0mL: Solution A.....................................................................................1.0L Solution B ................................................................................10.0mL Vitamin solution.........................................................................1.0mL pH 7.2–7.4 at 25°C

Solution A: Composition per liter: NaHCO3 ........................................................................................ 4.5g Na2SO4 ........................................................................................ 2.84g Sodium 3-hydroxybutyrate ........................................................... 1.3g NaCl............................................................................................ 1.17g Yeast extract.................................................................................. 1.0g MgCl2·6H2O ................................................................................. 0.4g KCl................................................................................................ 0.3g NH4Cl ......................................................................................... 0.27g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg Trace elements solution .............................................................1.0mL

Preparation of Solution A: Add components, except NaHCO3 and vitamin solution, and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 to 4 min. Allow to cool to room temperature while gassing under O2-free 80% N2 + 20% CO2. Add NaHCO3 and continue gassing with O2-free 80% N2 + 20% CO2 until pH reaches 6.9–7.1. Seal the flask under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 120.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 68.0mg H3BO3 ...................................................................................... 62.0mg Na2MoO4·2H2O ...................................................................... 24.0mg NiCl2·6H2O.............................................................................. 24.0mg CuCl2·2H2O ............................................................................. 17.0mg HCl (25% solution)..................................................................10.0mL

872

Ilyobacter tartaricus Medium

Preparation of Trace Elements Solution: Add FeCl2·4H2O to

10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Gas under 100% N2.

Vitamin Solution: Composition per 100.0mL: Thiamine·HCl .......................................................................... 10.0mg p-Aminobenzoic acid ................................................................. 4.0mg D(+)-Biotin ................................................................................. 1.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Gas under 100% N2. Solution B: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.36g

Preparation of Solution B: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: To 1.0L of sterile solution A, add 10.0mL of sterile solution B and 1.0mL of sterile vitamin solution. Mix thoroughly. Adjust final pH to 7.2–7.4. Use: For the cultivation and maintenance of Ilyobacter polytropus.

Ilyobacter tartaricus Medium Composition per liter: NaCl ............................................................................................ 20.0g MgCl2·6H2O.................................................................................. 3.0g KCl................................................................................................ 0.5g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................20.0mL Sodium L-tartrate solution........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 20.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N2 + 20% CO2.

Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate .......................................................................... 2.0g

Preparation of Sodium L-Tartrate Solution: Add sodium L-tartrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N2 + 20% CO2.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.36g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Gas under 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Add components, except NaHCO3 solution, sodium L-tartrate solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Gas under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of sterile NaHCO3 solution, 10.0mL of sterile sodium L-tartrate solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Ilyobacter tartaricus.

Ilyobacter tartaricus Medium Composition per liter: NaCl............................................................................................ 20.0g MgCl2·6H2O ................................................................................. 3.0g KCl................................................................................................ 0.5g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................20.0mL Sodium L-tartrate solution .......................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Yeast extract solution...............................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 20.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N2 + 20% CO2.

Sodium L-Tartrate Solution: Composition per 10.0mL: Sodium L-tartrate .......................................................................... 2.0g

Preparation of Sodium L-Tartrate Solution: Add sodium L-tartrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Gas under 80% N2 + 20% CO2.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................. 0.36g

Imidazole Utilization Medium Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Yeast Extract Solution: Composition per 10.0mL: Yeast extract.................................................................................. 1.0g

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Gas under 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Preparation of Medium: Add components, except NaHCO3 solu-

tion, sodium L-tartrate solution, yeast extract solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gas under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of sterile NaHCO3 solution, 10.0mL of sterile sodium L-tartrate solution, 10.0mL of sterile yeast extract solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Propionigenium modestum.

IM See: Infection Medium

873

Preparation of Sodium Sulfide Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C under N2. Maintain under 100% N2. SLA Trace Elements Solution: Composition per liter: FeCl2·4H2O................................................................................... 1.8g H3BO3 ........................................................................................... 0.5g CoCl2·6H2O ................................................................................ 0.25g ZnCl2 ............................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.07g Na2MoO4·2H2O .......................................................................... 0.03g CuCl2·2H2O ................................................................................ 0.01g Na2SeO3·5H2O............................................................................ 0.01g NiCl2·6H2O................................................................................. 0.01g

Preparation of SLA Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 2–3. Filter sterilize. VA Vitamin Solution: Composition per 500.0mL: Nicotinamide............................................................................... 0.17g Thiamine·HCl ............................................................................. 0.15g p-Aminobenzoic acid.................................................................... 0.1g Biotin .......................................................................................... 0.05g Calcium pantothenate ................................................................. 0.05g Pyridoxine·2HCl ......................................................................... 0.05g Cyanocobalamin ......................................................................... 0.02g

Preparation of VA Vitamin Solution: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components—except sodium sulfide solution, SLA trace elements solution, and VA vitamin solution—to distilled/deionized water and bring volume to 988.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 1.0mL of sterile SLA trace elements solution and 1.0mL of sterile VA vitamin solution. Aseptically add 10.0mL of sterile sodium sulfide solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Rhodobacter adriaticus

Imhoff’s Medium, Modified Composition per liter: NaCl ............................................................................................ 30.0g NaHCO3 ........................................................................................ 3.0g KH2PO4 ......................................................................................... 1.0g NH4Cl ........................................................................................... 1.0g Sodium acetate .............................................................................. 1.0g Na2SO4 .......................................................................................... 0.7g MgCl2·6H2O.................................................................................. 0.5g Sodium ascorbate .......................................................................... 0.5g CaCl2·2H2O................................................................................... 0.1g Yeast extract.................................................................................. 0.1g Sodium sulfide solution ...........................................................10.0mL SLA trace elements solution ......................................................1.0mL VA vitamin solution ...................................................................1.0mL pH 6.9–7.0 at 25°C

and Rhodobacter sulfidophilus.

Imidazole Utilization Medium Composition per liter: Imidazole ...................................................................................... 5.0g KH2PO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g CaCl2 .......................................................................................... 3.0mg FeSO4·7H2O............................................................................... 3.0mg Molybdenum solution................................................................1.0mL Trace elements solution .............................................................1.0mL pH 6.0 ± 0.2 at 25°C

Molybdenum Solution: Composition per 18.0mL: Na2MoO4·2H2O ......................................................................... 0.5mg

Preparation of Molybdenum Solution: Add components to distilled/deionized water and bring volume to 18.0mL. Mix thoroughly. Filter sterilize.

874

Indole Medium

Trace Elements Solution: Composition per 18.0mL:

tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

H3BO3 ...................................................................................... 11.0mg MnCl2·4H2O............................................................................... 7.0mg Al2(SO4)3·18 H2O .................................................................... 1.94mg Co(NO3)2·6H2O ......................................................................... 1.0mg CuSO4·5H2O .............................................................................. 1.0mg NiSO4·6H2O............................................................................... 1.0mg ZnSO4·H2O .............................................................................. 0.62mg KBr............................................................................................. 0.5mg KI ............................................................................................... 0.5mg LiCl ............................................................................................ 0.5mg SnCl2·2H2O................................................................................ 0.5mg

Use: For the differentiation of microorganisms by means of the indole

Preparation of Trace Elements Solution: Add components to

test.

Indole Nitrate HiVeg Medium (Tryptone Nitrate HiVeg Medium) Composition per liter: Plant hydrolysate ........................................................................ 20.0g Na2HPO4 ....................................................................................... 2.0g Agar .............................................................................................. 1.0g Glucose ......................................................................................... 1.0g Potassium nitrate........................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

distilled/deionized water and bring volume to 18.0mL. Mix thoroughly. Filter sterilize.

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components, except molybdenum

Preparation of Medium: Add components to distilled/deionized

solution and trace elements solution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 1.0mL of molybdenum solution and 1.0mL of trace elements solution. Mix thoroughly. Adjust pH to 6.0 with phosphoric acid. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Pseudomonas species.

Indole Medium Composition per 200.0mL:

Media. water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the identification of microorganisms by means of the nitrate reduction and indole tests.

Indole Nitrate Medium (Trypticase™ Nitrate Broth) Composition per liter:

K2HPO4 ....................................................................................... 3.13g L-Tryptophan ................................................................................. 1.0g NaCl .............................................................................................. 1.0g KH2PO4 ....................................................................................... 0.27g pH 7.2 ± 0.2 at 25°C

Pancreatic digest of casein.......................................................... 20.0g Na2HPO4 ....................................................................................... 2.0g Agar .............................................................................................. 1.0g Glucose ......................................................................................... 1.0g KNO3 ............................................................................................ 1.0g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from BD Di-

water and bring volume to 200.0mL. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

agnostic Systems.

Use: For the differentiation of microorganisms by means of indole production from the tryptophan test.

Indole Medium Composition per liter: Pancreatic digest of casein .......................................................... 20.0g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add pancreatic digest of casein to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the differentiation of microorganisms by means of the indole test.

Indole Medium, CDC (BAM M65) Composition per liter: Pancreatic digest of casein .......................................................... 20.0g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the identification of microorganisms by means of the nitrate reduction and indole tests.

Infection Medium (IM) Composition per 100.0mL: Pancreatic digest of gelatin......................................................... 0.05g Bile salts No. 3............................................................................ 0.05g Brain heart, solids from infusion ................................................ 0.02g Peptic digest of animal tissue ..................................................... 0.02g NaCl.......................................................................................... 0.017g Glucose ....................................................................................... 0.01g Na2HPO4 .................................................................................... 8.0mg Earle’s balanced salts solution .................................................80.0mL Fetal bovine serum, heat inactivated (2 hr at 55°C) ................20.0mL pH 7.4 ± 0.2 at 25°C

Earle’s Balanced Salts Solution: Composition per liter: NaCl.............................................................................................. 6.8g NaHCO3 ........................................................................................ 2.2g

Inorganic Salts-Maltose Medium

875

Glucose ......................................................................................... 1.0g KCl................................................................................................ 0.4g CaCl2·2H2O............................................................................... 0.265g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4·H2O............................................................................. 0.14g

L-Cystine....................................................................................... 1.0g Hemoglobin solution, 2% ......................................................100.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Earle’s Balanced Salts Solution: Add compo-

Preparation of Medium: Add components, except hemoglobin so-

nents to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Combine components. Mix thoroughly. Filter sterilize. Store at 4°–10°C.

Use: For the screening of Escherichia coli for pathogenicity using the HeLa cell test for invasiveness.

Infusion Agar See: Blood Agar Base

Source: This medium, without hemoglobin solution, is available as a premixed powder from HiMedia. lution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 100.0mL of sterile hemoglobin solution. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the cultivation of Gram-negative cocci and other pathogenic organisms.With added hemoglobin it is used for cultivation of Francisella tularensis.

Inhibitory Mold Agar Infusion Broth Composition per liter: Pancreatic digest of casein .......................................................... 13.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Heart muscle, solids from infusion ............................................... 2.0g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of a wide variety of microorganisms.

Infusion Cystine Agar Base, HiVeg Composition per liter:

Composition per liter: Agar ............................................................................................ 15.0g Glucose ......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Pancreatic digest of casein............................................................ 3.0g Na2HPO4 ....................................................................................... 2.0g Peptic digest of animal tissue ....................................................... 2.0g Starch ............................................................................................ 2.0g Dextrin .......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.8g Chloramphenicol....................................................................... 0.125g FeSO4 .......................................................................................... 0.04g NaCl............................................................................................ 0.04g MnSO4 ........................................................................................ 0.16g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Plant infusion .............................................................................. 10.0g Plant peptone No. 3..................................................................... 10.0g NaCl .............................................................................................. 5.0g L-Cystine ....................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from Hi-

Inorganic Salts-Maltose Medium (DSMZ Medium 754)

Media.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation of pathogenic fungi.

Composition per liter:

water and bring volume to1.0L. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Yeast extract.................................................................................. 4.0g Peptone ......................................................................................... 2.0g Inorganic salt solution............................................................980.0mL Maltose solution.......................................................................20.0mL

Use: For the cultivation of Gram-negative cocci and other pathogenic

Maltose Solution: Composition per liter:

organisms.

Infusion Cystine Agar Base, HiVeg with Hemoglobin

Maltose ......................................................................................... 5.0g

Preparation of Maltose Solution: Add maltose to 20.0mL dis-

Composition per liter:

tilled/deionized water. Mix thoroughly. Filter sterilize.

Inorganic Salt Solution: Composition per liter:

MgSO4·7H2O ............................................................................ 49.37g NaCl............................................................................................ 43.8g CaCl2·2H2O ................................................................................ 1.29g

876

Inorganic Salts Maltose Medium

Preparation of Inorganic Salt Solution: Add components in the

Preparation of Medium: Add components to distilled/deionized

order CaCl2·2H2O, NaCl, MgSO4·7H2O to 900.0mL distilled/deionized water. After addition of each, mix thorougly to prevent precipitation. Add distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components except maltose solution

Use: For the cultivation of soil microorganisms such as Rhizobium

to 980.0mL inorganic salt solution. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 20.0mL sterile maltose solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

spp.

Inorganic Salts Starch Agar See: ISP Medium 4

Use: For the cultivation of Spirochaeta halophila.

Inorganic Salts Maltose Medium Composition per liter: Yeast extract.................................................................................. 4.0g Peptone.......................................................................................... 2.0g Inorganic salts solution ..........................................................980.0mL Maltose solution.......................................................................20.0mL pH 7.5 ± 0.2 at 25°C

Inorganic Salts Solution: Composition per liter: MgSO4·7H2O ............................................................................ 49.37g NaCl ............................................................................................ 43.8g CaCl2·2H2O................................................................................. 1.29g

Preparation of Inorganic Salts Solution: Add NaCl first and then other components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Maltose Solution: Composition per 100.0mL: Maltose........................................................................................ 25.0g

Preparation of Maltose Solution: Add maltose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except maltose solution, to inorganic salts solution and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 7.5 with KOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 20.0mL of sterile maltose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Spirochaeta halophila.

Inorganic Salt Medium (Modified Raggios Medium) Composition per liter: Na2CO3 ......................................................................................... 3.0g KI ................................................................................................ 0.75g MgSO4·7H2O ................................................................................ 0.7g CaCl2·2H2O............................................................................... 0.446g KH2PO4 ......................................................................................... 0.2g Na2SO4 .......................................................................................... 0.2g KCl............................................................................................ 0.165g MnSO4·2H2O ........................................................................... 6.64mg ZnSO4·7H2O ............................................................................ 2.67mg FeCl3·4H2O ................................................................................ 2.5mg H3BO3 ........................................................................................ 1.5mg Na2MoO4·2H2O ....................................................................... 0.25mg CuSO4·5H2O ............................................................................ 0.07mg

Inositol Assay Medium Composition per liter: Glucose ..................................................................................... 100.0g Potassium citrate......................................................................... 10.0g Citric acid...................................................................................... 2.0g KH2PO4......................................................................................... 1.1g KCl.............................................................................................. 0.85g L-Asparagine ................................................................................. 0.8g L-Glutamic acid............................................................................. 0.6g L-Isoleucine ................................................................................... 0.5g L-Leucine ...................................................................................... 0.5g L-Lysine......................................................................................... 0.5g L-Valine ......................................................................................... 0.5g L-Arginine ................................................................................... 0.48g DL-Alanine .................................................................................... 0.4g DL-Threonine................................................................................. 0.4g CaCl2 ........................................................................................... 0.25g MgSO4·7H2O .............................................................................. 0.25g DL-Aspartic acid............................................................................ 0.2g DL-Phenylalanine .......................................................................... 0.2g Glycine.......................................................................................... 0.2g L-Methionine ................................................................................. 0.2g L-Tyrosine ..................................................................................... 0.2g L-Proline........................................................................................ 0.2g L-Histidine................................................................................. 0.124g DL-Serine ....................................................................................... 0.1g L-Cystine ....................................................................................... 0.1g DL-Tryptophan............................................................................. 0.08g FeCl3 ........................................................................................... 0.05g MnSO4·7H2O .............................................................................. 0.05g Calcium pantothenate ................................................................ 5.0mg Pyridoxine·HCl .......................................................................... 1.0mg Thiamine·HCl ............................................................................ 0.5mg Biotin ....................................................................................... 0.01mg pH 5.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 5 min at 15 psi pressure–121°C.

Use: For the microbiological assaying of inositol using Saccharomyces uvarum as the test organism.

Inositol Assay Medium KB Composition per liter: Glucose ....................................................................................... 40.0g (NH4)2SO4 .................................................................................... 4.0g DL-Asparagine ............................................................................... 4.0g KH2PO4......................................................................................... 3.0g MgSO4·7H2O ................................................................................ 1.0g

Inositol Urea Caffeic Acid Medium

877

Use: For the isolation of Aeromonas and Plesiomonas species.

CaCl2 ........................................................................................... 0.98g FeSO4·7H2O............................................................................... 0.5mg Calcium pantothenate ................................................................ 0.4mg Nicotinic acid ............................................................................. 0.4mg Pyridoxine .................................................................................. 0.4mg Thiamine .................................................................................... 0.4mg H3BO3 ........................................................................................ 0.2mg KI ............................................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.09mg MnSO4·7H2O ........................................................................... 0.08mg ZnSO4·7H2O ............................................................................ 0.08mg (NH4)6Mo7O24·4H2O ............................................................... 0.04mg Riboflavin ................................................................................ 0.02mg Biotin ..........................................................................................0.4μg pH 5.0 ± 0.2 at 25°C

Gelatin....................................................................................... 120.0g Inositol ........................................................................................ 10.0g Na2HPO4 ....................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Phenol Red.................................................................................. 0.05g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Plesiomonas shigelloides from foods.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 5 min at 15 psi pressure–121°C.

Inositol Gelatin Deeps Composition per liter:

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.4. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 10 psi pressure–115°C.

Inositol Urea Caffeic Acid Medium

Use: For the microbiological assaying of inositol using Kloeckera api-

Composition per liter:

culata as the test organism.

Agar solution .........................................................................900.0mL Base solution..........................................................................100.0mL

Inositol Brilliant Green Bile Salts Agar (IBB Agar) (Plesiomonas Differential Agar) Composition per liter: Agar ............................................................................................ 15.0g meso-Inositol............................................................................... 10.0g Proteose peptone ......................................................................... 10.0g Bile salts No. 3.............................................................................. 8.5g Meat extract .................................................................................. 5.0g NaCL............................................................................................. 5.0g Neutral Red (2% solution) .......................................................1.25mL Brilliant Green (0.1% solution) ...............................................0.33mL pH 7.2 ± 0.1 at 25°C

Use: For the isolation of Aeromonas and Plesiomonas species.

Inositol Brilliant Green HiVeg Agar (Plesiomonas Differential HiVeg Agar) Composition per liter: Plant peptone No. 3..................................................................... 15.0g Agar ............................................................................................ 13.5g meso-Inositol............................................................................... 10.0g Plant extract No. 1 ........................................................................ 6.5g NaCl .............................................................................................. 5.0g Synthetic detergent No. I .............................................................. 2.0g Neutral Red ............................................................................... 0.025g Brilliant Green ......................................................................... 0.33mg pH 7.2 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Agar Solution: Composition per 900.0mL: Agar ............................................................................................ 15.0g

Preparation of Agar Solution: Add agar to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Base Solution: Composition per 100.0mL: Inositol ........................................................................................ 10.0g Urea............................................................................................... 5.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g Caffeic acid ................................................................................... 0.2g NaCl.............................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g Gentamicin sulfate ...................................................................... 0.04g H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg KI ............................................................................................... 0.1mg CuSO4·5H2O............................................................................ 0.04mg Folic acid .................................................................................... 2.0μg Biotin .......................................................................................... 2.0μg Ferric citrate solution (1% solution) ..........................................1.0mL

Preparation of Base Solution: Add components, except urea, to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Gently heat just until components are dissolved. Cool to 75°–80°C. Add urea. Mix thoroughly. Do not heat after addition of urea. Do not autoclave. Filter sterilize.

878

Intracellular Growth Phase Medium

Preparation of Medium: Aseptically combine the cooled, sterile agar solution with the sterile base solution. Mix thoroughly. Pour into sterile Petri dishes.

Dulbecco’s phosphate-buffered saline .....................................20.0mL Fetal bovine serum.....................................................................8.0mL pH 7.2–7.4 at 25°C

Use: For the selective isolation and differentiation of Cryptococcus

Eagle MEM: Composition per liter:

species based on inositol and urea utilization and pigment production from caffeic acid. On this medium, only Cryptococcus species utilize inositol as sole carbon source and urea as sole nitrogen source. Cryptococcus neoformans appears as dark brown colonies. Other Cryptococcus species are unpigmented.

International Streptomyces Project Medium 1 See: ISP Medium 1 International Streptomyces Project Medium 2 See: ISP Medium 2 International Streptomyces Project Medium 3 See: ISP Medium 3 International Streptomyces Project Medium 4 See: ISP Medium 4 International Streptomyces Project Medium 4 with Glucose See: ISP Medium 4 with Glucose International Streptomyces Project Medium 4 with Yeast Extract See: ISP Medium 4 with Yeast Extract International Streptomyces Project Medium 5 See: ISP Medium 5 International Streptomyces Project Medium 6 See: ISP Medium 6 International Streptomyces Project Medium 7 See: Tyrosine Agar International Streptomyces Project Medium 8 See: Nitrate Broth International Streptomyces Project Medium 9 See: ISP Medium 9

Intracellular Growth Phase Medium (IGP Medium) Composition per 100.0mL: Gentamicin sulfate ................................................................. 500.0mg Lysozyme ................................................................................. 30.0mg Eagle MEM..............................................................................72.0mL © 2010 by Taylor and Francis Group, LLC

NaCl.............................................................................................. 8.0g Glucose ......................................................................................... 1.0g KCl................................................................................................ 0.4g CaCl2·2H2O ................................................................................ 0.14g MgSO4·7H2O ................................................................................ 0.1g KH2PO4....................................................................................... 0.06g Na2HPO4 ..................................................................................... 0.05g L-Isoleucine ............................................................................... 0.026g L-Leucine .................................................................................. 0.026g L-Lysine..................................................................................... 0.026g L-Threonine ............................................................................... 0.024g L-Valine ................................................................................... 0.0235g L-Tyrosine ................................................................................. 0.018g L-Arginine ............................................................................... 0.0174g L-Phenylalanine....................................................................... 0.0165g L-Cystine ................................................................................... 0.012g L-Histidine.................................................................................. 8.0mg L-Methionine .............................................................................. 7.5mg Phenol Red................................................................................. 5.0mg L-Tryptophan.............................................................................. 4.0mg Inositol ....................................................................................... 1.8mg Biotin ......................................................................................... 1.0mg Folic acid ................................................................................... 1.0mg Calcium pantothenate ................................................................ 1.0mg Choline chloride......................................................................... 1.0mg Nicotinamide.............................................................................. 1.0mg Pyridoxal·HCl ............................................................................ 1.0mg Thiamine·HCl ............................................................................ 1.0mg Riboflavin .................................................................................. 0.1mg

Preparation of Eagle MEM: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Dulbecco’s Phosphate-Buffered Saline: Composition per liter: NaCl.............................................................................................. 8.0g Na2HPO4·7H2O........................................................................... 2.16g KCl................................................................................................ 0.2g KH2PO4......................................................................................... 0.2g CaCl2 ............................................................................................. 0.1g MnCl2·6H2O ................................................................................. 0.1g

Preparation of Dulbecco’s Phosphate-Buffered Saline: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Combine components. Mix thoroughly. Filter sterilize. Aseptically distribute into sterile tubes or flasks.

Use: For the screening of Escherichia coli for pathogenicity using the HeLa cell test for invasiveness.

Ion Agar for Ureaplasma Composition per 101.45mL: HEPES (N-[2-hydroxyethyl]piperazine-N´-[2-ethanesulfonic acid]) buffer ............................................................ 1.19g Ionagar No. 2 .............................................................................. 0.75g Pancreatic digest of casein............................................................ 0.7g NaCl.............................................................................................. 0.5g Beef extract................................................................................... 0.3g

Irgasan® Ticarcillin Chlorate Broth

Yeast extract.................................................................................. 0.3g Beef heart, solids from infusion.................................................... 0.2g Yeast extract.................................................................................. 0.1g Horse serum, normal sterile .....................................................10.0mL Ampicillin solution ....................................................................1.0mL Urea solution............................................................................0.25mL Nystatin solution ........................................................................0.1mL Tripeptide solution .....................................................................0.1mL pH 7.2 ± 0.2 at 25°C

Ampicillin Solution: Composition per 10.0mL:

879

Preparation of Ionic Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Salt Solution: Composition per liter: MgSO4·7H2O .............................................................................. 14.8g FeSO4·7H2O................................................................................ 0.55g MnSO4 ...................................................................................... 0.045g H2SO4, concentrated ..................................................................0.2mL

Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Ampicillin ..................................................................................... 1.0g

Pipecolic Acid·HCl Solution: Composition per 100.0mL:

Preparation of Ampicillin Solution: Add ampicillin to distilled/

Pipecolic acid·HCl ...................................................................... 4.14g

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Pipecolic Acid·HCl Solution: Add pipecolic

Urea Solution: Composition per 100.0mL: Urea............................................................................................. 10.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 100.0mL. Filter sterilize. Store at –20°C. Nystatin Solution: Composition per 1.0mL: Nystatin.................................................................................. 50,000U

Preparation of Nystatin Solution: Add nystatin to distilled/deionized water and bring volume to 1.0mL. Filter sterilize.

Tripeptide Solution: Composition per 10.0mL: Glycyl-L-histidyl-L-lysine acetate .............................................. 0.2mg

Preparation of Tripeptide Solution: Add glycyl-L-histidyl-Llysine acetate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Store at –20°C.

Preparation of Medium: Add components—except horse serum, ampicillin solution, urea solution, nystatin solution, and tripeptide solution—to distilled/deionized water and bring volume to 90.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile horse serum, 1.0mL of sterile ampicillin solution, 0.25mL of sterile urea solution, 0.1mL of sterile nystatin solution, and 0.1mL of sterile tripeptide solution. Mix thoroughly. Pour into sterile Petri dishes. Use: For the cultivation of Ureaplasma species from clinical specimens.

Ionic Medium with Pipecolate Composition per liter: Agar ............................................................................................ 30.0g Ionic medium .........................................................................950.0mL Pipecolic acid·HCl solution .....................................................50.0mL

Ionic Medium: Composition per liter: K2HPO4 ......................................................................................... 4.1g Na2HPO4 ..................................................................................... 3.34g KH2PO4 ....................................................................................... 2.26g NaH2PO4 ..................................................................................... 2.24g Salt solution .............................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

acid·HCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.0.

Preparation of Medium: Add agar to 950.0mL of ionic medium. Mix thoroughly. Gently heat and bring to boiling. Add 50.0mL of pipecolic acid·HCl. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Pseudomonas putida.

Irgasan® Ticarcillin Chlorate Broth (ITC Broth) Composition per liter: MgCl2·6H20 ................................................................................ 60.0g Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g KClO4 ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Malachite Green (0.2% solution)...............................................5.0mL Irgasan solution..........................................................................1.0mL Ticarcillin solution.....................................................................1.0mL pH 7.6 ± 0.2 at 25°C

Irgasan Solution: Composition per 10.0mL: Irgasan (triclosan) ...................................................................... 1.0mg

Preparation of Irgasan Solution: Add Irgasan to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Ticarcillin Solution: Composition per 10.0mL: Ticarcillin.................................................................................. 1.0 mg

Preparation of Ticarcillin Solution: Add ticarcillin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Irgasan solution and ticarcillin solution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Adjust to pH 7.6. Aseptically add 1.0mL of Irgasan solution and 1.0mL of ticarcillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the selective isolation and cultivation of Yersinia species.

880

Iron Agar, Lyngby

Iron Agar, Lyngby (Lyngby Iron Agar) Composition per liter: Peptone........................................................................................ 20.0g Agar ............................................................................................ 12.0g NaCl .............................................................................................. 5.0g Beef extract ................................................................................... 3.0g Yeast extract.................................................................................. 3.0g L-Cysteine...................................................................................... 0.6g Ferric citrate .................................................................................. 0.3g Na2S2O3 ........................................................................................ 0.3g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Use: For the cultivation and enumeration of H2S-producing bacteria and total counts of heterotrophic bacteria from fish and fish products.

Iron Bacteria Isolation Medium Composition per liter:

Use: For the cultivation of lactic acid bacteria. For the cultivation and differentiation of Clostridium species. The medium turns black if H2S is produced. The medium turns red if acid is produced from milk carbohydrate fermentation. Acid and gas production is characteristic of Clostridium perfringens and Clostridium butyricum.

Iron Milk Medium, Modified Composition per 1050.0mL: Whole milk, fresh .........................................................................1.0L FeSO4·7H2O.................................................................................. 1.0g

Preparation of Medium: Add FeSO4·7H2O to distilled/deionized water and bring volume to 50.0mL. Add slowly to 1.0L of whole milk. Mix thoroughly. Distribute into tubes in 11.0mL volumes. Autoclave for 12 min at 13 psi pressure–118°C. Use: For the cultivation and enumeration of Clostridium perfringens in foods.

Iron-Oxidizing Medium Composition per liter: (NH4)2SO4 .................................................................................... 3.0g K2HPO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g KCl................................................................................................ 0.1g Ca(NO3)2..................................................................................... 0.01g FeSO4·7H2O solution.............................................................300.0mL H2SO4 (10N) ..............................................................................1.0mL pH 3.0–3.6 at 25°C

Agar ............................................................................................ 10.0g (NH4)2SO4 ..................................................................................... 0.5g Glucose ....................................................................................... 0.15g CaCO3 ........................................................................................... 0.1g K2HPO4 ....................................................................................... 0.05g MgSO4·7H2O .............................................................................. 0.05g KCl.............................................................................................. 0.05g Ca(NO3)2 ..................................................................................... 0.01g Vitamin solution.......................................................................10.0mL

FeSO4·7H2O Solution: Composition per 300.0mL:

Vitamin Solution: Composition per 10.0mL:

Preparation of Medium: Add components, except FeSO4·7H2O so-

Thiamine .................................................................................... 0.4mg Cyanocobalamin ...................................................................... 0.01mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of vitamin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation of iron bacteria.

Iron Milk Medium

FeSO4·7H2O.............................................................................. 44.22g

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 300.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. lution, to distilled/deionized water and bring volume to 700.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 300.0mL of sterile FeSO4·7H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the enumeration, isolation, and cultivation of iron and sulfur bacteria, such as Thiobacillus ferrooxidans.

Iron Sulfite Agar Composition per liter: Agar ............................................................................................ 12.0g Pancreatic digest of casein.......................................................... 10.0g Ferric citrate.................................................................................. 0.5g Na2S·9H2O.................................................................................... 0.5g pH 7.1 ± 0.2 at 25°C

Composition per liter:

Source: This medium is available as a premixed powder from Oxoid

Iron filings..................................................................................... 1.0g Whole milk ...................................................................................1.0L pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium: Add iron filings, which may be small balls of steel wool, to whole milk and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC

Unipath. water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Use: For the detection of thermophilic anaerobic organisms.

Iso-Sensitest Agar

Iron Sulfite HiVeg Agar Composition per liter: Agar ............................................................................................ 15.0g Plant hydrolysate......................................................................... 10.0g Iron (III) citrate ............................................................................. 0.5g Na2SO3 ......................................................................................... 0.5g pH 7.1 ± 0.2 at 25°C

881

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 10 psi pressure–115°C. Cool to 50°C. Aseptically add horse serum. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the identification and cultivation of Group B streptococci from clinical specimens.

Source: This medium is available as a premixed powder from HiMedia.

ISM Broth

Preparation of Medium: Add components to distilled/deionized

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

MgSO4·7H2O .............................................................................. 49.2g NaCl............................................................................................ 43.5g Yeast extract.................................................................................. 4.0g Peptone ......................................................................................... 2.0g CaCl2·2H2O .................................................................................. 1.5g Maltose solution.....................................................................100.0mL

Use: For the detection of thermophilic anaerobic organisms. Irradiated Tryptone Soya Broth See: Cold Filterable Tryptone Soya Broth Irradiated Vegetable Peptone Broth See: Cold Filterable Vegetable Peptone Broth Islam GBS Agar See: GBS Agar Base, Islam

ISM Agar Composition per liter: MgSO4·7H2O .............................................................................. 49.2g NaCl ............................................................................................ 43.5g Agar .............................................................................................. 7.5g Yeast extract.................................................................................. 4.0g Peptone.......................................................................................... 2.0g CaCl2·2H2O................................................................................... 1.5g Maltose solution.....................................................................100.0mL

Maltose Solution: Composition per 100.0mL: Maltose.......................................................................................... 5.0g

Preparation of Maltose Solution: Add maltose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except maltose solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile maltose solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Spirochaeta halophila.

Islams Medium Base for Group B Streptococci Composition per liter: Proteose peptone ......................................................................... 23.0g Agar ............................................................................................ 10.0g Na2HPO4 ................................................................................... 5.749g Starch, soluble............................................................................... 5.0g KH2PO4 ..................................................................................... 1.482g Horse serum, sterile inactivated...............................................50.0mL pH 7.4 ± 0.2 at 25°C

Maltose Solution: Composition per 100.0mL: Maltose ......................................................................................... 5.0g

Preparation of Maltose Solution: Add maltose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except maltose solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile maltose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Spirochaeta halophila.

Iso-Sensitest Agar Composition per liter: Casein, hydrolyzed ..................................................................... 11.0g Agar .............................................................................................. 8.0g Peptones........................................................................................ 3.0g NaCl.............................................................................................. 3.0g Na2HPO4 ....................................................................................... 2.0g Glucose ......................................................................................... 2.0g Sodium acetate.............................................................................. 1.0g Soluble starch................................................................................ 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-Cysteine·HCl............................................................................ 0.02g L-Tryptophan............................................................................... 0.02g Adenine....................................................................................... 0.01g Guanine....................................................................................... 0.01g Xanthine...................................................................................... 0.01g Uracil .......................................................................................... 0.01g Nicotinamide.............................................................................. 3.0mg Pantothenate............................................................................... 3.0mg Pyridoxine.................................................................................. 3.0mg MnCl2·4H2O .............................................................................. 2.0mg CoSO4 ........................................................................................ 1.0mg CuSO4·5H2O.............................................................................. 1.0mg FeSO4·7H2O............................................................................... 1.0mg Menadione ................................................................................. 1.0mg Cyanocobalamin ........................................................................ 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg

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Iso Sensitest Broth

Biotin ......................................................................................... 0.3mg Thiamine .................................................................................. 0.04mg pH 7.4 ± 0.2 at 25°C

Use: For antimicrobial susceptibility testing.

Iso Sensitest Broth Composition per liter: Casein, hydrolyzed...................................................................... 11.0g Peptones ........................................................................................ 3.0g NaCl .............................................................................................. 3.0g Glucose ......................................................................................... 2.0g Na2HPO4 ....................................................................................... 2.0g Sodium acetate .............................................................................. 1.0g Soluble starch................................................................................ 1.0g Magnesium glycerophosphate ...................................................... 0.2g Calcium gluconate ........................................................................ 0.1g L-cysteine·HCl ............................................................................ 0.02g L-Tryptophan ............................................................................... 0.02g Adenine ....................................................................................... 0.01g Guanine ....................................................................................... 0.01g Xanthine...................................................................................... 0.01g Uracil .......................................................................................... 0.01g Nicotinamide.............................................................................. 3.0mg Pantothenate............................................................................... 3.0mg Pyridoxine .................................................................................. 3.0mg MnCl2·4H2O............................................................................... 2.0mg CoSO4 ........................................................................................ 1.0mg CuSO4·5H2O .............................................................................. 1.0mg FeSO4·7H2O............................................................................... 1.0mg Menadione ................................................................................. 1.0mg Cyanocobalamin ........................................................................ 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.3mg Thiamine .................................................................................. 0.04mg pH 7.4 ± 0.2 at 25°C

Use: For antimicrobial susceptibility testing.

Isoleucine Hydroxamate Medium Composition per liter: Agar ............................................................................................ 15.0g K2HPO4 ......................................................................................... 7.0g Glucose ......................................................................................... 5.0g KH2PO4 ......................................................................................... 3.0g L-Isoleucine hydroxamate ............................................................. 1.0g (NH4)2SO4 ..................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Isonema Medium Composition per liter: Pancreatic digest of casein............................................................ 1.0g Seawater.................................................................................990.0mL Horse serum, heat inactivated..................................................10.0mL

Preparation of Medium: Add pancreatic digest of casein to seawater and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL of heat-inactivated horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Isonema papillatum.

Isosphaera Agar Composition per 1000.5mL: Solution A..............................................................................800.0mL Solution B ..............................................................................100.0mL Solution C ..............................................................................100.0mL Vitamin solution.........................................................................0.5mL pH 7.6 ± 0.2 at 25°C

Solution A: Composition per 800.0mL: Agar, noble.................................................................................. 15.0g NaCl............................................................................................ 0.25g (NH4)2SO4 ................................................................................ 0.125g KCl............................................................................................ 0.125g MgSO4·7H2O ................................................................................ 0.1g CaCl2·2H2O ............................................................................. 80.0mg KH2PO4.................................................................................... 75.0mg FeCl3 ......................................................................................... 73.0μg Trace elements solution SL-7 ....................................................2.5mL

Trace Elements Solution SL-7: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg H3BO3 ...................................................................................... 62.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................. 17.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-7: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly.

Preparation of Solution A: Add agar to 400.0mL of distilled/deionized water. In another flask, add remaining components to 400.0mL of distilled/deionized water. Mix thoroughly. Adjust pH to 7.6 with NaOH. Remove any precipitate that forms by filtering through Whatman #1 filter paper. Autoclave both solutions separately for 15 min at 15 psi pressure–121°C. Aseptically combine the two sterile solutions. Cool to 50°–55°C.

Solution B: Composition per 100.0mL: NaHCO3 ...................................................................................... 0.42g

Preparation of Solution B: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 50°–55°C.

Isosphaera pallida Medium

883

Solution C: Composition per 100.5mL:

Whatman #1 filter paper. Autoclave for 15 min at 15 psi pressure– 121°C.

Glucose ....................................................................................... 0.25g Casamino acids ........................................................................... 0.25g

Solution B: Composition per 100.0mL:

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 50°–55°C. Vitamin Solution: Composition per 100.0mL: Nicotinic acid ......................................................................... 200.0mg Thiamine HCl ........................................................................ 200.0mg p-Aminobenzoic acid............................................................... 20.0mg Biotin ......................................................................................... 2.0mg Vitamin B12 ...............................................................................25.0μg

Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Warm to 50°–55°C.

Preparation of Medium: Aseptically combine 800.0mL of sterile solution A with 100.0mL of sterile solution B, 100.0mL of sterile solution C, and 0.5mL of sterile vitamin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Isosphaera pallida.

Isosphaera Broth Composition per 1000.5mL: Solution A ..............................................................................800.0mL Solution B ..............................................................................100.0mL Solution C ..............................................................................100.0mL Vitamin solution.........................................................................0.5mL pH 7.6 ± 0.2 at 25°C

Solution A: Composition per 800.0mL: NaCl ............................................................................................ 0.25g (NH4)2SO4 ................................................................................. 0.125g KCl............................................................................................ 0.125g MgSO4·7H2O ................................................................................ 0.1g CaCl2·2H2O.............................................................................. 80.0mg KH2PO4 .................................................................................... 75.0mg FeCl3 .........................................................................................73.0μg Trace elements solution SL-7 ....................................................2.5mL

Trace Elements Solution SL-7: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg H3BO3 ...................................................................................... 62.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................. 17.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-7: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly.

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Adjust pH to 7.6 with NaOH. Remove any precipitate that forms by filtering through © 2010 by Taylor and Francis Group, LLC

NaHCO3 ...................................................................................... 0.42g

Preparation of Solution B: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Solution C: Composition per 100.5mL: Glucose ....................................................................................... 0.25g Casamino acids ........................................................................... 0.25g

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Vitamin Solution: Composition per 100.0mL: Nicotinic acid......................................................................... 200.0mg Thiamine HCl ........................................................................ 200.0mg p-Aminobenzoic acid............................................................... 20.0mg Biotin ......................................................................................... 2.0mg Vitamin B12 ............................................................................... 25.0μg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Vitamin Solution: Aseptically combine 800.0mL of sterile solution A with 100.0mL of sterile solution B, 100.0mL of sterile solution C, and 0.5mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Isosphaera pallida.

Isosphaera pallida Medium (DSMZ Medium 765) Composition per 1005.0mL: Solution A..............................................................................900.0mL NaHCO3 solution ...................................................................100.0mL Vitamin solution.........................................................................5.0mL pH 7.9 ± 0.2 at 25°C Solution A: Composition per 900.0mL: KCl............................................................................................... 4.0g MgSO4·7H2O ................................................................................ 2.0g (NH4)2SO4 .................................................................................... 1.0g NaCl.............................................................................................. 1.0g KH2PO4......................................................................................... 0.3g Glucose ....................................................................................... 0.25g Casamino acids ........................................................................... 0.25g CaCl2·2H2O .................................................................................. 0.2g FeCl3 ...................................................................................... 0.292mg Trace elements solution SL-7a ..................................................1.0mL

Trace Elements Solution SL-7a: CoCl2·6H2O ........................................................................... 200.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg H3BO3 ...................................................................................... 60.0mg Na2MoO4·4H2O ....................................................................... 40.0mg NiCl2·6H2O.............................................................................. 20.0mg CuCl2·2H2O ............................................................................. 20.0mg HCl (25%)..................................................................................1.0mL

884

IsoVitaleX® Enrichment

Preparation of Trace Elements Solution SL-7a: Add components

Preparation of Medium: Add components to distilled/deionized

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Sparge with 95% air + 5% CO2.

Use: For the cultivation of Streptomyces species according to the International Streptomyces Project.

Vitamin Solution: Composition per 100.0mL: Nicotinic acid ........................................................................... 20.0mg Thiamine-HCl·2H2O ................................................................ 10.0mg p-Aminobenzoic acid ................................................................. 0.2mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

NaHCO3 Solution: Composition per 100.0mL:

ISP HiVeg Medium No. 2 (Yeast Malt HiVeg Agar) Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 10.0g Plant peptone ................................................................................ 5.0g Malt extract................................................................................... 3.0g Yeast extract.................................................................................. 3.0g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

NaHCO3 ........................................................................................ 4.2g

Media.

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Seal in serum bottle under 100% CO2.

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium: Dispense under 95% air + 5% CO2. Fill

Use: For the cultivation of Streptomyces species according to the

serum bottles so that the gas to liquid ratio is about 5:1 (v/v). Sparge with 95% air + 5% CO2. Seal bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Using a syringe, inject 10.0mL sterile NaHCO3 solution/90.0mL solution A. Then inject 0.5 mL sterile vitamin solution per 100.0mL of the resulting medium.

International Streptomyces Project.

Use: For the cultivation of Isosphaera pallida=Isocystis pallida.

Agar ............................................................................................ 15.0g Plant peptone .............................................................................. 15.0g Plant peptone No. 3....................................................................... 5.0g K2HPO4......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ...................................................................................... 0.08g pH 6.7 ± 0.2 at 25°C

IsoVitaleX® Enrichment Composition per liter: Glucose ..................................................................................... 100.0g L-Cysteine·HCl............................................................................ 25.9g L-Glutamine................................................................................. 10.0g Adenine ......................................................................................... 1.0g Thiamine pyrophosphate............................................................... 0.1g Vitamin B12 ................................................................................... 0.1g Guanine·HCl ............................................................................... 0.03g Fe(NO3)3·9H2O ........................................................................... 0.02g p-Aminobenzoic acid ................................................................ 0.013g Thiamine·HCl ........................................................................... 0.003g

Preparation of IsoVitaleX® Enrichment: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Use: As a nutrient supplement for the isolation and cultivation of fastidious microorganisms.

ISP HiVeg Medium No. 1 (Tryptone Yeast Extract HiVeg Broth) Composition per liter: Plant hydrolysate........................................................................... 5.0g Yeast extract.................................................................................. 3.0g pH 7.0–7.2 at 25°C

ISP HiVeg Medium No. 6 (Peptone Yeast Extract Iron HiVeg Agar) Composition per liter:

Use: For the cultivation and maintenance of Streptomyces species.

ISP2 Medium (DSMZ Medium 987) Composition per liter: Agar ............................................................................................ 20.0g Malt extract................................................................................. 10.0g Yeast extract.................................................................................. 4.0g Glucose ......................................................................................... 4.0g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into Petri dishes or leave in tubes.

Use: For the cultivation of Streptacidiphilus jiangxiensis.

ISP Medium 4

ISP 5 Medium (DSMZ Medium 993) Composition per liter: Agar ............................................................................................ 20.0g Glycerol ...................................................................................... 10.0g L-asparagine, anhydrous ............................................................... 1.0g K2HPO4, anhydrous ...................................................................... 1.0g Trace elements solution .............................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O.................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Use: For the cultivation of Acrocarpospora macrocephala.

ISP Medium 1 (International Streptomyces Project Medium 1) (Tryptone Yeast Extract Broth) Composition per liter: Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 3.0g pH 7.0–7.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Streptomyces species according to the International Streptomyces Project.

ISP Medium 2 (International Streptomyces Project Medium 2) (Yeast Extract Malt Extract Agar) Composition per liter: Agar ............................................................................................ 20.0g Malt extract ................................................................................. 10.0g Yeast extract.................................................................................. 4.0g Glucose ......................................................................................... 4.0g pH 7.3 ± 0.2 at 25°C

Use: For the cultivation of Streptomyces species according to the International Streptomyces Project.

ISP Medium 2 with 5% Sodium Chloride Composition per liter: NaCl ............................................................................................ 50.0g Agar ............................................................................................ 20.0g © 2010 by Taylor and Francis Group, LLC

885

Malt extract................................................................................. 10.0g Glucose ......................................................................................... 4.0g Yeast extract.................................................................................. 4.0g pH 7.3–7.5 at 25°C

Use: For the cultivation of Streptomyces species.

ISP Medium 3 (International Streptomyces Project Medium 3) (Oatmeal Agar) Composition per liter: Oatmeal....................................................................................... 20.0g Agar ............................................................................................ 18.0g Trace salts solution ....................................................................1.0mL

Trace Salts Solution: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O ................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g

Preparation of Trace Salts Solution: Add components to distilled/deionized water and bring the volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add oatmeal to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Steam for 20 min. Filter through cheesecloth. Add agar. Add sufficient distilled/deionized water to bring volume to 999.0mL. Gently heat and bring to boiling. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 1.0mL of sterile trace salts solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Streptomyces species according to the International Streptomyces Project.

ISP Medium 4 (International Streptomyces Project Medium 4) (Inorganic Salts Starch Agar) Composition per liter: Agar ............................................................................................ 20.0g Soluble starch.............................................................................. 10.0g CaCO3 ........................................................................................... 2.0g (NH4)2SO4 .................................................................................... 2.0g K2HPO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g NaCl.............................................................................................. 1.0g FeSO4·7H2O............................................................................... 1.0mg MnCl2·7H2O .............................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

886

ISP Medium 4 with Glucose

Use: For characterizing Streptomyces species. For the cultivation and

L-Asparagine ................................................................................. 1.0g

maintenance of Actinomadura fastidiosa, Actinomadura roseoviolacea, Actinomadura species, Actinoplanes species, Amycolatopsis mediterranei, Kitasatosporia grisea, Kitasatosporia papulosa, Saccharomonospora internatus, Streptomyces species, Streptosporangium species, Saccharomonospora hirsuta, and Streptoverticillium species.

K2HPO4......................................................................................... 1.0g Trace salts solution ....................................................................1.0mL pH 7.4 ± 0.2 at 25°C

ISP Medium 4 with Glucose (International Streptomyces Project Medium 4 with Glucose)

FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O ................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g

Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g Soluble starch.............................................................................. 10.0g CaCO3 ........................................................................................... 2.0g (NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g NaCl .............................................................................................. 1.0g FeSO4·7H2O............................................................................... 1.0mg MnCl2·7H2O............................................................................... 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Streptomyces purpureus.

ISP Medium 4 with Yeast Extract (International Streptomyces Project Medium 4 with Yeast Extract) Composition per liter: Agar ............................................................................................ 20.0g Soluble starch.............................................................................. 10.0g CaCO3 ........................................................................................... 2.0g (NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g NaCl .............................................................................................. 1.0g Yeast extract.................................................................................. 1.0g FeSO4·7H2O............................................................................... 1.0mg MnCl2·7H2O............................................................................... 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Thermomonospora mesouviformis.

ISP Medium 5 (International Streptomyces Project Medium 5) (Glycerol Asparagine Agar) Composition per liter: Agar ............................................................................................ 20.0g Glycerol ...................................................................................... 10.0g © 2010 by Taylor and Francis Group, LLC

Trace Salts Solution: Composition per 100.0mL:

Preparation of Trace Salts Solution: Add components to distilled/deionized water and bring the volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except trace salts solution, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of sterile trace salts solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Pseudonocardia species and Streptomyces peucetius.

ISP Medium 6 (International Streptomyces Project Medium 6) (Peptone Yeast Extract Iron Agar) Composition per liter: Agar ............................................................................................ 15.0g Peptone ....................................................................................... 15.0g Proteose peptone........................................................................... 5.0g K2HPO4......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ...................................................................................... 0.08g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Streptomyces species. ISP Medium 7 See: Tyrosine Agar ISP Medium 8 See: Nitrate Broth

ISP Medium 9 (International Streptomyces Project Medium 9) Composition per liter: K2HPO4·3H2O ............................................................................ 5.65g (NH4)2SO4 .................................................................................. 2.64g KH2PO4....................................................................................... 2.38g MgSO4·7H2O ................................................................................ 1.0g Carbohydrate solution............................................................100.0mL Pridham and Gottlieb trace salts ................................................1.0mL pH 6.8–7.0 at 25°C

Carbohydrate Solution: Composition per 100.0mL: Carbohydrate............................................................................... 10.0g

IUT Medium Base with Glycerol and Egg Emulsion

887

Preparation of Carbohydrate Solution: Add carbohydrate to

Preparation of Medium: Add components to distilled/deionized

distilled/deionized water and bring volume to 100.0mL.Use glucose, arabinose, sucrose, xylose, inositol, mannitol, fructose, rhamnose, raffinose, or cellulose. Mix thoroughly. Filter sterilize.

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0 using H2SO4. Distribute into anaerobe tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Provide an atmosphere of 78% H2 + 20% CO2 + 2% O2.

Pridham And Gottlieb Trace Salts: Composition per 100.0mL: MnCl2·7H2O................................................................................ 0.79g CuSO4·5H2O ............................................................................... 0.64g ZnSO4·7H2O ............................................................................... 0.15g FeSO4·7H2O................................................................................ 0.11g

Preparation of Pridham and Gottlieb Trace Salts: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling with frequent agitation. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add sterile carbohydrate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and differentiation of Streptomyces purpureus and other Streptomyces species based on carbohydrate utilization.

ISS1 Medium (DSMZ Medium 889) Composition per liter: NaCl ............................................................................................ 20.0g MgSO4·7H2O ................................................................................ 7.0g MgCl2·6H2O.................................................................................. 5.5g NaHCO3 ........................................................................................ 2.0g KCl.............................................................................................. 0.65g CaCl2·2H2O................................................................................... 0.5g NH4Cl ........................................................................................... 0.3g K2HPO4 ......................................................................................... 0.2g Yeast extract.................................................................................. 0.2g NaBr.............................................................................................. 0.1g Trace elements solution ...........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of unclassified bacterium DSM 12045. ITC Broth See: Irgasan® Ticarcillin Chlorate Broth

ITC HiVeg Broth Base with Ticarcillin and Potassium Chlorate (TTC HiVeg Broth Base) Composition per liter: MnCl2·6H2O ............................................................................... 60.0g Plant hydrolysate ........................................................................ 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 1.0g Malachite Green.......................................................................... 0.01g Irgansan (Trichlosan) ................................................................. 1.0mg Ticarcillin solution...................................................................10.0mL Potassium chlorate solution pH 6.9 ± 0.2 at 25°C

Source: This medium, without ticarcillin and potassium chlorate, is available as a premixed powder from HiMedia. Ticarcillin Solution: Composition per 10.0mL: Ticarcillin................................................................................... 1.0mg

Preparation of Ticarcillin Solution: Add ticarcillin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Poassium Chlorate Solution: Composition per 10.0mL: KClO3 ........................................................................................... 1.0g

Preparation of Poassium Chlorate Solution: Add KClO3 to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Caution: Potassium chlorate is a highly oxidizable agent and can cause explosions. Take proper precautions when handling.

Preparation of Medium: Add components, except ticarcillin and potassium chlorate solutions, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL ticarcillin solution and 10.0mL potassium chlorate solution. Mix thoroughly. Distribute into tubes or flasks.

Use: For the selective isolation and cultivation of Yersinia species. For the selective enrichment and enumeration of Yersinia enterocolitica.

IUT Medium Base with Glycerol and Egg Emulsion Composition per 1600.0mL: L-Asparagine................................................................................. 3.6g KH2PO4....................................................................................... 2.46g Magnesium citrate ........................................................................ 0.6g Malachite Green............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g

888

J Agar

Egg emulsion ................................................................................1.0L Glycerol ...................................................................................12.0mL pH 7.0 ± 0.2 at 25°C

K2HPO4......................................................................................... 3.0g Glucose ......................................................................................... 2.0g pH 7.3–7.5 at 25°C

Source: This medium is available from HiMedia.

Preparation of Medium: Add components to distilled/deionized

Egg Emulsion: Composition per 1.0L:

Eggs ........................................................................................Variable

Preparation of Egg Emulsion: Soak whole eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs. Beat to form emulsion. Avoiding the formation of air bubbles. Sterilize by inspissation at 85°C for 1 hr.

Preparation of Medium: Add glycerol to distilled/deionized water and bring volume to 600.0mL. Mix thoroughly. Add remaining components other than egg emulsion. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Add sterile whole egg emulsion. Aseptically distribute into culture vessels.

Use: For the cultivation of Mycobacterium tuberculosis.

J Agar Composition per liter: Agar ............................................................................................ 20.0g Yeast extract................................................................................ 15.0g Pancreatic digest of casein ............................................................ 5.0g K2HPO4 ......................................................................................... 3.0g Glucose solution ......................................................................10.0mL pH 7.3–7.5 at 25°C

Use: For the cultivation and maintenance of Bacillus popilliae.

JD1 Medium Composition per liter: Beef heart, solids from infusion.................................................. 25.0g Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g NaCl.............................................................................................. 2.5g Bovine albumin............................................................................. 0.5g Hemin chloride ........................................................................... 0.04g pH 7.8 ± 0.2 at 25°C

Use: For the isolation and cultivation of PD-ALS (Pierce’s diseasealmond leaf scorch) bacteria.

JD3 Medium Composition per liter:

Glucose Solution: Composition per 10.0mL: Glucose ......................................................................................... 2.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution. Mix thoroughly. Pour into sterile Petri dishes. Use: For the cultivation of Bacillus species and Sporolactobacillus species.

Pancreatic digest of casein............................................................ 4.0g Papaic digest of soybean meal...................................................... 2.0g Trisodium citrate........................................................................... 2.0g K2HPO4......................................................................................... 1.5g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g Disodium succinate..................................................................... 0.01g Bovine serum albumin solution .............................................100.0mL Hemin chloride solution ..........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Bovine Serum Albumin Solution: Composition per 100.0mL: Bovine serum albumin fraction V................................................. 2.0g

J Broth Composition per liter: Yeast extract................................................................................ 15.0g Pancreatic digest of casein ............................................................ 5.0g pH 7.3–7.5 at 25°C

Preparation of Bovine Serum Albumin Solution: Add 2.0g of bovine serum albumin fraction V to 100.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Hemin Chloride Solution: Composition per 10.0mL:

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3–7.5. Distribute into tubes or flasks. Autoclave for 20 min at 15 psi pressure–121°C.

Hemin chloride ........................................................................... 0.01g

Use: For the cultivation of Bacillus species and Sporolactobacillus

Preparation of Medium: Add components, except bovine serum albumin solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Adjust to pH 7.0. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 100.0mL of sterile bovine serum albumin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

species for performing the Voges-Proskauer test.

JB Medium with Glucose Composition per liter: Yeast extract................................................................................ 15.0g Pancreatic digest of casein ............................................................ 5.0g © 2010 by Taylor and Francis Group, LLC

Preparation of Hemin Chloride Solution: Add 0.01g of hemin chloride to 10.0 mL of 0.5N NaOH. Mix thoroughly.

Use: For the cultivation of ATCC strain 33107.

K101 Flexibacter Medium

Jensen’s Medium Composition per liter: Sucrose........................................................................................ 20.0g Agar ............................................................................................ 15.0g CaCO3 ........................................................................................... 2.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl .............................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g Na2MoO4·2H2O ......................................................................... 5.0mg

Use: For the detection and cultivation of nitrogen fixing bacteria.

JO Composition per liter: Agar ............................................................................................ 20.0g Sucrose........................................................................................ 6.35g NaNO3........................................................................................... 1.5g Yeast extract................................................................................ 0.50g K2HPO4 ....................................................................................... 0.35g MgSO4 ........................................................................................ 0.25g

Use: For the cultivation and maintenance of fungi with inhibition of Mucor species.

Johnson’s Marine Medium Composition per liter: Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g Na2S2O3 ........................................................................................ 0.3g FeSO4·7H2O.................................................................................. 0.2g Filtered, aged seawater ..........................................................750.0mL

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of marine bacteria.

Jones–Kendrick Pertussis Transport Medium Composition per liter: Beef heart, solids from infusion................................................ 500.0g Agar ............................................................................................ 20.0g Soluble starch.............................................................................. 10.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Charcoal powder, activated........................................................... 4.0g Yeast extract.................................................................................. 3.5g Penicillin solution ....................................................................10.0mL pH 7.4 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

889

Penicillin Solution: Composition per 10.0mL: Penicillin ..................................................................................... 300U

Preparation of Penicillin Solution: Add penicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except penicillin solution, starch, yeast extract, heart infusion, and agar, to water. Boil to dissolve. Add charcoal, mix well, and autoclave. Cool to 50°C, add penicillin, and dispense into small bottles as slants. Cool and seal tightly. Store at 5°C. Stable for 2 to 3 months.

Use: For the cultivation and transport of Bordetella pertussis between clinical isolation and laboratory cultivation.

Jordan’s Tartrate Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g Sodium potassium tartrate .......................................................... 10.0g NaCl.............................................................................................. 5.0g Phenol Red................................................................................ 0.024g pH 7.7 ± 0.3 at 25°C

Source: This medium is available as a prepared medium in tubes from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.7. Distribute into tubes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the differentiation and identification of members of the Enterobacteriaceae, especially Salmonella species, based upon the ability to utilize tartrate. Utilization of tartrate turns the medium yellow. Salmonella enteritidis utilizes tartrate. Salmonella paratyphi A does not utilize tartrate.

K101 Flexibacter Medium Composition per liter: Agar ............................................................................................ 10.0g Casamino acids ............................................................................. 1.0g Glucose ......................................................................................... 1.0g Tris(hydroxymethyl)aminomethane buffer................................... 1.0g CaCl2 ............................................................................................. 0.1g KNO3 ............................................................................................ 0.1g MgSO4·7H2O ................................................................................ 0.1g Sodium glycerophosphate............................................................. 0.1g Thiamine·HCl ............................................................................ 1.0mg Cyanocobalamin ......................................................................... 1.0μg Trace elements solution HO-LE ................................................1.0mL pH 7.5 ± 0.2 at 25°C

Trace Elements Solution HO-LE: Composition per liter: H3BO3 ......................................................................................... 2.85g MnCl2·4H2O ................................................................................. 1.8g Sodium tartrate............................................................................ 1.77g FeSO4·7H2O................................................................................ 1.36g CoCl2·6H2O ................................................................................ 0.04g CuCl2·2H 2O ............................................................................. 0.027g Na2MoO4·2H2O ........................................................................ 0.025g ZnCl2 ........................................................................................... 0.02g

890

K7 Medium

Preparation of Trace Elements Solution HO-LE: Add compo-

Source: This medium is available as a premixed powder from Oxoid

nents to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Unipath.

Preparation of Medium: Add components, except trace elements solution HO-LE, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of trace elements solution HO-LE. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Cytophaga species, Flexibacter species, Herpetosiphon geysericola, and Myxococcus fulvus.

K7 Medium (DSMZ Medium 1199)

Kanamycin Sulfate Solution: Composition per 10.0mL: Kanamycin sulfate ................................................................... 20.0mg

Preparation of Kanamycin Sulfate Solution: Add kanamycin sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation of enterococci from foods.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.5. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into Petri dishes or leave in tubes.

Use: For the cultivation of Novosphingobium acidiphilum.

Kado’s Agar Composition per liter: Agar ............................................................................................ 15.0g Sucrose........................................................................................ 10.0g Pancreatic digest of casein ............................................................ 8.0g Yeast extract.................................................................................. 4.0g K2HPO4 ......................................................................................... 2.0g MgSO4·7H2O ........................................................................... 30.0mg

Kanamycin Esculin Azide Broth Composition per liter: Pancreatic digest of casein.......................................................... 20.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.15g Kanamycin sulfate solution .....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium is available as a premixed powder from Oxoid Unipath.

Kanamycin Sulfate Solution: Composition per 10.0mL: Kanamycin sulfate ...................................................................... 0.02g

Preparation of Kanamycin Sulfate Solution: Add kanamycin sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation of enterococci from foods.

Use: For the cultivation of Lactococcus lactis subspecies hordniae.

Kanamycin Esculin Azide Agar Composition per liter: Pancreatic digest of casein .......................................................... 20.0g Agar ............................................................................................ 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate ............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3............................................................................................ 0.15g Kanamycin sulfate solution .....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Kanamycin Esculin Azide HiVeg Agar Composition per liter: Plant hydrolysate ........................................................................ 20.0g Agar ............................................................................................ 12.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.15g Kanamycin sulfate ...................................................................... 0.02g pH 7.0 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Caution: Sodium azide is toxic. Azides also react with metals and

Source: This medium is available as a premixed powder from Hi-

disposal must be highly diluted.

Media.

Kanamycin Luria Agar Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation of enterococci from foods.

Kanamycin Esculin Azide HiVeg Agar Base with Kanamycin Composition per liter: Plant hydrolysate......................................................................... 20.0g Agar ............................................................................................ 10.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate ............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.15g Kanamycin sulfate solution .....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without kanamycin sulfate solution, is available as a premixed powder from HiMedia.

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Kanamycin Sulfate Solution: Composition per 10.0mL: Kanamycin sulfate ...................................................................... 0.02g

891

Kanamycin Esculin Azide HiVeg Broth Base with Kanamycin Composition per liter: Plant hydrolysate ........................................................................ 20.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.15g Kanamycin sulfate solution .....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium, without kanamycin sulfate solution, is available as a premixed powder from HiMedia.

Kanamycin Sulfate Solution: Composition per 10.0mL: Kanamycin sulfate ...................................................................... 0.02g

Preparation of Kanamycin Sulfate Solution: Add kanamycin sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of enterococci from foods.

Preparation of Kanamycin Sulfate Solution: Add kanamycin sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Use: For the isolation of enterococci from foods.

Kanamycin Esculin Azide HiVeg Broth

Kanamycin L Broth Medium Composition per liter: Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 1.0g Kanamycin solution .................................................................10.0mL

Kanamycin Solution: Composition per 10.0mL:

Composition per liter:

Kanamycin............................................................................... 50.0mg

Plant hydrolysate......................................................................... 20.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Esculin .......................................................................................... 1.0g Sodium citrate ............................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.15g Kanamycin sulfate ...................................................................... 0.02g pH 7.0 ± 0.2 at 25°C

Preparation of Kanamycin Solution: Add kanamycin to dis-

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except kanamycin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Bring pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL of sterile kanamycin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Escherichia coli.

Kanamycin Luria Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 0.5g Glucose solution ......................................................................20.0mL Kanamycin solution .................................................................10.0mL

892

Kanamycin Vancomycin Blood Agar

Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 10.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Kanamycin Solution: Composition per 10.0mL: Kanamycin ............................................................................... 10.0mg

Preparation of Kanamycin Solution: Add kanamycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except glucose solution and kanamycin solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Bring pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile glucose solution and 10.0mL of sterile kanamycin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Escherichia coli.

Kanamycin Vancomycin Blood Agar (KVBA) Composition per liter: Agar ............................................................................................ 17.5g Pancreatic digest of casein .......................................................... 15.0g Papaic digest of soybean meal ...................................................... 5.0g NaCl .............................................................................................. 5.0g Kanamycin .................................................................................... 0.1g Sheep blood, defibrinated ........................................................50.0mL Vancomycin solution................................................................10.0mL Vitamin K1 solution ...................................................................1.0mL

Vancomycin Solution: Composition per 10.0mL: Vancomycin ............................................................................... 7.5mg

Preparation of Vancomycin Solution: Add vancomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Vitamin K1 Solution: Composition per 100.0mL: Vitamin K1 .................................................................................... 1.0g

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except sheep blood, vancomycin solution, and vitamin K1 solution, to distilled/deionized water and bring volume to 939.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sheep blood, vancomycin solution, and 1.0mL vitamin K1 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation of anaerobes, particularly Bacteroides, from clinical specimens.

Kanamycin Vancomycin Laked Blood Agar

NaCl.............................................................................................. 5.0g Kanamycin................................................................................ 0.075g Sheep blood, laked...................................................................50.0mL Vancomycin solution ...............................................................10.0mL Vitamin K1 solution ...................................................................1.0mL

Vancomycin Solution: Composition per 10.0mL: Vancomycin ............................................................................... 7.5mg

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly. Filter sterilize.

Preparation of Medium: The blood is laked (hemolyzed) by freezing whole blood overnight and then thawing. Add components, except sheep blood, vancomycin solution, and vitamin K1 solution, to distilled/deionized water and bring volume to 939.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sheep blood, vancomycin solution, and 1.0mL of vitamin K1 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For isolation of the Bacteroides melaninogenicus group. Karmali’s Campylobacter Medium See: Campylobacter Selective Medium, Karmali’s

Kasai Medium Composition per liter: Pancreatic digest of casein.......................................................... 20.0g Soluble starch.............................................................................. 20.0g L-Cysteine·HCl·H2O ..................................................................... 5.0g K2HPO4......................................................................................... 5.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 2.0g

Use: For the isolation and cultivation of Leptotrichia buccalis from saliva and plaque.

KC Bottom Agar Composition per liter: Agar ............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g KCl................................................................................................ 2.5g NaCl.............................................................................................. 2.5g CaCl2 solution............................................................................1.0mL

CaCl2 Solution: Composition per 10.0mL:

Composition per liter:

CaCl2·2H2O ................................................................................ 1.47g

Agar ............................................................................................ 17.5g Pancreatic digest of casein .......................................................... 15.0g Papaic digest of soybean meal ...................................................... 5.0g

Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Kelly Medium, Nonselective Modified Preparation of Medium: Add components, except CaCl2 solution,

to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of CaCl2 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Escherichia coli.

893

KDM-2 Medium Composition per liter: Peptone ....................................................................................... 10.0g L-Cysteine·HCl ............................................................................. 1.0g Yeast extract.................................................................................. 0.5g Calf serum..............................................................................100.0mL pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

KC Broth Composition per liter: Pancreatic digest of casein .......................................................... 10.0g KCl................................................................................................ 5.0g CaCl2 solution ............................................................................0.5mL

CaCl2 Solution: Composition per 10.0mL: CaCl2·2H2O................................................................................. 1.47g

Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except CaCl2 solution,

to distilled/deionized water and bring volume to 999.5mL. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 0.5mL of CaCl2 solution. Mix thoroughly. Aseptically distribute into sterile tubes.

Use: For the cultivation of Escherichia coli.

KC Top Agar Composition per liter: Pancreatic digest of casein .......................................................... 10.0g Agar .............................................................................................. 8.0g NaCl .............................................................................................. 5.0g

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Renibacterium salmoninarum.

Keister's Modified TYI-S-33 Medium Composition per liter: Pancreatic digest of casein.......................................................... 20.0g Glucose ....................................................................................... 10.0g Yeast extract................................................................................ 10.0g L-Cysteine·HCl ............................................................................. 2.0g NaCl.............................................................................................. 2.0g K2HPO4......................................................................................... 1.0g Bovine bile.................................................................................. 0.75g KH2PO4......................................................................................... 0.6g Ascorbic acid ................................................................................ 0.2g Ferric ammonium citrate.......................................................... 22.8mg Bovine serum, heat inactivated..............................................100.0mL

Preparation of Medium: Add components, except bovine serum, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 1.0L of sterile, heat-inactivated bovine serum. Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks. Use: For the cultivation of Giardia cati, Giardia intestinalis, and Hexamita species.

Kelly Medium, Nonselective Modified

Preparation of Medium: Add components to distilled/deionized

Composition per 1430.0mL:

HEPES buffer (N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid) ........................................................ 6.0g Proteose peptone No. 2 ................................................................. 5.0g D-Glucose ...................................................................................... 3.0g NaHCO3 ........................................................................................ 2.2g Pancreatic digest of casein............................................................ 1.0g Yeast, autolyzed ............................................................................ 1.0g Sodium pyruvate........................................................................... 0.8g Sodium citrate............................................................................... 0.7g N-Acetylglucosamine ................................................................... 0.4g MgCl2·6H2O ................................................................................. 0.3g Gelatin solution......................................................................200.0mL Bovine serum albumin solution .............................................143.0mL CMRL-1066 medium with glutamine, 10X...........................100.0mL Rabbit serum, heat inactivated.................................................86.0mL Hemin solution...........................................................................1.0mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Escherichia coli.

KCN Broth Composition per liter: Na2HPO4 ..................................................................................... 5.64g NaCl .............................................................................................. 5.0g Peptone.......................................................................................... 3.0g KH2PO4 ..................................................................................... 0.225g KCN (0.5% solution) ...............................................................15.0mL pH 7.6 ± 0.2 at 25°C

Caution: Cyanide is toxic. Preparation of Medium: Add components, except KCN solution, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add KCN solution. Mix thoroughly. Aseptically distribute into sterile tubes. Stopper immediately.

CMRL-1066 Medium with Glutamine, 10X: Composition per liter: NaCl.............................................................................................. 6.8g NaHCO3 ........................................................................................ 2.2g D-Glucose ...................................................................................... 1.0g KCl................................................................................................ 0.4g L-Cysteine·HCl·H2O ................................................................... 0.26g

894

Kelly Medium, Selective Modified

CaCl2, anhydrous .......................................................................... 0.2g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4·H2O............................................................................. 0.14g L-Glutamine................................................................................... 0.1g Sodium acetate·3H2O................................................................ 0.083g L-Glutamic acid ......................................................................... 0.075g L-Arginine·HCl............................................................................ 0.07g L-Lysine·HCl ............................................................................... 0.07g L-Leucine..................................................................................... 0.06g Glycine........................................................................................ 0.05g Ascorbic acid .............................................................................. 0.05g L-Proline ...................................................................................... 0.04g L-Tyrosine.................................................................................... 0.04g L-Aspartic acid ............................................................................ 0.03g L-Threonine ................................................................................. 0.03g L-Alanine................................................................................... 0.025g L-Phenylalanine......................................................................... 0.025g L-Serine ..................................................................................... 0.025g L-Valine ..................................................................................... 0.025g L-Cystine ..................................................................................... 0.02g L-Histidine·HCl·H2O ................................................................... 0.02g L-Isoleucine ................................................................................. 0.02g Phenol Red .................................................................................. 0.02g L-Methionine ............................................................................. 0.015g Deoxyadenosine.......................................................................... 0.01g Deoxycytidine ............................................................................. 0.01g Deoxyguanosine.......................................................................... 0.01g Glutathione, reduced ................................................................... 0.01g Thymidine ................................................................................... 0.01g Hydroxy-L-proline....................................................................... 0.01g L-Tryptophan ............................................................................... 0.01g Nicotinamide adenine dinucleotide ........................................... 7.0mg Tween™ 80 ................................................................................ 5.0mg Sodium glucuronate·H2O ........................................................... 4.2mg Coenzyme A .............................................................................. 2.5mg Cocarboxylase............................................................................ 1.0mg Flavin adenine dinucleotide ....................................................... 1.0mg Nicotinamide adenine dinucleotide phosphate ........................................................ 1.0mg Uridine triphosphate .................................................................. 1.0mg Choline chloride......................................................................... 0.5mg Cholesterol ................................................................................. 0.2mg 5-Methyldeoxycytidine .............................................................. 0.1mg Inositol ..................................................................................... 0.05mg p-Aminobenzoic acid ............................................................... 0.05mg Niacin..................................................................................... 0.025mg Niacinamide ........................................................................... 0.025mg Pyridoxine .............................................................................. 0.025mg Pyridoxal·HCl ........................................................................ 0.025mg Biotin ....................................................................................... 0.01mg D-Calcium pantothenate ........................................................... 0.01mg Folic acid.................................................................................. 0.01mg Riboflavin ................................................................................ 0.01mg Thiamine·HCl .......................................................................... 0.01mg pH 7.2 ± 0.2 at 25°C

Source: This solution is available as a premixed powder from BD Diagnostics.

Gelatin Solution: Composition per 200.0mL: Gelatin......................................................................................... 14.0g

Preparation of Gelatin Solution: Add gelatin to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Hemin Solution: Composition per 100.0mL: Hemin ........................................................................................... 1.0g NaOH (1N solution).................................................................20.0mL

Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water.

Bovine Serum Albumin Solution: Composition per 200.0mL: Bovine serum albumin................................................................ 70.0g

Preparation of Bovine Serum Albumin Solution: Add bovine serum albumin to distilled/deionized water and bring volume to 200.0mL. Filter sterilize. Preparation of Medium: Add components, except gelatin solution, bovine serum albumin solution, and rabbit serum, to distilled/deionized water and bring volume to 1001.0mL. Mix thoroughly. Bring pH to 7.6 with 5N NaOH. Filter sterilize. Aseptically add 200.0mL of sterile gelatin solution, 143.0mL of sterile bovine serum albumin, and 86.0mL of sterile heat-inactivated rabbit serum. Mix thoroughly. Aseptically dispense into sterile tubes or flasks.

Use: For the isolation of Borrelia burgdorferi and other spirochetes.

Kelly Medium, Selective Modified Composition per 1270.0mL: Bovine serum albumin fraction V............................................... 50.0g HEPES buffer (N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid) ........................................................ 6.0g Glucose ......................................................................................... 5.0g Neopeptone ................................................................................... 5.0g NaHCO3 ........................................................................................ 2.2g Sodium pyruvate........................................................................... 0.8g Sodium citrate............................................................................... 0.7g N-Acetylglucosamine ................................................................... 0.4g Kanamycin................................................................................. 8.0mg 5-Fluorouracil ............................................................................ 2.3mg Gelatin solution......................................................................200.0mL CMRL-1066 medium with glutamine, 10X...........................100.0mL Rabbit serum, partially hemolyzed ..........................................70.0mL pH 7.7 ± 0.2 at 25°C

Gelatin Solution: Composition per 200.0mL: Gelatin......................................................................................... 14.0g

Preparation of Gelatin Solution: Add gelatin to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Preparation of CMRL-1066 Medium with Glutamine, 10X:

CMRL-1066 Medium with Glutamine, 10X: Composition per liter:

Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Filter sterilize.

NaCl.............................................................................................. 6.8g NaHCO3 ........................................................................................ 2.2g

Ketogluconate Broth

D-Glucose ...................................................................................... 1.0g KCl................................................................................................ 0.4g L-Cysteine·HCl·H2O.................................................................... 0.26g CaCl2, anhydrous .......................................................................... 0.2g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4·H2O............................................................................. 0.14g L-Glutamine................................................................................... 0.1g Sodium acetate·3H2O................................................................ 0.083g L-Glutamic acid ......................................................................... 0.075g L-Arginine·HCl............................................................................ 0.07g L-Lysine·HCl ............................................................................... 0.07g L-Leucine..................................................................................... 0.06g Glycine........................................................................................ 0.05g Ascorbic acid .............................................................................. 0.05g L-Proline ...................................................................................... 0.04g L-Tyrosine.................................................................................... 0.04g L-Aspartic acid ............................................................................ 0.03g L-Threonine ................................................................................. 0.03g L-Alanine................................................................................... 0.025g L-Phenylalanine......................................................................... 0.025g L-Serine ..................................................................................... 0.025g L-Valine ..................................................................................... 0.025g L-Cystine ..................................................................................... 0.02g L-Histidine·HCl·H2O ................................................................... 0.02g L-Isoleucine ................................................................................. 0.02g Phenol Red .................................................................................. 0.02g L-Methionine ............................................................................. 0.015g Deoxyadenosine.......................................................................... 0.01g Deoxycytidine ............................................................................. 0.01g Deoxyguanosine.......................................................................... 0.01g Glutathione, reduced ................................................................... 0.01g Thymidine ................................................................................... 0.01g Hydroxy-L-proline....................................................................... 0.01g L-Tryptophan ............................................................................... 0.01g Nicotinamide adenine dinucleotide ........................................... 7.0mg Tween™ 80 ................................................................................ 5.0mg Sodium glucuronate·H2O ........................................................... 4.2mg Coenzyme A .............................................................................. 2.5mg Cocarboxylase............................................................................ 1.0mg Flavin adenine dinucleotide ....................................................... 1.0mg Nicotinamide adenine dinucleotide phosphate .......................... 1.0mg Uridine triphosphate .................................................................. 1.0mg Choline chloride......................................................................... 0.5mg Cholesterol ................................................................................. 0.2mg 5-Methyldeoxycytidine .............................................................. 0.1mg Inositol ..................................................................................... 0.05mg p-Aminobenzoic acid............................................................... 0.05mg Niacin..................................................................................... 0.025mg Niacinamide ........................................................................... 0.025mg Pyridoxine .............................................................................. 0.025mg Pyridoxal·HCl ........................................................................ 0.025mg Biotin ....................................................................................... 0.01mg D-Calcium pantothenate ........................................................... 0.01mg Folic acid.................................................................................. 0.01mg Riboflavin ................................................................................ 0.01mg Thiamine·HCl .......................................................................... 0.01mg pH 7.2 ± 0.2 at 25°C

895

Preparation of CMRL-1066 Medium with Glutamine, 10X: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Filter sterilize.

Preparation of Medium: Add components, except gelatin solution, partially hemolyzed rabbit serum, kanamycin, and 5-fluorouracil, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Bring pH to 7.6 with 5N NaOH. Filter sterilize. Aseptically add 200.0mL of sterile gelatin solution, 70.0mL of partially hemolyzed rabbit serum, 8.0mg of kanamycin, and 230.0mg of 5-fluorouracil. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the isolation of Borrelia burgdorferi. Kempler–McKay Agar See: KM Agar

Kenknight and Munaier’s Medium Composition per liter: Agar ............................................................................................ 15.0g Glucose ......................................................................................... 1.0g K2HPO4......................................................................................... 0.1g NaNO3 .......................................................................................... 0.1g KCl................................................................................................ 0.1g MgSO4·7H2O ................................................................................ 0.1g

Use: For isolating Actinomyces spp. from soil.

Kerosene Mineral Salts Medium Composition per liter: KH2PO4......................................................................................... 1.0g K2HPO4......................................................................................... 1.0g NH4NO3 ........................................................................................ 1.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2 ........................................................................................... 0.02g FeCl3 ........................................................................................... 0.05g Kerosene ..................................................................................20.0mL pH 6.9–7.0 at 25°C

Preparation of Medium: Add components, except kerosene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.9–7.0 with dilute NaOH. Distribute into tubes in 10.0mL volumes or flasks in 100.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Overlay tubes with 0.2mL of kerosene per tube. Overlay flasks with 2.0mL of kerosene per flask.

Use: For the cultivation and maintenance of Pseudomonas aeruginosa.

Ketogluconate Broth Composition per liter: Potassium gluconate ................................................................... 20.0g KH2PO4......................................................................................... 5.4g KNO3 ............................................................................................ 2.0g pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Aseptically distribute into sterile tubes.

896

Ketolactonate Broth

Use: For use in identifying bacteria that can utilize 2-ketogluconate.

Ketolactonate Broth Composition per liter: Agar ............................................................................................ 20.0g Lactose ........................................................................................ 10.0g Yeast extract................................................................................ 10.0g

NaCl.............................................................................................. 5.0g Lactose.......................................................................................... 1.0g NaN3 ............................................................................................. 0.4g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without 2,3,5-triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

Caution: Sodium azide is toxic. Azides also react with metals and

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL:

Use: For use in the identification of agrobacteria and other bacteria

2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

based upon production of 3-ketogluconate. After incubation, Benedict’s solution is added to the plates. Yellow zones around colonies indicate positive production of 3-ketogluconate.

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized

KF Streptococcal Agar Base with Triphyenyltetrazolium Chloride Composition per liter: Agar ............................................................................................ 15.0g Maltose........................................................................................ 20.0g Plant special peptone .................................................................. 10.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl .............................................................................................. 5.0g Lactose .......................................................................................... 1.0g NaN3 ............................................................................................. 0.4g Bromcresol Purple .................................................................... 0.018g 2,3,5-Triphenyltetrazolium chloride solution ................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the detection and enumeration of fecal streptococci in waters and examination of feces and other materials.

disposal must be highly diluted.

water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

KF Streptococcal HiVeg Broth Base with BCP and Triphyenyltetrazolium Chloride Composition per liter: Maltose ....................................................................................... 20.0g Proteose peptone......................................................................... 10.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl.............................................................................................. 5.0g Lactose.......................................................................................... 1.0g Na2CO3 ..................................................................................... 0.636g NaN3 ............................................................................................. 0.4g Bromcresol Purple .................................................................... 0.018g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without 2,3,5-triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia.

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

KF Streptococcal HiVeg Agar Base Composition per liter: Agar ............................................................................................ 20.0g Maltose........................................................................................ 20.0g Plant special peptone .................................................................. 10.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the selective cultivation of fecal streptococci.

KF Streptococcus Agar

KF Streptococcal HiVeg Broth Base with Triphyenyltetrazolium Chloride Composition per liter: Maltose........................................................................................ 20.0g Plant special peptone .................................................................. 10.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl .............................................................................................. 5.0g Lactose .......................................................................................... 1.0g Na2CO3 ..................................................................................... 0.636g NaN3 ............................................................................................. 0.4g Phenol Red ................................................................................ 0.018g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without 2,3,5-Triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia. Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the selective cultivation of fecal streptococci.

KF Streptococcus Agar Composition per liter: Agar ............................................................................................ 20.0g Maltose........................................................................................ 20.0g Proteose peptone ......................................................................... 10.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl .............................................................................................. 5.5g Lactose .......................................................................................... 1.0g NaN3 ............................................................................................. 0.4g Bromcresol Purple .................................................................... 0.015g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium is available as a premixed powder from BD Diagnostic Systems and Oxoid Unipath.

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized

897

volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and enumeration of enterococci.

KF Streptococcus Agar Composition per liter: Agar ............................................................................................ 20.0g Maltose ....................................................................................... 20.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl.............................................................................................. 5.0g Pancreatic digest of casein............................................................ 5.0g Peptic digest of animal tissue ....................................................... 5.0g Lactose.......................................................................................... 1.0g NaN3 ............................................................................................. 0.4g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the selective cultivation and enumeration of fecal streptococci.

KF Streptococcus Broth Composition per liter: Maltose ....................................................................................... 20.0g Sodium glycerophosphate........................................................... 10.0g Yeast extract................................................................................ 10.0g NaCl.............................................................................................. 5.0g Pancreatic digest of casein............................................................ 5.0g Peptic digest of animal tissue ....................................................... 5.0g Lactose.......................................................................................... 1.0g Na2CO3 ..................................................................................... 0.636g NaN3 ............................................................................................. 0.4g Phenol Red................................................................................ 0.018g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and

water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

disposal must be highly diluted.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring

Source: This medium is available as a premixed powder from BD Di-

agnostic Systems.

898

KG HiVeg Agar Base

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the selective cultivation of fecal streptococci. KG Agar See: Kim-Goepfert Agar

KG HiVeg Agar Base Composition per liter: Agar ............................................................................................ 18.0g Plant peptone................................................................................. 1.0g Yeast extract.................................................................................. 0.5g Phenol Red ................................................................................ 0.025g Egg yolk emulsion, 50% ........................................................100.0mL Polymyxin B solution ................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium, without egg yolk emulsion and polymyxin B solution, is available as a premixed powder from HiMedia.

Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Polymyxin B Solution: Add polymyxin B sulfate to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except egg yolk emulsion and polymyxin B solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile egg yolk emulsion, 50%, and 1.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes. Allow plates to dry in the dark at 30°C for 24 hr before using.

Use: For the cultivation and differentiation of Bacillus cereus.

Kidney Bean Agar Composition per liter: Kidney beans, dry ....................................................................... 30.0g Agar ............................................................................................ 15.0g

Preparation of Medium: Add dry kidney beans to distilled/deionized water and bring volume to 1.0L. Autoclave for 30 min at 15 psi pressure–121°C. Filter solids through cheesecloth. Add agar to filtrate. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto© 2010 by Taylor and Francis Group, LLC

clave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Conidiobolus stromoideus, Phialophora gregata, Phytophthora cactorum, Phytophthora cryptogea, Phytophthora erythroseptica, Phytophthora fragariae, Phytophthora heveae, and Phytophthora syringae.

Kievskaya Agar (Medium K) Composition per liter: Agar ............................................................................................ 15.0g KNO3 ............................................................................................ 1.0g KH2PO4 ........................................................................................ 1.0g K2HPO4 ........................................................................................ 1.0g NaCl.............................................................................................. 1.0g MgSO4 .......................................................................................... 0.2g CaCl2 .......................................................................................... 0.02g FeCl3 .......................................................................................... 1.0mg pH 6.8–7.0 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 30 min at 3 psi pressure–105°C. Pour into sterile Petri dishes or leave in tubes. After inoculation, incubate in an atmosphere of natural gas.

Use: For the cultivation of Rhodococcus luteus, Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus species, and other bacteria that can grow on natural gas.

Kievskaya Broth (Medium K) Composition per liter: KNO3 ............................................................................................ 1.0g KH2PO4 ........................................................................................ 1.0g K2HPO4 ........................................................................................ 1.0g NaCl.............................................................................................. 1.0g MgSO4 .......................................................................................... 0.2g CaCl2 .......................................................................................... 0.02g FeCl3 .......................................................................................... 1.0mg pH 6.8–7.0 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 30 min at 15 psi pressure–121°C. After inoculation, sparge medium with natural gas.

Use: For the cultivation of Rhodococcus luteus, Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus species, and other bacteria that can grow on natural gas.

Kievskaya Broth with n-Hexadecane Composition per liter: KNO3 ............................................................................................ 1.0g KH2PO4 ........................................................................................ 1.0g K2HPO4 ........................................................................................ 1.0g NaCl.............................................................................................. 1.0g MgSO4 .......................................................................................... 0.2g CaCl2 .......................................................................................... 0.02g FeCl3 .......................................................................................... 1.0mg n-Hexadecane ..........................................................................10.0mL pH 6.8–7.0 at 25°C

Kimmig Fungi HiVeg Agar Base with George Kimmig Supplement Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Gordona terrae, Rhodococcus erythropolis, Rhodococcus luteus, and Rhodococcus maris.

899

Preparation of Medium: Add glycerol and then other components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the assay of fungistatic agents. For agar dilution testing of

Kim-Goepfert Agar (KG Agar) Composition per 1001.0mL: Solution A ..............................................................................900.0mL Egg yolk emulsion, 50% ........................................................100.0mL Polymyxin B solution ................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Solution A: Composition per 900.0mL:

antifungal agents. For the cultivation and preservation of various fungi.

Kimmig Fungi HiVeg Agar Base with Kimmig Supplement Composition per liter: Glucose ....................................................................................... 19.0g Plant peptone .............................................................................. 15.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 1.0g Cycloheximide.............................................................................. 0.4g Kimmig selective supplement..................................................10.0mL Glycerol .....................................................................................5.0mL pH 6.9 ± 0.2 at 35°C

Agar ............................................................................................ 18.0g Peptone.......................................................................................... 1.0g Yeast extract.................................................................................. 0.5g Phenol Red ................................................................................ 0.025g

Source: This medium, without glycerol or Kimmig selective supplement, is available as a premixed powder from HiMedia.

Preparation of Solution A: Add components to distilled/deionized

mation and inhalation.

water and bring volume to 900.0mL. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol forKimmig Selective Supplement: Composition per 10.0mL:

Egg Yolk Emulsion, 50%: Composition per 100.0mL:

Novobiocin ............................................................................ 200.0mg Colistin sulfate ......................................................................... 80.0mg

Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Kimmig Selective Supplement: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol and then other components,

Polymyxin B Solution: Composition per 5.0mL:

Use: For the assay of fungistatic agents. For agar dilution testing of

Polymyxin B sulfate............................................................. 500,000U

Preparation of Polymyxin B Solution: Add polymyxin B to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: To 900.0mL of cooled, sterile solution A, aseptically add 100.0mL of sterile egg yolk emulsion, 50%, and 1.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes. Allow plates to dry in the dark at 30°C for 24 hr before using.

Use: For the cultivation and differentiation of Bacillus cereus.

Kimmig’s Agar Composition per liter: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Pancreatic digest of gelatin ........................................................... 9.5g Beef extract ................................................................................... 5.5g NaCl .............................................................................................. 5.0g Peptone.......................................................................................... 5.0g Glycerol .....................................................................................5.0mL pH 6.9 ± 0.2 at 35°C © 2010 by Taylor and Francis Group, LLC

except Kimmig selective supplement, to distilled/deionized water and bring volume to 990.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL sterile Kimmig selective supplement. Mix thoroughly Pour into sterile Petri dishes or leave in tubes. antifungal agents. For the cultivation and preservation of various fungi.

Kimmig Fungi HiVeg Agar Base with George Kimmig Supplement Composition per liter: Glucose ....................................................................................... 19.0g Plant peptone .............................................................................. 15.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 1.0g Cycloheximide.............................................................................. 0.4g George Kimmig selective supplement.....................................10.0mL Glycerol .....................................................................................5.0mL pH 6.9 ± 0.2 at 35°C

Source: This medium, without glycertol or George Kimmig selective supplement, is available as a premixed powder from HiMedia.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

George Kimmig Selective Supplement: Composition per 10.0mL: Penicillin G ............................................................................ 40,000U Streptomycin.......................................................................... 40,000U

900

King’s Medium A

Preparation of George Kimmig Selective Supplement: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol and then other components, except George Kimmig selective supplement, to distilled/deionized water and bring volume to 990.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL sterile George Kimmig selective supplement. Mix thoroughly Pour into sterile Petri dishes or leave in tubes.

Use: For the assay of fungistatic agents. For agar dilution testing of antifungal agents. For the cultivation and preservation of various fungi.

King’s Medium A

King’s OF Basal Medium (Oxidation-Fermentation Medium, King’s) (King’s OF Medium) (BAM M70) Composition per liter: Agar .............................................................................................. 3.0g Pancreatic digest of casein............................................................ 2.0g Carbohydrate solution............................................................100.0mL Phenol Red (1.5% solution).......................................................2.0mL pH to 7.3 ± 0.2

Carbohydrate Solution: Composition per 100.0mL:

Composition per liter:

Carbohydrate............................................................................... 10.0g

Proteose peptone ......................................................................... 20.0g Agar ............................................................................................ 15.0g Glycerol ...................................................................................... 10.0g K2SO4 .......................................................................................... 10.0g MgCl2·6H2O.................................................................................. 3.5g pH 7.2–7.4 ± 0.2 at 25°C

Preparation of Carbohydrate Solution: Add carbohydrate to

Use: For the nonselective isolation, cultivation, and pigment production of Pseudomonas.

King’s Medium B

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile carbohydrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For differentiating bacteria based upon determining the oxidative and fermentative metabolism of carbohydrates. Bacteria that ferment the carbohydrate turn the medium yellow.

King’s OF HiVeg Medium Base with Carbohydrate

Composition per liter:

Agar ............................................................................................ 20.0g Proteose peptone No. 3 ............................................................... 20.0g K2HPO4, anhydrous ...................................................................... 1.5g MgSO4·7H2O ................................................................................ 1.5g Glycerol ...................................................................................15.0mL pH 7.2 ± 0.2 at 25°C

Agar .............................................................................................. 0.3g Plant hydrolysate .......................................................................... 0.2g Phenol Red................................................................................. 3.0mg Carbohydrate solution............................................................100.0mL pH 6.9 ± 0.2 at 35°C

Source: This medium, without carbohydrate solution, is available as

Preparation of Medium: Add components to distilled/deionized

a premixed powder from HiMedia.

Carbohydrate Solution: Composition per 100.0mL:

Use: For the nonselective isolation, cultivation, and pigment produc-

Preparation of Carbohydrate Solution: Add carbohydrate to

tion of Pseudomonas species.

King’s Medium B Composition per liter: Proteose peptone No. 3 ............................................................... 20.0g Agar ............................................................................................ 15.0g K2HPO4 ......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 1.5g Glycerol ...................................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Carbohydrate............................................................................... 10.0g distilled/deionized water and bring volume to 100.0mL. Adonitol, arabinose, cellobiose, glucose, dulcitol, fructose, galactose, inositol, lactose, maltose, mannitol, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose, xylose, or other carbohydrates may be used. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 100.0mL of sterile carbohydrate solution. Mix thoroughly. Aseptically distribute into tubes. Use: For studyling oxidation fermentation of carbohydrates by Campylobacter species. King’s OF Medium See: Oxidation-Fermentation Medium, King’s

K-L Virulence Agar

Kirchner Enrichment Medium Composition per liter: Na2HPO4·12H2O......................................................................... 19.0g Asparagine .................................................................................... 5.0g KH2PO4 ......................................................................................... 2.5g Sodium citrate ............................................................................... 2.5g MgSO4 .......................................................................................... 0.6g Serum .....................................................................................100.0mL Glycerol ...................................................................................20.0mL Penicillin solution ....................................................................10.0mL Phenol Red (0.4% solution) .......................................................3.0mL pH 7.4–7.6 at 25°C

Penicillin Solution: Composition per 10.0mL: Penicillin .............................................................................. 100,000U

Preparation of Penicillin Solution: Add penicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except serum and penicillin solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile serum and penicillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and enrichment of Mycobacterium species.

Kirchner Medium Base, Modified Composition per liter: L-Asparagine ................................................................................. 5.0g

KH2PO4 ......................................................................................... 4.0g Na2HPO4 ....................................................................................... 3.0g Sodium citrate ............................................................................... 2.5g MgSO4·7H2O ................................................................................ 0.6g Horse serum, sterile inactivated.............................................100.0mL Selective supplement solution .................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Penicllin ............................................................................... 100,000U Amphotericin B.......................................................................... 5.0mg

Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except horse serum and selective supplement solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add horse serum and selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the cultivation of Mycobacterium tuberculosis.

901

K2TeO3 solution.....................................................................100.0mL K-L filter strips .............................................................................. 100 pH 7.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

K-L Agar Base: Composition per liter: Meat peptone .............................................................................. 20.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 2.5g

Preparation of K-L Agar Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

K2TeO3 Solution: Composition per 100.0mL: K2TeO3 .......................................................................................... 0.3g

Preparation of K2TeO3 Solution: Add K2TeO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Caution: Potassium tellurite is toxic. K-L Filter Strips: Composition: Whatman No. 3 filter paper ..................................................as needed Diphtheria toxin solution .........................................................10.0mL

Preparation of K-L Strips: Cut Whatman No. 3 filter paper into

1.5cm × 7cm strips. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically dip each strip into a sterile solution containing 1000U of purified diphtheria toxin/mL. Drain off excess liquid.

Preparation of Medium: Filter sterilize rabbit serum. To 1.0L of cooled, sterile K-L agar base, aseptically add sterile rabbit serum and sterile K2TeO3 solution. Mix thoroughly. Pour into sterile Petri dishes in 13.0mL volumes. Before the agar solidifies, aseptically add one KL filter strip across the diameter of the plate. Allow the filter strip to sink to the bottom of the plate or press it down with sterile forceps. Allow the agar to solidify. Dry the surface of the plates by incubating at 35°C with lid of plate ajar for 2 hr.

Use: For in vitro toxigenicity testing of Corynebacterium diphtheriae by the agar diffusion technique. Corynebacterium diphtheriae that produce toxin form white precipitin lines at approximately 45° angles from the culture streak line.

K-L Virulence Agar (Klebs-Loeffler Virulence Agar) Composition per 1250.0mL: K-L agar base................................................................................1.0L K-L enrichment......................................................................200.0mL K2TeO3 solution.......................................................................50.0mL K-L filter strips .............................................................................. 100 pH 7.8 ± 0.2 at 25°C

K-L Virulence Agar (Klebs-Loeffler Virulence Agar) (Elek Agar) (Corynebacterium diphtheriae Virulence Test Medium)

Source: This medium is available as a premixed powder from BD Di-

Composition per 1300.0mL:

Meat peptone .............................................................................. 20.0g Agar ............................................................................................ 15.0g NaCl.............................................................................................. 2.5g

K-L agar base................................................................................1.0L Rabbit serum ..........................................................................200.0mL © 2010 by Taylor and Francis Group, LLC

agnostic Systems.

K-L Agar Base: Composition per liter:

902

Kleb Agar

Preparation of K-L Agar Base: Add components to distilled/de-

Preparation of Carbenicillin Solution: Add carbenicillin to dis-

ionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

K-L Enrichment: Composition per 200.0mL: Casamino acids ............................................................................. 4.0g Glycerol .................................................................................100.0mL Tween™ 80 ............................................................................100.0mL

benicillin solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 20.0mL of ethanol and 10.0mL of carbenicillin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of K-L Enrichment: Combine components. Mix

Use: For the enumeration of bacteria from waters.

thoroughly. Filter sterilize.

K2TeO3 Solution: Composition per 100.0mL: K2TeO3 .......................................................................................... 1.0g

Preparation of K2TeO3 Solution: Add K2TeO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Caution: Potassium tellurite is toxic. K-L Filter Strips: Composition: Diphtheria toxin solution .........................................................10.0mL Whatman No. 3 filter paper ................................................. as needed

Preparation of K-L Strips: Cut Whatman #3 filter paper into 1.5cm × 7cm strips. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically dip each strip into a sterile solution containing 1000U of purified diphtheria toxin/mL. Drain off excess liquid. Preparation of Medium: To 1.0L of cooled, sterile K-L agar base, aseptically add sterile K-L enrichment and sterile K2TeO3 solution. Mix thoroughly. Pour into sterile Petri dishes in 13.0mL volumes. Before the agar solidifies, aseptically add one K-L filter strip across the diameter of the plate. Allow the filter strip to sink to the bottom of the plate or press it down with sterile forceps. Allow the agar to solidify. Dry the surface of the plates by incubating at 35°C with lid of plate ajar for 2 hr. Use: For in vitro toxigenicity testing of Corynebacterium diphtheriae by the agar diffusion technique. Corynebacterium diphtheriae that produce toxin form white precipitin lines at approximately 45° angles from the culture streak line.

Kleb Agar (m-Kleb Agar) Composition per liter: Agar ............................................................................................ 15.0g Proteose peptone No. 3 ............................................................... 10.0g NaCl .............................................................................................. 5.0g Adonitol ........................................................................................ 5.0g Beef extract ................................................................................... 1.0g Aniline Blue .................................................................................. 0.1g Sodium lauryl sulfate .................................................................... 0.1g Phenol Red ................................................................................ 0.025g Ethanol (95% solution) ............................................................20.0mL Carbenicillin solution...............................................................10.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components, except ethanol and car-

Klebs-Loeffler Virulence Agar See: K-L Virulence Agar

Klebsiella Medium (m-Klebsiella Medium) Composition per 1041.0mL: Agar ............................................................................................ 15.0g Adonitol ........................................................................................ 4.0g 2X Salt solution .....................................................................500.0mL Uric acid solution...................................................................200.0mL Phenol Red solution.................................................................10.0mL Sodium taurocholate solution ..................................................30.0mL Carbenicillin solution.................................................................1.0mL

2X Salt Solution: Composition per liter: KCl................................................................................................ 8.0g K2HPO4......................................................................................... 3.0g NaCl.............................................................................................. 2.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g

Preparation of 2X Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Uric Acid Solution: Composition per 200.0mL: Uric acid........................................................................................ 0.3g

Preparation of Uric Acid Solution: Dissolve uric acid in a small volume of 1N NaOH. Bring volume to 200.0mL with distilled/deionized water. Adjust pH to 7.1 with 1N HCl. Filter sterilize.

Phenol Red Solution: Composition per 10.0mL: Phenol Red.................................................................................... 0.1g

Preparation of Phenol Red Solution: Add Phenol Red to sterile distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Sodium Taurocholate Solution: Composition per 30.0mL: Sodium taurocholate ..................................................................... 0.4g

Preparation of Sodium Taurocholate Solution: Add sodium taurocholate to sterile distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Carbenicillin Solution: Composition per 1.0mL: Carbenicillin .............................................................................. 5.0mg

Preparation of Carbenicillin Solution: Add carbenicillin to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize.

Kligler Iron Agar Preparation of Medium: Add adonitol and agar to 500.0mL of 2X salt solution. Bring volume to 800.0mL with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 200.0mL of uric acid solution, 30.0mL of sodium taurocholate solution, 10.0mL of Phenol Red solution, and 1.0mL of carbenicillin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the enumeration of Klebsiella species by the membrane filter method.

Klebsiella Selective Agar Composition per liter: Agar ............................................................................................ 26.0g DL–Phenylalanine........................................................................ 10.0g L-Ornithine·HCl........................................................................... 10.0g Raffinose ....................................................................................... 7.0g Pancreatic digest of casein ............................................................ 2.5g Yeast extract.................................................................................. 2.5g K2HPO4 ......................................................................................... 2.0g Phenol Red solution .................................................................10.0mL Carbenicillin solution...............................................................10.0mL pH 5.6 ± 0.2 at 25°C

Phenol Red Solution: Composition per 10.0mL: Phenol Red .................................................................................... 0.5g

Preparation of Phenol Red Solution: Add Phenol Red to 50% ethanol and bring volume to 10.0mL. Mix thoroughly. Carbenicillin Solution: Composition per 1.0mL: Carbenicillin............................................................................... 5.0mg

Preparation of Carbenicillin Solution: Add carbenicillin to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except carbenicillin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL carbenicillin solution. Mix thoroughly. Adjust pH to 5.6–5.7 with sterile 1N HCl. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and identification of Klebsiella pneumoniae from clinical specimens.

Kligler Iron Agar Composition per liter: Peptone........................................................................................ 20.0g Agar ............................................................................................ 12.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Beef extract ................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Ferric citrate .................................................................................. 0.3g Na2S2O3 ........................................................................................ 0.3g Phenol Red .................................................................................. 0.05g pH 7.4 ± 0.2 at 25°C

903

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the differentiation and identification of Enterobacteriaceae based upon sugar fermentation and hydrogen sulfide production. Sugar fermentation is indicated by the medium turning yellow. H2S production results in the medium turning black.

Kligler Iron Agar Composition per liter: Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Peptic digest of animal tissue ..................................................... 10.0g NaCl.............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ........................................................................................ 0.5g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the differentiation and identification of Enterobacteriaceae based upon sugar fermentation and hydrogen sulfide production. Sugar fermentation is indicated by the medium turning yellow. H2S production results in the medium turning black.

Kligler Iron Agar (FDA M71) Composition per liter: Lactose........................................................................................ 20.0g Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g Peptic digest of animal tissue ..................................................... 10.0g NaCl.............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ........................................................................................ 0.5g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Kligler Iron Agar (BAM M71) Composition per liter: Lactose........................................................................................ 20.0g Polypeptone ................................................................................ 20.0g

904

Kligler Iron Agar with Sodium Chloride

Agar ............................................................................................ 15.0g Peptic digest of animal tissue...................................................... 10.0g NaCl .............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ........................................................................................ 0.5g Phenol Red ................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Kligler Iron Agar with Sodium Chloride (BAM M71) Composition per liter: NaCl ............................................................................................ 25.0g Lactose ........................................................................................ 20.0g Polypeptone ................................................................................ 20.0g Agar ............................................................................................ 15.0g Peptic digest of animal tissue...................................................... 10.0g NaCl .............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ........................................................................................ 0.5g Phenol Red ................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Use: For the differentiation and identification of Vibrio spp. based upon sugar fermentation and hydrogen sulfide production. Sugar fermentation is indicated by the medium turning yellow. H2S production results in the medium turning black.

Kligler Iron HiVeg Agar Composition per liter: Plant special peptone .................................................................. 15.0g Lactose ........................................................................................ 10.0g Agar ............................................................................................ 15.0g Plant peptone No. 3....................................................................... 5.0g NaCl .............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Na2S2O3 ........................................................................................ 0.3g FeSO4 ............................................................................................ 0.2g Phenol Red ................................................................................ 0.024g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

to boiling. Distribute into tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

Kligler Iron HiVeg Agar, Modified Composition per liter: Plant hydrolysate ........................................................................ 20.0g Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Yeast extract.................................................................................. 3.0g Glucose, anhydrous....................................................................... 1.0g Na2S2O3·5H2O.............................................................................. 0.3g FeSO4 ............................................................................................ 0.2g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the differentiation and identification of Enterobacteriaceae based upon sugar fermentation and hydrogen sulfide production. Recommended for identification of Yersinia enterocolitica.

KM Agar (Kempler-McKay Agar) Composition per liter: Agar ............................................................................................ 15.0g Milk, nonfat ................................................................................ 10.0g Glucose ......................................................................................... 5.0g Milk protein hydrolysate............................................................... 2.5g Solution 1.................................................................................10.0mL Solution 2.................................................................................10.0mL pH 6.6 ± 0.2 at 25°C

Solution 1: Composition per 100.0mL: K3Fe(CN)6 .................................................................................. 10.0g

Caution: Cyanide is toxic. Preparation of Solution 1: Add K3Fe(CN)6 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Solution 2: Composition per 40.0mL: Ferric citrate.................................................................................. 1.0g Sodium citrate............................................................................... 1.0g

Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except solution 1 and solution 2, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.6. Autoclave for 12 min at 10 psi pressure–115°C. Cool to 45°–50°C. Aseptically add sterile solution 1 and solution 2. Mix thoroughly. Pour into

Kohn Two Tube HiVeg Medium No. 1 Base with Urea

sterile Petri dishes. Allow plates to dry in the dark at 30°C for 24 hr before using.

Use: For the isolation and cultivation of acidogenic microorganisms from foods. For the differentiation of citrate-fermenting lactic bacteria, such as Lactobacillus lactis subspecies diacetylactis, from the noncitrate-fermenting Lactobacillus lactis subspecies cremoris.

to boiling. Distribute into tubes. Autoclave for 15 min at 10 psi pressure– 115°C. Cool to 60°C. Aseptically add 25mL of sterile urea solution. Mix thoroughly. Allow to solidify as a slant in tubes with a genrous butt.

Use: For the identification of Enterobacteriaceae on the basis of glucose and mannitol fermentation and urease production.

Knisely Medium for Bacillus anthracis Composition per liter: Beef heart, solids from infusion................................................ 500.0g Agar ............................................................................................ 15.0g Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g EDTA ..................................................................................... 200.0mg Lysozyme ................................................................................. 40.0mg Thallous acetate ....................................................................... 40.0mg Polymyxin .............................................................................. 30,000U

Preparation of Medium: Add components, except EDTA, lysozyme, thallous acetate, and polymyxin, to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add sterile EDTA, lysozyme, thallous acetate, and polymyxin. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Bacillus anthracis.

Koch’s K1 Medium Composition per liter: Glucose ......................................................................................... 1.8g Peptone.......................................................................................... 0.6g Yeast extract.................................................................................. 0.4g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of a variety of fungi.

Kohn Two Tube Medium No. 1 Base with Urea Composition per liter: Agar ............................................................................................ 16.0g Peptic digest of animal tissue...................................................... 15.0g Mannitol...................................................................................... 10.0g Beef extract ................................................................................... 2.0g Yeast extract.................................................................................. 2.0g Glucose ......................................................................................... 1.0g Phenol Red .................................................................................. 0.05g Urea solution............................................................................25.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without urea solution, is available as a premixed powder from HiMedia.

Urea Solution: Composition per 100.0mL: Urea............................................................................................... 5.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring © 2010 by Taylor and Francis Group, LLC

905

Kohn Two Tube Medium No. 2 Composition per liter: Casein enzymic hydrolysate ....................................................... 10.0g Peptic digest of animal tissue ..................................................... 10.0g Salicin ......................................................................................... 10.0g Sucrose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Agar .............................................................................................. 3.0g Na2HPO4 .................................................................................... 0.09g Bromthymol Blue ....................................................................... 0.02g Na2S2O3 .................................................................................... 0.016g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 10 psi pressure– 115°C. Allow to solidify in tubes in an upright position. Use: For the identification of members of Enterobacteriaceae on the basis of sucrose and salicin fermentation, motility, H2S production, and indole production.

Kohn Two Tube HiVeg Medium No. 1 Base with Urea Composition per liter: Agar ............................................................................................ 16.0g Plant peptone .............................................................................. 15.0g Mannitol...................................................................................... 10.0g Plant extract .................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Glucose ......................................................................................... 1.0g Phenol Red.................................................................................. 0.05g Urea solution............................................................................25.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without urea solution, is available as a premixed powder from HiMedia.

Urea Solution: Composition per 100.0mL: Urea............................................................................................... 5.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 10 psi pressure– 115°C. Cool to 60°C. Aseptically add 25.0mL of sterile urea solution. Mix htoroughly. Allow to solidify as a slant in tubes with a genrous butt. Use: For the identification of Enterobacteriaceae on the basis of glucose and mannitol fermentation and urease production.

906

Kohn Two Tube HiVeg Medium No. 2

Kohn Two Tube HiVeg Medium No. 2 Composition per liter: Plant hydrolysate......................................................................... 10.0g Plant peptone............................................................................... 10.0g Salicin ......................................................................................... 10.0g Sucrose........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Agar .............................................................................................. 3.0g Na2HPO4..................................................................................... 0.09g Bromthymol Blue ....................................................................... 0.02g Na2S2O3 .................................................................................... 0.016g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 10 psi pressure– 115°C. Allow to solidify in tubes in an upright position. Use: For the identification of members of Enterobacteriaceae on the basis of sucrose and salicin fermentation, motility, H2S production, and indole production.

KoKo Medium Composition per 1020.0mL: K2HPO4 ......................................................................................... 1.6g NaH2PO4·2H2O............................................................................. 1.0g Peptone, meat................................................................................ 1.0g Pancreatic digest of casein ............................................................ 1.0g Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 0.5g MgSO4·6H2O .............................................................................. 0.16g Resazurin ................................................................................... 0.5mg Glucose solution ....................................................................100.0mL NaHCO3 solution .....................................................................10.0mL CaCl2·2H2O solution................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-4 ..................................................10.0mL Wolfe’s vitamin solution ..........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: D-Glucose...................................................................................... 5.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. CaCl2 Solution: Composition per 10.0mL: CaCl2·2H2O................................................................................. 0.06g

Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

L-Cysteine·HCl·H2O

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine·HCl·H2O Solution: Add Lcysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl. Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Calcium DL-pantothenate........................................................... 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 100% N2. Add components, except glucose solution, NaHCO3 solution, CaCl2·2H2O solution, L-cysteine·HCl·H2O solution, Na2S·9H2O solution, and Wolfe’s vitamin solution, to distilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Adjust pH to 7.0. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 100.0mL of sterile glucose solution, 10.0mL of sterile NaHCO3 solution, 10.0mL of sterile CaCl2·2H2O solution, 10.0mL of sterile L-cysteine·HCl·H2O solution, 10.0mL of sterile Na2S·9H2O solution, and 10.0mL of sterile Wolfe’s vitamin

KPL Medium

solution. Mix thoroughly. The pH should be 7.0. A buffer solution of 1% MOPS from a 10% anaerobic solution at pH 6.9–7.0 may be added aseptically and anaerobically to enhance the buffer capacity of the medium. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Thermoanaerobacter italicus.

Korthof Medium Composition per 1088.0mL: NaCl .............................................................................................. 1.4g Na2HPO4·2H2O........................................................................... 0.88g Peptone.......................................................................................... 0.8g KH2PO4 ....................................................................................... 0.24g CaCl2 ........................................................................................... 0.04g KCl.............................................................................................. 0.04g NaHCO3 ...................................................................................... 0.02g Rabbit serum, inactivated ........................................................80.0mL Rabbit hemoglobin solution.......................................................8.0mL pH 7.2 ± 0.2 at 25°C

Rabbit Hemoglobin Solution: Composition per 20.0mL:

907

Source: This medium is available as a premixed powder from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the differentiation of Escherichia coli and Enterobacter aerogenes based on citrate utilization.

Koser Citrate Medium Composition per liter: Sodium citrate............................................................................... 3.0g NaNH4HPO4·4H2O....................................................................... 1.5g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Rabbit blood clot......................................................................10.0mL

agnostic Systems.

Preparation of Rabbit Hemoglobin Solution: Add rabbit blood

Preparation of Medium: Add components to distilled/deionized

clot to 10.0mL of distilled/deionized water. Lyse the clot by freezing and thawing.

Preparation of Medium: Add components, except rabbit serum and rabbit hemoglobin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Cool to 25°C. Filter through Whatman #1 filter paper. Distribute into flasks in 100.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 8.0mL of rabbit serum and 0.8mL of rabbit hemoglobin solution to each flask. Mix thoroughly. Use: For the cultivation of Leptospira species.

Korthof Medium, Modified Composition per liter: NaCl .............................................................................................. 1.4g Na2HPO4·2H2O........................................................................... 0.88g Peptone.......................................................................................... 0.8g KH2PO4 ....................................................................................... 0.24g CaCl2 ........................................................................................... 0.04g KCl.............................................................................................. 0.04g NaHCO3 ...................................................................................... 0.02g Rabbit serum, heat inactivated at 56°C..................................100.0mL pH 7.2–7.6 at 25°C

Use: For the differentiation of Escherichia coli and Enterobacter aerogenes based on citrate utilization.

Kosmachev’s Medium Composition per liter: Agar ............................................................................................ 15.0g CaCO3 ........................................................................................... 4.0g KNO3 ............................................................................................ 1.0g (NH4)2SO4 .................................................................................... 1.0g Na2HPO4 ....................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g FeSO4·7H2O................................................................................ 0.01g Yeast autolysate (30% solution) ..............................................15.0mL

Use: For the isolation and cultivation of Actinomadura species, Actinopolyspora species, Excellospora species, and Microspora species.

Preparation of Medium: Add components, except rabbit serum, to distilled/deionized water and bring volume to 900.0L. Mix thoroughly. Gently heat and bring to boiling. Boil for 20 min. Cool overnight at 4°C. Filter through Whatman #2 filter paper. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°– 56°C. Aseptically add 100.0mL of rabbit serum. Mix thoroughly.

Use: For the cultivation of Leptospira species.

Koser Citrate Broth (BAM M72) Composition per liter: Sodium citrate ............................................................................... 3.0g NaNH4HPO4·4H2O ....................................................................... 1.5g © 2010 by Taylor and Francis Group, LLC

KPL Medium Composition per 1141.0mL: Agar ............................................................................................ 15.0g Galactose..................................................................................... 10.0g Glucose ....................................................................................... 10.0g Lactic acid whey ...........................................................................1.0L White table wine ....................................................................140.0mL Tween™ 80................................................................................1.0mL pH 5.5 ± 0.2 at 25°C

Lactic Acid Whey: Composition per liter: Skim milk (10% solution).............................................................1.0L

908

Kracke Blood Culture HiVeg Medium

Preparation of Lactic Acid Whey: Adjust the pH of the skim milk to 5.5 with lactic acid. Gently heat and bring to boiling. Continue boiling for 30 min. Filter through Whatman #1 filter paper.

Cycloheximide.......................................................................... 0.041g Egg yolk emulsion .................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Combine components, except white table

Caution: Sodium azide is toxic. Azides also react with metals and

wine. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 5.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Filter sterilize white table wine. To cooled, sterile basal medium, aseptically add sterile white table wine. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

disposal must be highly diluted.

Use: For the cultivation and maintenance of Lactobacillus kefiranofaciens.

Kracke Blood Culture HiVeg Medium Composition per liter: NaCl ............................................................................................ 49.0g Glucose ....................................................................................... 10.0g Plant peptone No. 3..................................................................... 10.0g Na2HPO4 ....................................................................................... 2.0g Plant infusion ................................................................................ 2.0g Plant special infusion .................................................................... 1.0g Sodium citrate ............................................................................... 1.0g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of pathogens in cases of bacteremia. The medium is inoculated with blood from a patient. Approximately 1015mL of blood normally is inoculated into 50mL of medium.

Krainsky’s Asparagine Agar Composition per liter: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g K2HPO4 ......................................................................................... 0.5g L-Asparagine ................................................................................. 0.5g pH 7.0 ± 0.2 at 25°C

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Source: This medium is available as a premixed powder from Oxoid Unipath.

Egg Yolk Emulsion: Composition: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Preparation of Medium: Add components, except egg yolk emulsion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of egg yolk emulsion. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and enumeration of staphylococci from foods.

KRANEP HiVeg Agar Base with Egg Yolk Emulsion Composition per liter: Potassium thiocyanate ................................................................ 25.5g Agar ............................................................................................ 15.0g Sodium pyruvate........................................................................... 8.2g LiCl ............................................................................................... 5.1g Mannitol........................................................................................ 5.1g Plant peptone ................................................................................ 5.0g NaCl.............................................................................................. 5.0g Plant extract .................................................................................. 1.5g Yeast extract.................................................................................. 1.5g NaN3 ........................................................................................... 0.05g Cycloheximide.......................................................................... 0.041g Egg yolk emulsion .................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium, without egg yolk emulsion, is available as a

Preparation of Medium: Add components to distilled/deionized

premixed powder from HiMedia.

Caution: Sodium azide is toxic. Azides also react with metals and

Use: For the cultivation and maintenance of Streptomyces fragmen-

halation of vapors. On contact with skin wash with plenty of water immediately.

tans.

disposal must be highly diluted.

Caution: Lithium Chloride is harmful. Avoid bodily contact and in-

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-

KRANEP Agar Base Composition per liter: KSCN.......................................................................................... 25.5g Agar ............................................................................................ 18.3g Sodium pyruvate ........................................................................... 8.2g Pancreatic digest of gelatin ........................................................... 6.1g LiCl ............................................................................................... 5.1g Mannitol........................................................................................ 5.1g Beef extract ................................................................................... 3.7g NaN3............................................................................................ 0.05g © 2010 by Taylor and Francis Group, LLC

mation and inhalation.

Egg Yolk Emulsion: Composition per liter: Egg yolks .................................................................................30.0mL NaCl, 0.9% solution.................................................................70.0mL

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack 11 eggs and separate yolks from whites. Mix egg yolks. Measure 30.0mL of egg yolk emulsion and add to 70.0mL of 0.9% sterile NaCl solution. Mix thoroughly. Warm to 45°–50°C.

Kupferberg Trichomonas Broth Preparation of Medium: Add components, except egg yolk emulsion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of egg yolk emulsion. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and enumeration of staphylococci from foods.

Kreb’s Yeast Lactate Medium Composition per liter: Yeast extract................................................................................ 10.0g Na2HPO4·2H2O............................................................................. 3.0g KH2PO4 ......................................................................................... 1.0g Sodium lactate solution............................................................40.0mL pH 7.0 ± 0.2 at 25°C

909

Yeast extract.................................................................................. 5.0g Filtered tomato juice ..............................................................250.0mL Ethanol (96% solution) ..........................................................176.0mL Tween™ 80................................................................................0.5mL pH 5.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except ethanol, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 0.88mL of ethanol solution to each tube containing 5.0mL of medium.

Use: For the cultivation of Lactobacillus fructivorans.

Kupferberg Trichomonas Base Composition per liter:

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Pancreatic digest of casein.......................................................... 20.0g L-Cysteine·HCl·H2O ..................................................................... 1.5g Agar .............................................................................................. 1.0g Maltose ......................................................................................... 1.0g Methylene Blue.......................................................................... 3.0mg Bovine serum ...........................................................................50.0mL pH 6.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except sodium lactate

Source: This medium is available as a premixed powder from BD Di-

Sodium Lactate Solution: Composition per 100.0mL: Sodium lactate............................................................................. 70.0g

Preparation of Sodium Lactate Solution: Add sodium lactate to

solution, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Bring pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 40.0mL of sterile sodium lactate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Propionibacterium acidipropionici, Propionibacterium freudenreichii, Propionibacterium intermedium, Propionibacterium jensenii, Propionibacterium species, and Propionibacterium thoenii.

Kundrant Agar Composition per liter: Agar ............................................................................................ 10.0g Meat peptone................................................................................. 7.8g Casein peptone .............................................................................. 7.8g Starch ............................................................................................ 4.0g Gelatin........................................................................................... 4.0g NaCl .............................................................................................. 3.0g Yeast extract.................................................................................. 2.8g Glucose ......................................................................................... 1.0g Bromcresol Purple ..................................................................... 1.6mg pH 6.8 ± 0.2 at 25°C

agnostic Systems.

Preparation of Medium: Add components, except bovine serum, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of bovine serum. If desired, additional selectivity can be obtained by aseptically adding 250,000U of penicillin and 1.0g of streptomycin or 1.0g of chloramphenicol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of the Trichomonas species from clinical specimens.

Kupferberg Trichomonas Broth Composition per liter:

Source: This medium is available from HiMedia.

Enzymatic digest of protein ........................................................ 20.0g L-Cysteine·HCl·H2O ..................................................................... 1.5g Agar .............................................................................................. 1.0g Maltose ......................................................................................... 1.0g Chloramphenicol........................................................................... 0.1g Methylene Blue.......................................................................... 3.0mg Bovine serum ...........................................................................50.0mL pH 6.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from BD Di-

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the qualitative detection of residues from sulfonamides and other antimicrobics in animal-derived foods.

Kunkee Medium Composition per 1000.88mL: Pancreatic digest of casein .......................................................... 20.0g Glucose ......................................................................................... 5.0g Peptone.......................................................................................... 5.0g © 2010 by Taylor and Francis Group, LLC

agnostic Systems.

Preparation of Medium: Add components, except bovine serum, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add bovine serum. If desired, additional selectivity can be obtained by aseptically adding 250,000U penicillin and 1.0g streptomycin or 1.0g chloramphenicol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of the Trichomonas species from clinical specimens.

910

Kupferberg Trichomonas HiVeg Broth Base with Serum and Selective Supplement

Kupferberg Trichomonas HiVeg Broth Base with Serum and Selective Supplement (Trichomonas HiVeg Broth Base, Kupferberg)

Composition per liter:

Plant hydrolysate......................................................................... 20.0g Agar .............................................................................................. 1.0g L-Cysteine·HCl.............................................................................. 1.5g Maltose.......................................................................................... 1.0g Methylene Blue.......................................................................... 3.0mg Bovine serum ...........................................................................50.0mL Selective supplement ...............................................................10.0mL pH 6.0 ± 0.2 at 25°C

Plant hydrolysate ........................................................................ 20.0g Agar .............................................................................................. 1.0g L-Cysteine·HCl ............................................................................. 1.5g Maltose ......................................................................................... 1.0g Methylene Blue.......................................................................... 3.0mg Bovine serum ...........................................................................50.0mL Selective supplement ...............................................................10.0mL pH 6.0 ± 0.2 at 25°C

Source: This medium, without bovine serum and selective supplement, is available as a premixed powder from HiMedia.

Source: This medium, without bovine serum and selective supple-

Selective Supplement Solution: Composition per 10.0mL:

Penicillin .............................................................................. 250,000U

Preparation of Selective Supplement Solution: Add penicilliln to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except bovine serum adn selective supplement, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of bovine serum and 10.0mL selective supplement.

Use: For the cultivation of Trichomonas species from clinical specimens.

Kupferberg Trichomonas HiVeg Broth Base with Serum and Selective Supplement (Trichomonas HiVeg Broth Base, Kupferberg) Composition per liter: Plant hydrolysate......................................................................... 20.0g Agar .............................................................................................. 1.0g L-Cysteine·HCl.............................................................................. 1.5g Maltose.......................................................................................... 1.0g Methylene Blue.......................................................................... 3.0mg Bovine serum ...........................................................................50.0mL Selective supplement ...............................................................10.0mL pH 6.0 ± 0.2 at 25°C

Source: This medium, without bovine serum and selective supplement, is available as a premixed powder from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Streptomycin ................................................................................. 1.0g

Preparation of Selective Supplement Solution: Add streptomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except bovine serum and selective supplement, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of bovine serum and 10.0mL selective supplement. Use: For the cultivation of Trichomonas species from clinical specimens. © 2010 by Taylor and Francis Group, LLC

ment, is available as a premixed powder from HiMedia.

Chloramphenicol........................................................................... 1.0g

Preparation of Selective Supplement Solution: Add chloramphenicol to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except bovine serum adn selective supplement, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of bovine serum and 10.0mL selective supplement. Use: For the cultivation of Trichomonas species from clinical specimens.

Kushneria aurantia Medium (DSMZ Medium 1195) Composition per liter: NaCl ........................................................................................... 78.0g MgSO4·7H2O .............................................................................. 20.3g MgCl2·6H2O ............................................................................... 13.0g Yeast extract ................................................................................. 5.0g KCl ............................................................................................... 2.0g CaCl2·2H2O ................................................................................ 0.33g NaBr ........................................................................................... 0.23g NaHCO3 ...................................................................................... 0.06g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Kushneria aurantia. KVBA See: Kanamycin Vancomycin Blood Agar

KYE Agar Composition per liter: Agar ............................................................................................ 15.0g NaNO3 .......................................................................................... 2.5g KH2PO4......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.3g

L Diphasic Blood Agar Medium

911

CaCl2·6H2O................................................................................. 0.15g NaCl .............................................................................................. 0.1g FeCl3 ........................................................................................ 10.0mg pH 10.0 ± 0.2 at 25°C

Diaminopimelic Acid Solution: Composition per 10.0mL:

Preparation of Medium: Add components to distilled/deionized

opimelic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Diaminopimelic acid..................................................................... 0.1g

Preparation of Diaminopimelic Acid Solution: Add diamin-

Thymidine Solution: Composition per 10.0mL:

Use: For the cultivation of a variety of alkaliphilic bacteria.

Thymidine................................................................................... 0.01g

L Agar (Luria Agar) Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 0.5g Glucose solution ......................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 10.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Bring pH to 7.0. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile glucose solution. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Escherichia coli.

L Broth (Luria Broth) Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Escherichia coli.

L Broth DAP Thymidine Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Diaminopimelic acid solution ..................................................10.0mL Thymidine solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except diaminopimelic acid solution and thymidine solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Bring pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile diaminopimelic acid solution and 10.0mL of sterile thymidine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Escherichia coli.

L Diphasic Blood Agar Medium (ATCC Medium 947) Composition per 1150.0mL: Blood agar, diphasic base medium ........................................700.0mL Rabbit blood, defibrinated .....................................................300.0mL Locke’s solution.....................................................................150.0mL pH 7.2–7.4 at 25°C

Blood Agar, Diphasic Base Medium: Composition per 750.0mL: Beef............................................................................................. 25.0g Agar ............................................................................................ 10.0g Neopeptone ................................................................................. 10.0g NaCl.............................................................................................. 2.5g

Preparation of Blood Agar, Diphasic Base Medium: Trim beef to remove fat. Add 25.0g of lean beef to 250.0mL of distilled/deionized water. Gently heat and bring to boiling. Boil for 2–3 min. Filter through Whatman #2 filter paper. Add agar, neopeptone, and NaCl to filtrate. Bring volume to 750.0mL with distilled/deionized water. Mix thoroughly. Adjust pH to 7.2–7.4. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Locke’s Solution: Composition per liter: NaCl.............................................................................................. 8.0g Glucose ......................................................................................... 2.5g KH2PO4........................................................................................ 0.3g CaCl2 ............................................................................................ 0.2g KCl................................................................................................ 0.2g

Preparation of Locke's Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C.

Preparation of Medium: Aseptically combine 700.0mL of sterile blood agar, diphasic base medium, with 300.0mL of sterile defibrinated rabbit blood warmed to 50°–55°C. Mix thoroughly. Aseptically distribute 5.0mL volumes into 16 × 125mm screw-capped test tubes. Allow to cool in a slanted position. Overlay the agar in each tube with 1.0mL of sterile Locke’s solution.

912

L Diphasic Blood Agar Medium

Use: For the cultivation and maintenance of Trypanosoma species, Leishmania donovani, Herpetomonas species, and Trypanosoma neveulemairei.

L Diphasic Blood Agar Medium (ATCC Medium 1011) Composition per 1150.0mL: Blood agar, diphasic base medium ........................................700.0mL Rabbit blood, defibrinated .....................................................300.0mL Locke’s solution .....................................................................150.0mL pH 7.2–7.4 at 25°C

Blood Agar, Diphasic Base Medium: Composition per 750.0mL: Beef ............................................................................................. 25.0g Agar ............................................................................................ 10.0g Neopeptone ................................................................................. 10.0g NaCl .............................................................................................. 2.5g

Preparation of Blood Agar, Diphasic Base Medium: Trim beef to remove fat. Add 25.0g of lean beef to 250.0mL of distilled/deionized water. Gently heat and bring to boiling. Boil for 2–3 min. Filter through Whatman #2 filter paper. Add agar, neopeptone, and NaCl to filtrate. Bring volume to 750.0mL with distilled/deionized water. Mix thoroughly. Adjust pH to 7.2–7.4. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Locke’s Solution: Composition per liter: NaCl .............................................................................................. 8.0g Glucose ......................................................................................... 2.5g KH2PO4 ........................................................................................ 0.3g CaCl2 ............................................................................................ 0.2g KCl................................................................................................ 0.2g

Preparation of Locke’s Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Preparation of Medium: Aseptically combine 700.0mL of sterile blood agar, diphasic base medium, with 300.0mL of sterile defibrinated rabbit blood warmed to 50°–55°C. Mix thoroughly. Aseptically distribute 5.0mL volumes into 16 × 125mm screw-capped test tubes. Allow to cool in a slanted position. Overlay the agar in each tube with 3.0mL of sterile Locke’s solution.

Use: For the cultivation and maintenance of Trypanosoma species.

L and F Basal Salts, Modified with Heptadecane Composition per liter: NH4Cl ........................................................................................... 2.0g Na2HPO4 ..................................................................................... 0.21g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.09g KCl.............................................................................................. 0.04g CaCl2 ......................................................................................... 0.015g FeSO4·7H2O............................................................................... 1.0mg ZnSO4·7H2O ............................................................................ 0.07mg H3BO3 ...................................................................................... 0.01mg MnSO4·5H2O ........................................................................... 0.01mg MoO3........................................................................................ 0.01mg CuSO4·5H2O ...............................................................................5.0μg Heptadecane...............................................................................2.0mL pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components, except heptadecane, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute equally into four 250.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C. Filter sterilize heptadecane. To one 250.0mL fraction of basal salts, aseptically add 0.5mL of sterile heptadecane. Pour mixture into a sterile blender. Homogenize slowly to mix heptadecane with basal salts and not to create excess bubbles. Rapidly distribute medium to sterile screw-capped tubes. Chill tubes quickly in an ice pack or in the refrigerator. Allow tubes to solidify in a slanted position.

Use: For the cultivation and maintenance of Thermoleophilum album and Thermoleophilum minutum. .L Medium (ATCC Medium 167)

Composition per liter: Agar ............................................................................................ 20.0g NaNO3 .......................................................................................... 2.0g Na2HPO4 ..................................................................................... 0.21g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.09g KCl.............................................................................................. 0.04g CaCl2 ......................................................................................... 0.015g FeSO4·7H2O............................................................................... 1.0mg Salts solution..............................................................................1.0mL

Salts Solution: Composition per 100.0mL: ZnSO4·7H2O .............................................................................. 7.0mg H3BO3 ........................................................................................ 1.0mg MnSO4·5H2O ............................................................................. 1.0mg MoO3 ......................................................................................... 1.0mg CuSO4·5H2O .............................................................................. 0.5mg

Preparation of Salts Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Use: For the cultivation and maintenance of Methylococcus capsulatus and Pseudomonas methanica.

L Medium (ATCC Medium 1154) Composition per liter: Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 25 min at 15 psi pressure– 121°C.

Use: For the cultivation and maintenance of Escherichia coli.

L Medium with Ampicillin Composition per liter: Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g

L Medium with Methanol

Yeast extract.................................................................................. 5.0g Ampicillin solution .................................................................. 20.0mg pH 7.0 ± 0.2 at 25°C

Ampicillin Solution: Composition per 10.0mL: Ampicillin ................................................................................... 0.02g

L Medium with DAP, THY, and AMP (L Medium with Diaminopimelic Acid, Thymidine, and Ampicillin) Composition per liter: Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Diaminopimelic acid solution..................................................10.0mL Thymidine solution..................................................................10.0mL Ampicillin solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Diaminopimelic Acid Solution: Composition per 10.0mL:

Use: For the cultivation and maintenance of Escherichia coli.

Preparation of Diaminopimelic Acid Solution: Add

L Medium with DAP and THY (L Medium with Diaminopimelic Acid and Thymidine)

Thymidine Solution: Composition per 10.0mL:

Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Glucose solution ......................................................................10.0mL Diaminopimelic acid solution ..................................................10.0mL Thymidine solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 1.0g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Diaminopimelic Acid Solution: Composition per 10.0mL: DL-Diaminopimelic

acid................................................................ 0.1g

Preparation of Diaminopimelic Acid Solution: Add diaminopimelic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thymidine Solution: Composition per 10.0mL: Thymidine ................................................................................... 0.02g

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except glucose solution, diaminopimelic acid solution, and thymidine solution—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution, diaminopimelic acid solution, and thymidine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

913

DL-Diaminopimelic

acid ............................................................... 0.1g

DL-diaminopimelic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thymidine................................................................................... 0.04g

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Ampicillin Solution: Composition per 10.0mL: Ampicillin ................................................................................ 0.02mg

Preparation of Ampicillin Solution: Add ampicillin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except ampicillin solution, diaminopimelic acid solution, and thymidine solution—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile ampicillin solution, diaminopimelic acid solution, and thymidine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli. L Medium with Diaminopimelic Acid and Thymidine See: L Medium with DAP and THY L Medium with Diaminopimelic Acid, Thymidine, and Ampicillin See: L Medium with DAP, THY, and AMP

L Medium with Methanol Composition per liter: Agar ............................................................................................ 20.0g NaNO3 .......................................................................................... 2.0g Na2HPO4 ..................................................................................... 0.21g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.09g KCl.............................................................................................. 0.04g CaCl2 ......................................................................................... 0.015g FeSO4·7H2O............................................................................... 1.0mg Methanol ..................................................................................20.0mL Salts solution..............................................................................1.0mL

914

L Medium for Salmonella

Salts Solution: Composition per 100.0mL: ZnSO4·7H2O .............................................................................. 7.0mg H3BO3 ........................................................................................ 1.0mg MnSO4·5H2O ............................................................................. 1.0mg MoO3.......................................................................................... 1.0mg CuSO4·5H2O .............................................................................. 0.5mg

Preparation of Salts Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Filter sterilize methanol. Aseptically add 20.0mL of sterile methanol to cooled, sterile basal medium. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylobacillus glycogenes.

L Medium for Salmonella Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Salmonella choleraesuis.

L Medium with Tetracycline Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Tetracycline solution................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Tetracycline Solution: Composition per 10.0mL: Tetracycline.............................................................................. 0.02mg

Preparation of Tetracycline Solution: Add tetracycline to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except tetracycline solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile tetracycline solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

Sodium pyruvate........................................................................... 0.6g .................................................................................... 0.5g L-Arginine, free base..................................................................... 0.5g KCl................................................................................................ 0.4g L-Asparagine·H2O ......................................................................... 0.3g L-Histidine, free base .................................................................... 0.3g L-Glutamine .................................................................................. 0.3g L-Isoleucine ................................................................................... 0.3g L-Phenylalanine............................................................................. 0.3g L-Tyrosine ..................................................................................... 0.3g DL-Methionine............................................................................... 0.2g DL-Valine ....................................................................................... 0.2g Glycine.......................................................................................... 0.2g L-Serine ......................................................................................... 0.2g Na2HPO4, anhydrous .................................................................... 0.2g CaCl2, anhydrous .......................................................................... 0.1g L-Cysteine, free base ..................................................................... 0.1g L-Leucine·HCl............................................................................... 0.1g MgCl2, anhydrous..................................................................... 0.094g D-Galactose ................................................................................. 0.09g KH2PO4....................................................................................... 0.06g L-Tryptophan............................................................................... 0.02g Phenol Red.................................................................................. 0.01g i-Inositol..................................................................................... 2.0mg Choline chloride......................................................................... 1.0mg D-Calcium pantothenate ............................................................. 1.0mg Folic acid ................................................................................... 1.0mg Nicotinamide.............................................................................. 1.0mg Pyridoxine·HCl .......................................................................... 1.0mg Thiamine monophosphate·2H2O ............................................... 1.0mg Riboflavin-5-phosphate ............................................................. 0.1mg pH 7.5 ± 0.2 at 25°C DL-Alanine

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Store at 5°C.

Use: For the cultivation of oysters used for the growth of enteroviruses.

L. mono Confirmatory Agar Base (Listeria monocytogenes Confirmatory Agar, Base) Composition per liter: Special peptone........................................................................... 30.0g Agar ............................................................................................ 12.0g LiCl ............................................................................................. 10.0g B.C. indicator............................................................................... 8.6g Yeast extract.................................................................................. 6.0g NaCl.............................................................................................. 5.0g α-Methyl-D-mannoside ................................................................ 3.0g Na2HPO4, anhydrous.................................................................... 2.5g Listeria mono enrichment supplement II.................................10.0mL Listeria mono selective supplement I ......................................10.0mL Listeria mono selective supplement II.....................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without Listeria mono enrichment supplement Composition per liter:

II, Listeria mono selective supplement I, and Listeria mono selective supplement II, is available as a premixed powder from BioChemika.

NaCl .............................................................................................. 8.0g DL-Threonine................................................................................. 0.6g

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

L15 Medium, Modified Leibovitz

L. mono Differential HiVeg Agar Base

915

Listeria mono Enrichment Supplement II Solution: Composition per 10.0mL:

Listeria mono Selective Supplement I Solution: Composition per 10.0mL:

L-phosphatidylinositol

Polymyxin B sulfate .............................................................. 76,700U

.................................................................. 1.0g

Preparation of Listeria mono Enrichment Supplement II Solution: Add L-phosphatidylinositol to distilled/deionized water and

Preparation of Listeria mono Selective Supplement I Solution: Add polymyxin B sulfate to distilled/deionized water and bring

bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

volume to 10.0mL. Mix thoroughly. Filter sterilize.

Listeria mono Selective Supplement I Solution: Composition per 10.0mL:

Listeria mono Selective Supplement II Solution: Composition per 10.0mL:

Polymyxin B sulfate............................................................... 76,700U

Preparation of Listeria mono Selective Supplement I Solution: Add polymyxin B sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Listeria mono Selective Supplement II Solution: Composition per 10.0mL: Ceftazidime ............................................................................. 20.0mg Nalidixic acid, sodium salt ...................................................... 20.0mg Amphotericin B ....................................................................... 10.0mg

Preparation of Listeria mono Selective Supplement II Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Listeria mono enrichment supplement II, Listeria mono selective supplement I, and Listeria mono selective supplement II, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 10.0mL Listeria mono enrichment supplement II, 10.0mL Listeria mono selective supplement I, and 10.0mL Listeria mono selective supplement II. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective and differential isolation of Listeria monocytogenes from clinical and food specimens.

L. mono Confirmatory HiVeg Agar Base Composition per liter: Plant special peptone .................................................................. 30.0g Agar ............................................................................................ 12.0g LiCl ............................................................................................. 10.0g B.C. indicator................................................................................ 8.6g Yeast extract.................................................................................. 6.0g NaCl .............................................................................................. 5.0g α-Methyl-D-mannoside................................................................. 3.0g Na2HPO4, anhydrous .................................................................... 2.5g Listeria mono enrichment supplement II .................................10.0mL Listeria mono selective supplement I ......................................10.0mL Listeria mono selective supplement II .....................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without Listeria mono enrichment supplement

Ceftazidime ............................................................................. 20.0mg Nalidixic acid, sodium salt ..................................................... 20.0mg Amphotericin B ...................................................................... 10.0mg

Preparation of Listeria mono Selective Supplement II Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the selective and differential isolation of Listeria monocytogenes from clinical and food specimens.

L. mono Differential HiVeg Agar Base Composition per liter: Plant peptone No. 1..................................................................... 18.0g Agar ............................................................................................ 15.0g LiCl ............................................................................................. 10.0g Yeast extract................................................................................ 10.0g Plant hydrolysate .......................................................................... 6.0g NaCl.............................................................................................. 5.0g Na2HPO4, anhydrous .................................................................... 2.5g Glucose ......................................................................................... 2.0g Sodium pyruvate........................................................................... 2.0g Magnesium glycerophosphate ...................................................... 1.0g MgSO4 .......................................................................................... 0.5g Chromogenic substrate ............................................................... 0.05g Listeria mono enrichment supplement II.................................10.0mL Listeria mono selective supplement I ......................................10.0mL Listeria mono selective supplement II.....................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without Listeria mono enrichment supplement

II, Listeria mono selective supplement I, and Listeria mono selective supplement II, is available as a premixed powder from HiMedia.

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

Caution: LiC lis harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

Listeria mono Enrichment Supplement II Solution: Composition per 10.0mL:

L-phosphatidylinositol

.................................................................. 1.0g

................................................................. 1.0g

Preparation of Listeria mono Enrichment Supplement II Solution: Add L-phosphatidylinositol to distilled/deionized water and

bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

916

Lab-Lemco Agar

Listeria mono Selective Supplement I Solution: Composition per 10.0mL: Polymyxin B sulfate............................................................... 76,700U

Preparation of Listeria mono Selective Supplement I Solution: Add polymyxin B sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Listeria mono Selective Supplement II Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the selective and differential isolation of Listeria monocytogenes from clinical and food specimens.

L Salts for Thermophiles See: Mineral Salts for Thermophiles

Lachica’s Medium Base Composition per liter: Beef heart, infusion from.......................................................... 500.0g Agar ............................................................................................ 15.0g Tryptose ...................................................................................... 10.0g NaCl.............................................................................................. 5.0g Amylose Azure ............................................................................. 3.0g Selective supplement solution .................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Ampicillin ................................................................................ 10.0mg

Preparation of Selective Supplement Solution: Add ampicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the isolation and cultivation of Aeromonas hydrophila from foods stored under different temperature conditions.

Lactate Agar Composition per liter:

Yeast extract.................................................................................. 3.0g K2HPO4......................................................................................... 2.8g Agar .............................................................................................. 2.0g Peptone ......................................................................................... 2.0g KH2PO4....................................................................................... 0.52g Sodium lactate (60% solution).................................................10.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Lab-Lemco Agar Composition per liter:

Use: For the cultivation and maintenance of a variety of heterotrophic microorganisms.

Lab-Lemco Broth Composition per liter: Peptone.......................................................................................... 5.0g Lab Lemco meat extract ............................................................... 3.0g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of a variety of heterotrophic microorganisms, including microorganisms from wastewater.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Serpens flexibilis.

Lactate Broth Composition per liter: Yeast extract.................................................................................. 3.0g K2HPO4......................................................................................... 2.8g Peptone ......................................................................................... 2.0g KH2PO4....................................................................................... 0.52g Sodium lactate (60% solution).................................................10.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Serpens flexibilis.

Lactic Agar

Lactate Seawater Minimal Medium Composition per liter: Tris(hydroxymethyl)aminomethane·HCl.................................... 7.88g Sodium or potassium lactate ......................................................... 2.0g NH4Cl ........................................................................................... 1.0g K2HPO4·3H2O........................................................................... 0.075g FeSO4·7H2O.............................................................................. 0.028g Artificial seawater..................................................................500.0mL pH 7.5 ± 0.2 at 25°C

Artificial Seawater: Composition per liter: MgSO4·7H2O .............................................................................. 24.7g NaCl ............................................................................................ 23.4g CaCl2·2H2O................................................................................... 2.9g KCl................................................................................................ 1.5g

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of ATCC strains 27134 and 27136.

Lactic Acid Bacteria Broth Composition per liter: Sodium acetate ............................................................................ 12.0g Pancreatic digest of casein .......................................................... 10.0g Yeast autolysate............................................................................. 5.0g Glucose ....................................................................................... 10.0g Solution A ..................................................................................5.0mL Solution B ..................................................................................5.0mL pH 5.1 ± 0.2 at 25°C

Source: Yeast autolysate is available from Oxoid Unipath. Solution A: Composition per 100.0mL:

917

Sodium acetate·3H2O.................................................................... 5.0g Yeast extract.................................................................................. 5.0g Diammonium hydrogen citrate ..................................................... 2.0g Tween™ 80................................................................................... 1.0g Sorbic acid .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g MnSO4·4H2O ................................................................................ 0.2g FeSO4·7H2O................................................................................ 0.05g pH 5.3–5.4 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.3–5.4. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation and cultivation of lactic acid bacteria from wine.

Lactic Acid Bacteria Selective Agar Base Composition per liter: Casein enzymic hydrolysate ....................................................... 20.0g Agar ............................................................................................ 17.0g Maltose ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g Fructose......................................................................................... 5.0g Glucose ......................................................................................... 5.0g Potassium aspartate....................................................................... 2.5g Potassium glutamate ..................................................................... 2.5g Betaine hydrochloride................................................................... 2.0g Diammonium hydrogen citrate ..................................................... 2.0g MgSO4·7H2O ................................................................................ 2.0g KH2PO4......................................................................................... 2.0g Liver concentrate .......................................................................... 1.0g MnSO4·2H2O .............................................................................. 0.66g N-acetyl glucosamine.................................................................... 0.5g Selective supplement solution ................................................20.0mL pH 5.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

K2HPO4 ....................................................................................... 10.0g KH2PO4 ....................................................................................... 10.0g

Selective Supplement Solution: Composition per 20.0mL:

Preparation of Solution A: Add components to distilled/deionized

2-Phenylethanol ............................................................................ 3.0g Cycloheximide......................................................................... 10.0mg Polysorbate 80 .........................................................................10.0mL

water and bring volume to 100.0mL. Mix thoroughly.

Solution B: Composition per 100.0mL: MgSO4·7H2O ................................................................................ 4.0g FeSO4·7H2O.................................................................................. 0.2g MnSO4·H2O .................................................................................. 0.2g NaCl .............................................................................................. 0.2g

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus malefermentans.

Lactic Acid Bacteria Medium

Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the selective isolation of lactic acid bacteria from beer and brewing processes.

Lactic Agar Composition per liter: Casein enzymatic hydrolysate .................................................... 20.0g Agar ............................................................................................ 15.0g

918

Lactic Agar for Yogurt Bacteria, Modified

Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 5.0g Lactose .......................................................................................... 5.0g Sucrose.......................................................................................... 5.0g NaCl .............................................................................................. 4.0g Gelatin........................................................................................... 2.5g Sodium acetate .............................................................................. 1.5g Ascorbic acid ................................................................................ 0.5g

Use: For the enumeration and identification of lactic streptococci and lactobacilli by the pour plate method.

Lactic Agar for Yogurt Bacteria, Modified Composition per liter: Elliker agar....................................................................................1.0L Milk solution............................................................................70.0mL pH 6.8 ± 0.1 at 25°C

Elliker Agar: Composition per liter:

Solution A: Composition per 100.0mL: K2HPO4....................................................................................... 10.0g KH2PO4....................................................................................... 10.0g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Solution B: Composition per 100.0mL: MgSO4·7H2O ................................................................................ 4.0g FeSO4 ............................................................................................ 0.2g MnSO4·5H2O ................................................................................ 0.2g NaCl.............................................................................................. 0.2g

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Lactobacillus buchneri, Lactobacillus delbrueckii, and Pediococcus damnosus.

Lactic Bacteria Differential Agar Composition per liter:

Pancreatic digest of casein .......................................................... 20.0g Agar ............................................................................................ 15.0g Yeast extract................................................................................ 10.0g Gelatin........................................................................................... 4.0g Glucose ......................................................................................... 3.0g Ascorbic acid ................................................................................ 2.5g Lactose .......................................................................................... 2.5g NaCl .............................................................................................. 2.5g Sodium acetate .............................................................................. 2.5g Sucrose.......................................................................................... 2.5g

Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ....................................................... 10.0g Casein acid hydrolysate ................................................................ 3.0g Fructose......................................................................................... 2.5g KH2PO4......................................................................................... 2.5g Papaic digest of soybean meal...................................................... 1.5g Yeast extract.................................................................................. 1.0g Polysorbate 80 .............................................................................. 1.0g Bromcresol Green..................................................................... 0.055g pH 7.0 ± 0.2 at 25°C

Preparation of Elliker Agar: Add components to distilled/deion-

Source: This medium, without polysorbate 80, is available from Hi-

ized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Medium: Add components to distilled/deionized

Media.

Milk Solution: Composition per 100.0mL:

Nonfat dry milk solids ................................................................ 11.0g

Use: For the differentiation of homofermentative and heterofermenta-

Preparation of Milk Solution: Add nonfat dry milk solids to dis-

tive lactic acid bacteria.

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 12 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Lactic Bacteria Differential HiVeg Agar

Preparation of Medium: Add 70.0mL of sterile milk solution to

Composition per liter:

1.0L of cooled, sterile Elliker agar. Mix thoroughly. Pour into sterile Petri dishes. Allow plates to dry at 28°–30°C for 18–24 hr.

Agar ............................................................................................ 15.0g Plant hydrolysate ........................................................................ 10.0g Plant acid hydrolysate................................................................... 3.0g Fructose......................................................................................... 2.5g KH2PO4......................................................................................... 2.5g Papaic digest of soybean meal...................................................... 1.5g Yeast extract.................................................................................. 1.0g Bromcresol Green..................................................................... 0.055g Polysorbate 80 ...........................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of acidogenic microorganisms, especially Lactobacillus species and lactic streptococci, from foods.

Lactic Bacteria Broth Composition per liter: Sodium acetate ............................................................................ 12.0g Glucose ....................................................................................... 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast autolysate............................................................................. 5.0g Solution A ..................................................................................5.0mL Solution B ..................................................................................5.0mL pH 5.1–5.3 at 25°C © 2010 by Taylor and Francis Group, LLC

Source: This medium, without polysorbate 80, is available as a premixed powder from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.3–5.4.

Lactobacilli Agar, AOAC

919

Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

L-Arginine·HCl.............................................................................. 1.5g

Use: For the differentiation of homofermentative and heterofermentative lactic acid bacteria.

Preparation of Medium: Add components—except Bromcresol Purple solution, calcium citrate, and sodium carboxymethylcellulose—to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Gently heat and bring to boiling. In a blender, add the calcium citrate and sodium carboxymethylcellulose to 200.0mL of distilled/deionized water. Blend until mixed. Add the 200.0mL of citrate/ carboxymethylcellulose solution to the hot agar solution. Mix thoroughly. Adjust pH to 6.0. Distribute into flasks in 100.0mL volumes. Autoclave for 10 min at 10 psi pressure–115°C. Cool to 45°–50°C. Aseptically add 2.0mL of sterile Bromcresol Purple to each flask. Mix thoroughly. Pour into sterile Petri dishes. Allow plates to dry at 37°C for 1 hr before use.

Lactic Bacteria Differential HiVeg Broth Composition per liter: Plant hydrolysate......................................................................... 10.0g Plant acid hydrolysate ................................................................... 3.0g Fructose......................................................................................... 2.5g KH2PO4 ......................................................................................... 2.5g Papaic digest of soybean meal ...................................................... 1.5g Yeast extract.................................................................................. 1.0g Bromcresol Green ..................................................................... 0.055g Polysorbate 80............................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Bromcresol Purple (0.1% solution) .........................................20.0mL pH 6.0 ± 0.1 at 25°C

Use: For the differentiation of lactic streptococci. Bacteria that produce acid from lactose appear as yellow colonies.

Source: This medium, without polysorbate 80, is available as a pre-

Lactic Streak HiVeg Agar

mixed powder from HiMedia.

Use: For the differentiation of homofermentative and heterofermentative lactic acid bacteria.

Lactic HiVeg Agar Composition per liter: Plant hydrolysate......................................................................... 22.5g Agar ............................................................................................ 15.0g Glucose ......................................................................................... 5.0g Lactose .......................................................................................... 5.0g Sucrose.......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 4.0g Sodium acetate .............................................................................. 1.5g Ascorbic acid ................................................................................ 0.5g pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMe-

Composition per liter: Agar ............................................................................................ 15.0g Calcium citrate............................................................................ 10.0g Sodium carboxymethylcellulose................................................. 10.0g Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Papaic digest of soybean meal...................................................... 5.0g Yeast extract.................................................................................. 5.0g L-Arginine hydrochloride ............................................................. 1.5g Lactose.......................................................................................... 1.5g Bromcresol Purple ..................................................................... 2.0mg pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.0. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the differentiation of lactic streptococci. Bacteria that produce acid from lactose appear as yellow colonies.

dia.

Use: For the enumeration and identification of lactic streptococci and lactobacilli by the pour plate method.

Lactic Streak Agar Composition per liter: Agar ............................................................................................ 15.0g Sodium carboxymethylcellulose................................................. 10.0g Calcium citrate ............................................................................ 10.0g Beef extract ................................................................................... 5.0g Papaic digest of soybean meal ...................................................... 5.0g Polypeptone™............................................................................... 5.0g Yeast extract.................................................................................. 5.0g Lactose .......................................................................................... 1.5g © 2010 by Taylor and Francis Group, LLC

Lactobacilli Agar, AOAC (Lactobacilli Agar, Association of Official Analytical Chemists) Composition per liter: Milk, peptonized ......................................................................... 15.0g Agar ............................................................................................ 10.0g Glucose ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 1.0g Tomato juice ..........................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 2–3 min. Distribute into tubes in

920

Lactobacilli Broth, AOAC

10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in an upright position.

Use: For the cultivation and maintenance of stock cultures of Lactobacillus casei ATCC 7469, Lactobacillus fermentum ATCC 9338, Lactobacillus leichmannii ATCC 4797, and Lactobacillus viridescens ATCC 12706 used in the microbiological assay of vitamins.

Lactobacilli Broth, AOAC (Lactobacilli Broth, Association of Official Analytical Chemists)

Peptone ....................................................................................... 10.0g Sodium acetate.............................................................................. 5.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g Na2HPO4 ....................................................................................... 2.0g Tween™ 80................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·5H2O .............................................................................. 0.05g pH 6.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Composition per liter:

agnostic Systems.

Milk, peptonized ......................................................................... 15.0g Glucose ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g KH2PO4 ......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 1.0g Tomato juice...........................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 2–3 min. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and preparation of inocula of stock cultures of Lactobacillus casei ATCC 7469, Lactobacillus fermentum ATCC 9338, Lactobacillus leichmannii ATCC 4797, and Lactobacillus viridescens ATCC 12706 used in the microbiological assay of vitamins. Lactobacilli deManRogosa-Sharpe Broth See: Lactobacilli MRS Broth

Lactobacilli HiVeg Broth (Elliker Broth, HiVeg) Composition per liter: Plant extract ................................................................................ 20.0g Yeast extract................................................................................ 10.0g Gelatin........................................................................................... 4.0g Glucose ......................................................................................... 3.0g Ascorbic acid ................................................................................ 2.5g Lactose .......................................................................................... 2.5g NaCl .............................................................................................. 2.5g Sodium acetate .............................................................................. 2.5g Sucrose.......................................................................................... 2.5g pH 6.8 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus species and lactic streptococci, from foods.

Lactobacilli MRS Broth (Lactobacilli deMan-Rogosa-Sharpe Broth) Composition per liter: Glucose ....................................................................................... 20.0g Beef extract ................................................................................. 10.0g © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus species. Also used for the cultivation and maintenance of Aerococcus viridans, Bifidobacterium coryneforme, Lactococcus plantarum, Leuconostoc species, Pectinatus cerevisiiphilus, Pediococcus species, and Sporolactobacillus inulinus. Lactobacilli MRS Broth with Mevalonic Acid See: Pediococcus Medium with Mevalonic Acid Lactobacilli MRS Broth See: Pediococcus Medium

Lactobacilli MRS Broth with Cysteine Composition per liter: Glucose ....................................................................................... 20.0g Beef extract................................................................................. 10.0g Peptone ....................................................................................... 10.0g Sodium acetate.............................................................................. 5.0g Yeast extract.................................................................................. 5.0g L-Cysteine ..................................................................................... 2.0g Ammonium citrate ........................................................................ 2.0g Na2HPO4 ....................................................................................... 2.0g Tween™ 80................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·5H2O .............................................................................. 0.05g pH 6.5 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Lactobacillus species, including L. ruminis and L. vitulinus.

Lactobacilli MRS Broth with 0.5% Cysteine (ATCC 1844) Composition per liter: Glucose ....................................................................................... 20.0g Beef extract................................................................................. 10.0g Peptone ....................................................................................... 10.0g Sodium acetate.............................................................................. 5.0g Yeast extract.................................................................................. 5.0g L-Cysteine·HCl ............................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g

Lactobacillus bulgaricus Agar

Na2HPO4 ....................................................................................... 2.0g Tween™ 80 ................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·5H2O .............................................................................. 0.05g pH 6.5 ± 0.2 at 25°C

Use: For the cultivation of Lactobacillus sharpeae.

Lactobacilli MRS Broth with Ethanol Composition per liter: Glucose ....................................................................................... 20.0g Beef extract ................................................................................. 10.0g Peptone........................................................................................ 10.0g Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g L-Cysteine ..................................................................................... 2.0g Ammonium citrate ........................................................................ 2.0g Na2HPO4 ....................................................................................... 2.0g Tween™ 80 ................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·5H2O .............................................................................. 0.05g Ethanol (95% solution) ..........................................................100.0mL pH5.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except ethanol, to dis-

921

Lactobacillus bifidus Medium Composition per liter: Lactose........................................................................................ 70.0g Sodium acetate, anhydrous ......................................................... 50.0g Pancreatic digest of casein.......................................................... 10.0g K2HPO4......................................................................................... 5.0g Tween™ 80................................................................................... 1.0g Alanine.......................................................................................... 0.4g L-Cystine....................................................................................... 0.4g Tryptophan.................................................................................... 0.4g Asparagine .................................................................................... 0.2g Adenine....................................................................................... 0.02g Guanine....................................................................................... 0.02g Uracil .......................................................................................... 0.02g Xanthine...................................................................................... 0.02g Pyridoxine·HCl .......................................................................... 2.4mg Nicotinic acid............................................................................. 1.2mg Calcium pantothenate ................................................................ 0.8mg Riboflavin .................................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg p-Aminobenzoic acid............................................................... 0.02mg Folic acid ................................................................................. 0.02mg Biotin .......................................................................................... 8.0μg Ascorbic acid solution ...........................................................100.0mL Human milk, skimmed.............................................................20.0mL Salts B......................................................................................10.0mL pH 6.8 ± 0.2 at 25°C

Salts B: Composition per 250.0mL:

tilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Filter sterilize ethanol. Aseptically add 100.0mL of sterile ethanol. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

MgSO4·7H2O .............................................................................. 10.0g FeSO4·7H2O.................................................................................. 0.5g NaCl.............................................................................................. 0.5g MnSO4·2H2O ............................................................................ 0.337g

Use: For the cultivation and maintenance of Lactobacillus species.

ter and bring volume to 250.0mL. Mix thoroughly.

Lactobacillus Agar 2

Preparation of Salts B: Add components to distilled/deionized waAscorbic Acid Solution: Composition per 100.0mL:

Composition per liter:

Ascorbic acid ................................................................................ 1.0g

Glucose ....................................................................................... 20.0g Sodium acetate ............................................................................ 20.0g Agar ............................................................................................ 15.0g Tryptic digest of casein ............................................................... 10.0g Yeast extract.................................................................................. 5.0g Meat extract .................................................................................. 2.0g K2HPO4 ......................................................................................... 0.5g KH2PO4 ......................................................................................... 0.5g MgSO4·7H2O ......................................................................... 200.0mg DL-Mevalonic acid ................................................................... 30.0mg FeSO4·7H2O............................................................................. 10.0mg MnSO4·H2O .............................................................................. 7.5mg Ethanol .....................................................................................40.0mL Tween™ 80 ................................................................................1.0mL pH 5.2 ± 0.2 at 25°C

Preparation of Ascorbic Acid Solution: Add ascorbic acid to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 6.5. Filter sterilize. Preparation of Medium: Add components, except ascorbic acid solution and human milk, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile ascorbic acid solution. Mix thoroughly. This constitutes a double-strength medium. To prepare a single-strength medium, aseptically combine 500.0mL of sterile doublestrength medium, 480.0mL of sterile distilled/deionized water, and 20.0mL of sterile human milk. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Bifidobacterium (Lactobacillus) bifidum.

Lactobacillus bulgaricus Agar (LB Agar) Composition per 900.0mL: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g

922

Lactobacillus bulgaricus HiVeg Agar Base with Tomato Juice and Acetate Buffer

Beef extract ................................................................................. 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g K2HPO4 ......................................................................................... 2.0g Tween™ 80 ................................................................................... 1.0g Acetate buffer...........................................................................80.0mL Tomato juice, filtered ...............................................................40.0mL pH 6.8 ± 0.2 at 25°C

Acetate Buffer: Composition per liter: Sodium acetate ........................................................................ 113.55g Acetic acid ...............................................................................10.0mL

Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 1.0g Chloramphenicol........................................................................... 0.3g Tomato juice ..........................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Use: For the cultivation of Lactobacillus casei.

Preparation of Acetate Buffer: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except acetate buffer, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.8. Add 80.0mL of acetate buffer. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the isolation, cultivation, and enumeration of Lactobacillus bulgaricus from foods.

Lactobacillus bulgaricus HiVeg Agar Base with Tomato Juice and Acetate Buffer Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g Plant extract ................................................................................ 10.0g Plant hydrolysate......................................................................... 10.0g Yeast extract.................................................................................. 5.0g K2HPO4 ......................................................................................... 2.0g Tomato juice.................................................................................. 2.0g Polysorbate 80............................................................................... 1.0g Acetate buffer...........................................................................80.0mL Tomato juice, filtered ...............................................................40.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium, without tomato juice and acetate buffer, is available as a premixed powder from HiMedia.

Preparation of Acetate Buffer: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components, except acetate buffer, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.8. Add 80.0mL of acetate buffer. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the isolation, cultivation, and enumeration of Lactobacillus

Lactobacillus Chloramphenicol Broth 1 Composition per liter: Milk, peptonized ......................................................................... 15.0g Glucose ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 1.0g Chloramphenicol........................................................................... 0.3g Tomato juice ..........................................................................100.0mL pH 6.8 ± 0.2 at 25°C

Use: For the cultivation of Lactobacillus casei.

Lactobacillus Chloramphenicol Medium 2 Composition per liter: Glucose ....................................................................................... 18.5g Agar ............................................................................................ 13.5g Pancreatic digest of gelatin......................................................... 10.0g Beef extract................................................................................... 8.0g Yeast extract.................................................................................. 4.0g Sodium acetate.............................................................................. 3.0g K2HPO4......................................................................................... 2.0g Ammonium citrate ........................................................................ 2.0g Polysorbate 80 .............................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.2g Chloramphenicol........................................................................... 0.1g MnSO4·4H2O .............................................................................. 0.05g pH 6.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Lactobacillus casei and Pediococcus acidilactici.

bulgaricus from foods.

Lactobacillus Chloramphenicol Agar 1

Lactobacillus Heteroferm Screen Agar (MRS, Modified)

Composition per liter:

Milk, peptonized ......................................................................... 15.0g Agar ............................................................................................ 10.0g Glucose ....................................................................................... 10.0g

Lactobacillus Medium

Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g 2-Phenylethyl alcohol ................................................................... 3.0g Ammonium citrate ........................................................................ 2.0g K2HPO4 ......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·H2O ................................................................................ 0.05g Bromcresol Green ....................................................................... 0.04g Cycloheximide ........................................................................... 4.0mg Tween™ 80 ................................................................................1.0mL pH 5.5 ± 0.01 at 25°C

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Adjust pH to 5.5 with glacial acetic acid. Pour into sterile Petri dishes.

Use: For the isolation and cultivation of Lactobacillus species from salad dressings.

Lactobacillus Heteroferm Screen Broth (MRS, Modified) Composition per liter: Glucose ....................................................................................... 20.0g Proteose peptone No. 3 ............................................................... 10.0g Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g 2-Phenylethyl alcohol ................................................................... 3.0g Ammonium citrate ........................................................................ 2.0g K2HPO4 ......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.1g MnSO4·H2O ................................................................................ 0.05g Bromcresol Green ....................................................................... 0.04g Cycloheximide ........................................................................... 4.0mg Tween™ 80 ................................................................................1.0mL pH 4.3 ± 0.01 at 25°C

923

Ethanol.....................................................................................40.0mL Tween™ 80................................................................................1.0mL pH 5.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation and maintenance of Lactobacillus homohiochii.

Lactobacillus Medium (ATCC Medium 78) Composition per liter: Pancreatic digest of casein.......................................................... 20.0g Agar ............................................................................................ 15.0g Tryptose ........................................................................................ 5.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 3.0g Lactose.......................................................................................... 2.0g Liver extract concentrate .............................................................. 1.0g Tween™ 80................................................................................. 0.05g Tomato juice, filtered.............................................................200.0mL pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.5. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Lactobacillus fermentum and Lactobacillus salivarius.

Lactobacillus Medium (ATCC Medium 169) Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 4.3 with concentrated HCl. Distribute into test tubes that contain an inverted Durham tube. Autoclave for 15 min at 15 psi pressure–121°C.

Glucose ....................................................................................... 10.0g Proteose peptone........................................................................... 7.5g Yeast extract.................................................................................. 7.5g KH2PO4......................................................................................... 2.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g Tween™ 80................................................................................... 0.1g Tomato juice, filtered.............................................................100.0mL pH 7.0 ± 0.2 at 25°C

Use: For the isolation and cultivation of Lactobacillus species from

Preparation of Medium: Add components to distilled/deionized

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation. Preparation of Medium: Add components to distilled/deionized

foods.

Lactobacillus homohiochii Medium Composition per liter: D-Glucose.................................................................................... 20.0g

Tryptic digest of casein ............................................................... 10.0g Yeast extract.................................................................................. 5.0g Meat extract .................................................................................. 2.0g K2HPO4 ......................................................................................... 0.5g KH2PO4 ......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g DL-Mevalonic acid ................................................................... 30.0mg Sodium acetate ......................................................................... 20.0mg FeSO4·7H2O............................................................................. 10.0mg MnSO4·H2O ............................................................................... 7.5mg © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Lactobacillus casei.

Lactobacillus Medium (ATCC Medium 1006) Composition per liter: Agar ............................................................................................ 20.0g Pancreatic digest of casein.......................................................... 10.0g Glucose ......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 3.0g K2HPO4......................................................................................... 3.0g

924

Lactobacillus Medium

Tryptose ........................................................................................ 3.0g Sodium acetate .............................................................................. 1.0g L-Cysteine·HCl·H2O...................................................................... 0.2g Salt solution R............................................................................5.0mL Tween™ 80 ................................................................................1.0mL pH 6.3 ± 0.2 at 25°C

Salt Solution R: Composition per 100.0mL: MgSO4·7H2O .............................................................................. 11.5g MnSO4·2H2O ................................................................................ 2.4g FeSO4·7H2O................................................................................ 0.68g

Preparation of Salt Solution R: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Store at 4°C.

Use: For the cultivation and maintenance of Lactobacillus delbrueckii and Lactobacillus jensenii.

Lactobacillus Medium Composition per liter: Glucose ....................................................................................... 20.0g Sodium acetate ............................................................................ 20.0g Tryptone ...................................................................................... 10.0g Yeast extract.................................................................................. 5.0g Beef extract ................................................................................... 2.0g K2HPO4 ........................................................................................ 0.5g KH2PO4 ........................................................................................ 0.5g MgSO4·7H2O......................................................................... 200.0mg FeSO4·7H2O ............................................................................ 10.0mg MnSO4·H2O............................................................................... 7.5mg Ethanol .....................................................................................40.0mL Mevalonic acid solution...........................................................10.0mL Tween™ 80 ................................................................................1.0mL pH 5.2 ± 0.2 at 25°C

Mevalonic Acid Solution: Composition per 10.0mL: DL-Mevalonic

acid ................................................................... 30.0mg

Preparation of Mevalonic Acid Solution: Add DL-mevalonic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except mevalonic acid solution and ethanol, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 40.0mL of filter-sterilized ethanol and 10.0mL of sterile mevalonic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Lactobacillus homohiochii.

Lactobacillus Medium (Lactobacillus Sake Medium) Composition per liter: Agar ............................................................................................ 13.0g Yeast extract.................................................................................. 5.0g Liver extract concentrate .............................................................. 0.2g Sake........................................................................................700.0mL © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Lactobacillus fructivorans and Lactobacillus homohiochii.

Lactobacillus Medium II (DSMZ Medium 93) Composition per liter: Glucose ....................................................................................... 20.0g Na-acetate ................................................................................... 20.0g Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Meat extract .................................................................................. 2.0g KH2PO4......................................................................................... 0.5g K2HPO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g DL-Mevalonic acid ................................................................... 30.0mg FeSO4·7H2O............................................................................. 10.0mg MnSO4·2H2O ............................................................................. 7.5mg Ethanol.....................................................................................40.0mL Tween™ 80................................................................................1.0mL pH 5.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Lactobacillus fructivorans and Lactobacillus homohiochii.

Lactobacillus Medium III Composition per liter: Pancreatic digest of casein.......................................................... 10.0g Fructose......................................................................................... 7.0g Glucose ......................................................................................... 7.0g Maltose ......................................................................................... 7.0g Meat extract .................................................................................. 5.0g Sodium acetate·3H2O.................................................................... 5.0g Yeast extract.................................................................................. 5.0g K2HPO4·3H2O .............................................................................. 2.6g Diammonium citrate ..................................................................... 2.0g Sodium gluconate ......................................................................... 2.0g L-Cysteine·HCl ............................................................................. 0.5g MgSO4·7H2O ................................................................................ 0.1g MnSO4·4H2O .............................................................................. 0.05g Tween™ 80................................................................................1.0mL pH 6.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.3. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Lactobacillus species.

Lactobacillus Medium IV (DSMZ Medium 859) Composition per liter: Tryptone...................................................................................... 10.0g Yeast extract.................................................................................. 5.0g

Lactobacillus rimae Medium

Na-acetate·3H2O ........................................................................... 5.0g Glucose ......................................................................................... 4.0g Maltose.......................................................................................... 4.0g Meat extract .................................................................................. 3.0g K2HPO4·3H2O............................................................................... 2.6g (NH4)2 citrate ................................................................................ 2.0g Cysteine-HCl·H2O ........................................................................ 0.5g MgSO4·7H2O ................................................................................ 0.1g MnSO4·4H2O .............................................................................. 0.05g Tween™ 80 ................................................................................1.0mL pH 6.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus panis.

Lactobacillus 8664 Medium Composition per liter: Maltose........................................................................................ 20.0g Peptone........................................................................................ 10.0g Yeast extract................................................................................ 10.0g Glucose ......................................................................................... 5.0g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus brevis.

Lactobacillus MRS HiVeg Agar (MRS HiVeg Agar) Composition per liter:

925

Ammonium citrate ........................................................................ 2.0g K2HPO4......................................................................................... 2.0g Polysorbate 80 .............................................................................. 1.0g MgSO4 .......................................................................................... 0.1g MnSO4 ........................................................................................ 0.05g pH 6.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of Lactobacillus species.

Lactobacillus Orotic Acid Medium Composition per liter: Milk, peptonized ......................................................................... 15.0g Glucose ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 1.0g Orotic acid ............................................................................... 25.0mg D-Pantothenic acid ..................................................................... 0.2mg Tomato juice ..........................................................................100.0mL L-Cysteine·HCl·H2O solution ....................................................7.5mL pH 6.8 ± 0.2 at 25°C L-Cysteine·HCl·H2O Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.15g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Glucose ....................................................................................... 20.0g Agar ............................................................................................ 12.0g Plant extract ................................................................................ 10.0g Plant peptone No. 3..................................................................... 10.0g Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g K2HPO4 ......................................................................................... 2.0g Polysorbate 80............................................................................... 1.0g MgSO4 .......................................................................................... 0.1g MnSO4 ........................................................................................ 0.05g pH 6.5 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 2–3 min. Distribute into bottles in 20.0mL volumes. Add 0.15mL of L-cysteine·HCl·H2O solution to each bottle containing 20.0mL of medium. Autoclave for 15 min at 15 psi pressure–121°C. Screw caps tightly to maintain reduced conditions.

Source: This medium is available as a premixed powder from HiMe-

Composition per liter:

dia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.5. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the cultivation of Lactobacillus species.

Lactobacillus MRS HiVeg Broth (MRS HiVeg Broth) Composition per liter: Glucose ....................................................................................... 20.0g Plant extract ................................................................................ 10.0g Plant peptone No. 3..................................................................... 10.0g Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Lactobacillus helveticus.

Lactobacillus rimae Medium Yeast extract................................................................................ 10.0g Peptone ......................................................................................... 5.0g Pancreatic digest of casein............................................................ 5.0g Glucose ......................................................................................... 5.0g (NH4)2SO4 .................................................................................... 0.5g L-Cysteine·HCl ............................................................................. 0.5g Resazurin ................................................................................... 1.0mg Mineral solution.......................................................................40.0mL Fatty acid mixture ......................................................................3.1mL Hemin solution...........................................................................0.5mL Vitamin K1 .................................................................................0.2mL pH 6.9 ± 0.2 at 25°C

926

Lactobacillus rimae Medium with Tween™

KH2PO4 ......................................................................................... 1.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O .............................................................................. 0.48g CaCl2·2H2O................................................................................... 0.3g

Preparation of Mineral Solution: Add components to distilled/

Acetic acid ...............................................................................17.0mL Propionic acid ............................................................................6.0mL n-Butyric acid ............................................................................4.0mL n-Valeric acid .............................................................................1.0mL iso-Valeric acid ..........................................................................1.0mL iso-Butyric acid..........................................................................1.0mL DL-2-Methylbutyric acid............................................................1.0mL

deionized water and bring volume to 1.0L. Mix thoroughly.

Fatty Acid Mixture: Composition per 31.0mL: Acetic acid ...............................................................................17.0mL Propionic acid ............................................................................6.0mL n-Butyric acid ............................................................................4.0mL n-Valeric acid .............................................................................1.0mL iso-Valeric acid ..........................................................................1.0mL iso-Butyric acid..........................................................................1.0mL DL-2-Methylbutyric acid ............................................................1.0mL

Preparation of Fatty Acid Mixture: Combine components. Mix thoroughly. Adjust pH to 7.5 with concentrated NaOH.

deionized water and bring volume to 1.0L. Mix thoroughly.

Fatty Acid Mixture: Composition per 31.0mL:

Preparation of Fatty Acid Mixture: Combine components. Mix thoroughly. Adjust pH to 7.5 with concentrated NaOH. Hemin Solution: Composition per 1.0mL: Hemin ........................................................................................ 5.0mg NaOH (1N solution)...................................................................1.0mL

Hemin Solution: Composition per 1.0mL:

Preparation of Hemin Solution: Add hemin to 1.0mL of NaOH

Hemin......................................................................................... 5.0mg NaOH (1N solution)...................................................................1.0mL

Preparation of Medium: Add components, except L-cysteine·HCl,

Preparation of Hemin Solution: Add hemin to 1.0mL of NaOH solution. Mix thoroughly.

Preparation of Medium: Add components, except L-cysteine·HCl, hemin solution, and fatty acid mixture, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% CO2. Add L-cysteine·HCl, hemin solution, and fatty acid mixture. Adjust pH to 6.9 with 8N NaOH while continuing to sparge with 100% CO2. After pH has been reached, sparge with 100% N2. Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Acetivibrio ethanolgignens, Lactobacillus rimae, Lactobacillus uli, and Sphaerotilus natans.

Lactobacillus rimae Medium with Tween™ Composition per liter: Yeast extract................................................................................ 10.0g Peptone.......................................................................................... 5.0g Pancreatic digest of casein ............................................................ 5.0g Glucose ......................................................................................... 5.0g (NH4)2SO4 ..................................................................................... 0.5g L-Cysteine·HCl.............................................................................. 0.5g Tween™ 80 ................................................................................... 0.2g Resazurin ................................................................................... 1.0mg Mineral solution .......................................................................40.0mL Fatty acid mixture ......................................................................3.1mL Hemin solution...........................................................................0.5mL Vitamin K1 .................................................................................0.2mL pH 6.9 ± 0.2 at 25°C

Mineral Solution: Composition per liter: NaHCO3 ...................................................................................... 10.0g NaCl .............................................................................................. 2.0g KH2PO4 ......................................................................................... 1.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O .............................................................................. 0.48g CaCl2·2H2O................................................................................... 0.3g © 2010 by Taylor and Francis Group, LLC

solution. Mix thoroughly. hemin solution, and fatty acid mixture, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% CO2. Add L-cysteine·HCl, hemin solution, and fatty acid mixture. Adjust pH to 6.9 with 8N NaOH while continuing to sparge with 100% CO2. After pH has been reached, sparge with 100% N2. Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Lactobacillus rimae and Lactobacillus uli.

Lactobacillus Selection Agar See: LBS™ Agar

Lactobacillus Selection Agar Base Composition per liter: Sodium acetate............................................................................ 25.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ....................................................... 10.0g KH2PO4......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g Polysorbate 80 .............................................................................. 1.0g MgSO4·7H2O ............................................................................ 0.575g MnSO4·2H2O .............................................................................. 0.12g FeSO3 ........................................................................................ 0.034g Acetic acid, glacial...................................................................1.32mL pH 5.5 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add acetic acid to distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Distribute into tubes or flasks. Gently heat and bring to boiling. Boil for 1–2 min. Do not autoclave unless storage is needed. If storage is necessary sterilize by autoclaving for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation and enumeration of lactobacilli from foods.

Lactococcus piscium Medium

Lactobacillus Selection Broth See: LBS™ Broth

Lactobacillus Selection HiVeg Agar Base with Acetic Acid and Polysorbate Composition per liter: Sodium acetate·3H2O.................................................................. 25.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Plant hydrolysate......................................................................... 10.0g KH2PO4 ......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g MgSO4 ...................................................................................... 0.575g FeSO4 ........................................................................................ 0.034g MnSO4 ........................................................................................ 0.12g Acetic acid, glacial..................................................................1.32 mL Polysorbate 80............................................................................1.0mL pH 5.5 ± 0.2 at 25°C

Source: This medium, without polysorbate 80 or glacial acetic acid, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L.Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 5.5. Do not autoclave. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation, cultivation, and enumeration of lactobacilli from foods.

Lactobacillus Selection HiVeg Broth Base with Acetic Acid and Polysorbate Composition per liter: Sodium acetate ............................................................................ 25.0g Glucose ....................................................................................... 20.0g Plant hydrolysate......................................................................... 10.0g KH2PO4 ......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Polysorbate 80............................................................................... 1.0g MgSO4 ...................................................................................... 0.575g MnSO4 ........................................................................................ 0.12g FeSO4 ........................................................................................ 0.034g Acetic acid, glacial..................................................................1.32 mL Polysorbate 80............................................................................1.0mL pH 5.5 ± 0.2 at 25°C

Source: This medium, without polysorbate 80 or glacial acetic acid, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 5.5. Do not autoclave. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of lactobacilli.

Lactobacillus Selection Oxgall Agar (LBS™ Oxgall Agar) Composition per liter: Sodium acetate·3H2O.................................................................. 25.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g © 2010 by Taylor and Francis Group, LLC

927

Pancreatic digest of casein.......................................................... 10.0g KH2PO4......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g Oxgall ........................................................................................... 1.5g Polysorbate 80 .............................................................................. 1.0g MgSO4 ...................................................................................... 0.575g MnSO4 ........................................................................................ 0.12g FeSO4 ........................................................................................ 0.034g Acetic acid, glacial...................................................................1.32mL pH 5.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except acetic acid, to distilled/deionized water and bring volume to 998.7mL. Mix thoroughly. Gently heat and bring to boiling. Add glacial acetic acid. Mix thoroughly. Gently heat while stirring and bring to 90°–100°C for 2–3 min. Do not autoclave. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation, cultivation, and enumeration of lactobacilli.

Lactobacillus Selection Oxgall Agar Base (LBS Oxgall Agar) Composition per liter: Sodium acetate............................................................................ 25.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ....................................................... 10.0g KH2PO4......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g Oxgall ........................................................................................... 1.5g Polysorbate 80 .............................................................................. 1.0g MgSO4·7H2O ............................................................................ 0.575g MnSO4·2H2O .............................................................................. 0.12g FeSO4·7H2O.............................................................................. 0.034g Acetic acid, glacial...................................................................1.32mL pH 5.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add acetic acid to distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Distribute into tubes or flasks. Gently heat and bring to boiling. Boil for 1–2 min. Do not autoclave unless storage is needed. If storage is necessary, sterilize by autoclaving for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the selective isolation, cultivation, and enumeration of lactobacilli. Lactobacillus-Streptococcus Differential Medium See: L-S Differential Medium

Lactococcus piscium Medium Composition per liter: Glucose ....................................................................................... 10.0g Peptone ....................................................................................... 10.0g Beef extract................................................................................... 8.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g KH2PO4......................................................................................... 1.5g

928

Lactose Blue Agar

MgSO4·7H2O ................................................................................ 0.2g MnSO4·4H2O .............................................................................. 0.05g pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Lactococcus piscium.

Lactose Blue Agar (B.T.B. Lactose Agar, Modified) Composition per liter: Lactose ....................................................................................... 15.5g Agar ............................................................................................ 13.0g NaCl .............................................................................................. 5.0g Peptic digest of animal tissue....................................................... 3.5g Casein enzymic hydrolysate ........................................................ 3.5g Bromthymol Blue ...................................................................... 0.04g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the differentiation of lactose-fermenting and non-fermenting bacteria belonging to Enterobacteriaceae.

Lactose Blue HiVeg Agar (B.T.B. Lactose HiVeg Agar, Modified) Composition per liter: Lactose ....................................................................................... 15.5g Agar ............................................................................................ 13.0g NaCl .............................................................................................. 5.0g Plant extract ................................................................................. 3.5g Plant hydrolysate .......................................................................... 3.5g Bromthymol Blue ...................................................................... 0.04g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the differentiation of lactose-fermenting and non-fermenting bacteria belonging to Enterobacteriaceae.

Lactose Broth Composition per liter: Lactose .......................................................................................... 5.0g Pancreatic digest of gelatin ........................................................... 5.0g Beef extract ................................................................................... 3.0g pH 6.9 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes containing an inverted Durham tube in 10.0mL volumes. Autoclave for 12 min at 15 psi pressure–121°C. Cool broth quickly to 25°C. For testing water samples with 10.0mL volumes, prepare medium double strength.

Use: For the detection of lactose-fermenting, Gram-negative coliforms, as a preenrichment broth for Salmonella species, and in the study of lactose fermentation of bacteria in general.

Lactose Casein Hydrolysate Medium Composition per liter: Lactose........................................................................................ 37.5g Agar ............................................................................................ 15.0g Casein hydrolysate........................................................................ 3.0g KH2PO4......................................................................................... 1.0g MnSO4 .......................................................................................... 0.5g Microelements solution .............................................................2.0mL pH 6.0 ± 0.2 at 25°C

Microelements Solution: Composition per liter: Fe(NO3)3·9H2O...................................................................... 723.5mg ZnSO4·7H2O .......................................................................... 439.8mg MnSO4·4H2O ......................................................................... 203.0mg H2SO4 ..................................................................................... variable

Preparation of Microelements Solution: Add components, one at a time, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Be sure one component is dissolved before adding the next. Add sulfuric acid to yield a clear solution. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Adjust pH to 6.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Drechslera catenaria.

Lactose Distillers Solubles Medium Composition per liter: Lactose........................................................................................ 20.0g Distillers solubles........................................................................ 15.0g Yeast, autolyzed ............................................................................ 5.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Streptomyces avermitilis.

Lactose Egg Yolk Milk Agar Composition per 1206.0mL: Lactose........................................................................................ 12.0g Agar .............................................................................................. 1.0g Columbia blood agar base .....................................................800.0mL Skim milk...............................................................................150.0mL Egg yolk emulsion, 50%..........................................................36.0mL Inhibitor solution .....................................................................20.0mL Neutral Red (1% solution) .......................................................3.25mL pH 7.0 ± 0.2 at 25°C

Lactose Lecithin Agar

Columbia Blood Agar Base: Composition per 800.0mL: Agar ............................................................................................ 15.0g Pantone........................................................................................ 10.0g Bitone.......................................................................................... 10.0g NaCl .............................................................................................. 5.0g Tryptic digest of beef heart ........................................................... 3.0g Cornstarch ..................................................................................... 1.0g

929

Lactose Gelatin Medium, Modified Composition per liter:

Preparation of Columbia Blood Agar Base: Add components

Gelatin....................................................................................... 120.0g Tryptose ...................................................................................... 15.0g Yeast extract................................................................................ 10.0g Lactose........................................................................................ 10.0g Na2HPO4 ....................................................................................... 5.0g Phenol Red.................................................................................. 0.05g pH 7.8 ± 0.2 at 25°C

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling.

Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized

Egg Yolk Emulsion, 50%: Composition per 100.0mL:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into screw-cap tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Just before use heat to remove oxygen and cool rapidly.

Use: For the cultivation of Haemophilus spp.

Lactose HiVeg Broth Composition per liter: Plant peptone ................................................................................ 5.0g Lactose.......................................................................................... 5.0g Plant extract .................................................................................. 3.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMe-

Neomycin sulfate ........................................................................ 0.18g NaN3 ........................................................................................... 0.24g

dia.

Caution: Sodium azide is toxic. Azides also react with metals and

water and bring volume to 1.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

disposal must be highly diluted.

Preparation of Inhibitor Solution: Add neomycin sulfate and NaN3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Combine Columbia blood agar base, lactose, agar, and Neutral Red and bring volume to 1.0L. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 55°C. Filter sterilize skim milk. To 1.0L of cooled, sterile agar mixture, aseptically add 150.0mL of sterile skim milk, 36.0mL of sterile egg yolk emulsion, 50%, and 20.0mL of sterile inhibitor solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Clostridium species.

Lactose Gelatin Medium Composition per liter: Gelatin....................................................................................... 120.0g Tryptose ...................................................................................... 15.0g Lactose ........................................................................................ 10.0g Yeast extract................................................................................ 10.0g Phenol Red (0.5% solution) .....................................................10.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add tryptose, yeast extract, and lactose to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Add gelatin to distilled/deionized water and bring volume to 590.0mL. Gently heat gelatin solution while stirring and bring to 50°– 60°C. Add Phenol Red. Mix the two solutions together. Distribute into tubes in 10.0mL volumes. Autoclave for 10 min at 15 psi pressure– 121°C. If medium is not used in 8 hr, deoxygenate by heating to 50°– 70°C for 2–3 hr prior to inoculation.

Preparation of Medium: Add components to distilled/deionized

Use: For the detection of coliform bacteria in water, foods, and dairy products as per standard methods.

Lactose Lecithin Agar Composition per liter: Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ..................................................... 12.65g Lactose........................................................................................ 10.0g Peptic digest of animal tissue ....................................................... 5.5g NaCl.............................................................................................. 5.5g Yeast extract................................................................................ 3.85g Pancreatic digest of heart muscle ................................................. 3.3g Corn starch.................................................................................... 1.1g Egg lecithin................................................................................. 0.66g L-Cysteine·HCl·H2O ..................................................................... 0.5g NaN3 ............................................................................................. 0.2g Neomycin sulfate ........................................................................ 0.15g CaCl2·2H2O ................................................................................ 0.05g Bromcresol Purple .................................................................... 0.025g pH 6.8 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Caution: Sodium azide has a tendency to form explosive metal azides with plumbing materials. It is advisable to use enough water to flush off the disposables.

930

Lactose Medium

Use: For the isolation and differentiation of histotoxic clostridia from clinical specimens.

Lactose Medium

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Composition per liter:

Use: For the cultivation and maintenance of various fungi.

Agar ............................................................................................ 15.0g K2HPO4 ......................................................................................... 0.5g (NH4)2SO4 ..................................................................................... 0.5g NaCl ......................................................................................... 50.0mg Lactose solution .......................................................................50.0mL pH 7.2 ± 0.2 at 25°C

Composition per liter:

Lactose Solution: Composition per 50.0mL:

Preparation of Medium: Add components to distilled/deionized

Lactose .......................................................................................... 5.0g

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except lactose solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 50.0mL of sterile lactose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of unidentified bacterium ATCC 51468.

Lactose Minimal Medium Composition per liter: Agar ............................................................................................ 20.0g Lactose ........................................................................................ 15.0g K2HPO4 ......................................................................................... 5.0g NH4Cl ........................................................................................... 2.0g NaCl .............................................................................................. 1.0g MgSO4 .......................................................................................... 0.1g Yeast extract.................................................................................. 0.1g

Lactose Peptone Agar, Half Strength Agar ............................................................................................ 15.0g Lactose........................................................................................ 0.25g Peptone ....................................................................................... 0.25g water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of various fungi.

Lactose Peptone Broth Composition per liter: Casein enzymic hydrolysate ....................................................... 17.0g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Papaic digest of soybean meal...................................................... 3.0g Bromcresol Purple ...................................................................... 0.02g pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes with inverted Durham tubes. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the detection of coliform organisms in water.

Use: For the cultivation and maintenance of Xanthomonas campestris.

Lactose Peptone Agar Composition per liter:

Lactose Ricinoleate Broth Composition per liter: Lactose........................................................................................ 10.0g Peptone ......................................................................................... 5.0g Sodium ricinoleate ........................................................................ 1.0g pH 7.6 ± 0.2 at 25°C

Agar ............................................................................................ 15.0g Lactose .......................................................................................... 0.5g Peptone.......................................................................................... 0.5g

Preparation of Medium: Add components to distilled/deionized

Use: For the selective cultivation of members of the Enterobacteri-

water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

aceae.

Lactose Sulfite Broth Base

Use: For the cultivation and maintenance of Acytostelium subglobosum,

Composition per liter:

numerous Dictyostelium species, Didymium nigripes, Drechslera biseptata, several Mortierella species, Nodulisporium griseobrunneum, Penicillium chrysogenum, Polysphondylium pallidum, Polysphondylium violaceum, Pythium insidiosum, and Trichomycete species.

Lactose........................................................................................ 10.0g Casein enzymic hydrolysate ......................................................... 5.0g Yeast extract.................................................................................. 2.5g NaCl.............................................................................................. 2.5g L-Cysteine·HCl·H2O ..................................................................... 0.3g Sodium metabisulfite solution .................................................50.0mL Ferric ammonium citrate..........................................................50.0mL pH 7.1 ± 0.2 at 25°C

Lactose Peptone Agar, Double Strength Composition per liter: Agar ............................................................................................ 18.0g Lactose .......................................................................................... 1.0g Peptone.......................................................................................... 1.0g © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Source: This medium is available from HiMedia.

Lasseur Medium

Selective Sodium Metabisulfite Solution: Composition per 100.0mL: Sodium metabisulfite .................................................................... 1.2g

Preparation of Sodium Metabisulfite Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Selective Ferric Ammonium Citrate Solution: Composition per 100.0mL: Ferric ammonium citrate............................................................... 1.0g

Preparation of Ferric Ammonium Citrate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except sodium metabisulfite and ferric ammonium citrate solutions, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add sodium metabisulfite and ferric ammonium citrate solutions. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the detection and enumeration of Clostridium perfringens in pharmaceutical products.

931

Lange Medium Composition per liter: Agar ............................................................................................ 20.0g Maltose ......................................................................................... 5.0g Ca(NO3)2....................................................................................... 0.5g MgSO4 .......................................................................................... 0.5g K2HPO4....................................................................................... 0.25g Peptone ......................................................................................... 0.1g Horse dung extract.................................................................100.0mL

Horse Dung Extract: Composition per liter: Horse dung, fresh...................................................................... 100.0g

Preparation of Horse Dung Extract: Add fresh horse dung to distilled/deionized water and bring volume to 1.0L. Autoclave for 50 min at 15 psi pressure–121°C. Filter through Whatman #1 filter paper. Reserve filtrate. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Coprinus xanthothrix.

Lambda Broth Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 2.5g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 25 min at 15 psi pressure–121°C.

Use: For the cultivation of Escherichia coli in the preparation of bacteriophage lysates.

Lambda Plates Composition per liter: Agar ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 2.5g

Use: For use as a base agar to support the cultivation of Escherichia coli in the preparation of bacteriophages.

Lash Serum Medium Composition per liter: Casamino acids ........................................................................... 14.0g NaCl.............................................................................................. 6.0g Glucose ......................................................................................... 2.0g Maltose ......................................................................................... 1.5g Sodium lactate (60% solution)...................................................... 0.5g KCl................................................................................................ 0.1g CaCl2·2H2O .................................................................................. 0.1g Serum solution .......................................................................500.0mL pH 5.8 ± 0.2 at 25°C

Serum Solution: Composition per 500.0mL: NaHCO3 ........................................................................................ 0.1g Bovine serum .........................................................................200.0mL

Preparation of Serum Solution: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except serum solution, to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 5.0mL of sterile serum solution to each tube. Mix thoroughly. Use: For the cultivation of Trichomonas vaginalis from clinical spec-

Lambda Top Agar Composition per liter: Pancreatic digest of casein .......................................................... 10.0g Agar .............................................................................................. 7.0g NaCl .............................................................................................. 2.5g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Distribute into flasks in 100.0mL volumes. Reautoclave for 15 min at 15 psi pressure–121°C. Store at 25°C.

imens.

Lasseur Medium (LMG 170) Composition per liter: Glycerol ...................................................................................... 25.0g Agar ............................................................................................ 15.0g L-Asparagine................................................................................. 9.0g MgSO4·7H2O ................................................................................ 5.0g K2HPO4......................................................................................... 2.5g CaCl2·2H2O ................................................................................ 0.54g FeSO4·7H2O.................................................................................. 0.1g pH 6.7 ± 0.2 at 25°C

932

Lauryl Sulfate Broth

Preparation of Medium: Add K2HPO4 to 20.0mL of distilled/deionized water. Mix thoroughly. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Gently heat and bring to boiling. Adjust pH to 6.7. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Pseudomonas fluorescens.

Lauryl Sulfate Broth (m-Lauryl Sulfate Broth) Composition per liter: Peptone........................................................................................ 39.0g Lactose ........................................................................................ 30.0g Yeast extract.................................................................................. 6.0g Sodium lauryl sulfate .................................................................... 1.0g Phenol Red .................................................................................... 0.2g pH 7.4 ± 0.2 at 25°C

Sodium lauryl sulfate.................................................................... 0.1g 4-Methylumbelliferyl-ß-D-glucuronide........................................ 0.1g pH 6.8 ± 0.2 at 25°C

Source: This medium is available from Merck. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Cool. Distribute into test tubes containing inverted Durham tubes. Autoclave for 15 min at 15 psi pressure–121°C. The prepared broth is clear and yellowish-brown. Use: For the detection of E. coli in milk. The medium complies with the German-DIN-Norm 10183 for the examination of milk, with the regulations acc. to § 35 LMBG (01.00/54) for the examination of food, and according to ISO/DIS 11886-2.2 (1994) for milk and milk products. The lauryl sulfate largely inhibits the growth of undesirable microbial flora. The presence of E. coli is indicated by fluorescence under a long wavelength UV lamp. A positive indole reaction and gas formation due to fermentation of lactose confirm the results.

Source: This medium is available as a premixed powder from Oxoid

Lauryl Sulfate Broth with MUG

Unipath.

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Pancreatic digest of casein.......................................................... 20.0g Lactose.......................................................................................... 5.0g NaCl.............................................................................................. 5.0g K2HPO4....................................................................................... 2.75g KH2PO4....................................................................................... 2.75g Sodium lauryl sulfate.................................................................... 0.1g 4-Methylumbelliferyl-β-D-glucuronide (MUG) ......................... 0.05g pH 6.8 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Distribute into bottles or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and enumeration of coliform bacteria, especially Escherichia coli, in water by the membrane filter method.

Lauryl Sulfate Broth (Lauryl Tryptose Broth) Composition per liter:

Source: This medium is available as a premixed powder from BD Di-

Pancreatic digest of casein .......................................................... 20.0g Lactose .......................................................................................... 5.0g NaCl .............................................................................................. 5.0g K2HPO4 ....................................................................................... 2.75g KH2PO4 ....................................................................................... 2.75g Sodium lauryl sulfate .................................................................... 0.1g pH 6.8 ± 0.2 at 25°C

agnostic Systems.

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the detection of Escherichia coli in water and food samples by a fluorogenic procedure.

Use: For the detection of coliform bacteria in a variety of specimens. Also, for the enumeration of coliform bacteria by the multiple-tube fermentation technique.

Lauryl Sulfate Broth, Fluorocult (Fluorocult Lauryl Sulfate Broth) Composition per liter: Tryptose ...................................................................................... 20.0g Lactose .......................................................................................... 5.0g NaCl .............................................................................................. 5.0g K2HPO4 ....................................................................................... 2.75g KH2PO4 ....................................................................................... 2.75g L-tryptophan.................................................................................... 1.g © 2010 by Taylor and Francis Group, LLC

Lauryl Sulfate HiVeg Broth (Lauryl Tryptose HiVeg Broth) Composition per liter: Plant hydrolysate No. 1............................................................... 20.0g NaCl.............................................................................................. 5.0g Lactose.......................................................................................... 5.0g K2HPO4....................................................................................... 2.75g KH2PO4....................................................................................... 2.75g Sodium lauryl sulfate.................................................................... 0.1g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into bottles or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and enumeration of coliform bacteria in water, wastewater, dairy products, and other foods.

LB Broth, Modified

Lauryl Tryptose Broth See: Lauryl Sulfate Broth

Lauryl Tryptose Broth with MUG (Lauryl Sulfate Broth with MUG) (LST-MUG) (BAM M77) Composition per liter: Pancreatic digest of casein .......................................................... 20.0g Lactose .......................................................................................... 5.0g NaCl .............................................................................................. 5.0g K2HPO4 ....................................................................................... 2.75g KH2PO4 ....................................................................................... 2.75g Sodium lauryl sulfate .................................................................... 0.1g 4-Methylumbelliferyl-β-D-glucuronide (MUG).......................... 0.05g pH 6.8 ± 0.2 at 25°C

933

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5 with Na2CO3. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. The pH should be 8.0–8.1 after autoclaving. Use: For the cultivation of Caryophanon latum.

LB Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g 1N NaOH ...................................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 25 min at 15 psi pressure–121°C. Pour into sterile Petri dishes in 35–40.0mL volumes.

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Escherichia coli.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes containing an inverted Durham tube in 10.0mL volumes. Autoclave for 12 min at 15 psi pressure–121°C. Cool broth quickly to 25°C. For testing water samples with 10.0mL volumes, prepare medium double strength.

Use: For the detection of Escherichia coli in water and food samples by a fluorogenic procedure.

Lauryl Tryptose Mannitol Broth with Tryptophan Composition per liter: Pancreatic digest of casein .......................................................... 20.0g Lactose .......................................................................................... 5.0g NaCl .............................................................................................. 5.0g K2HPO4 ....................................................................................... 2.75g KH2PO4 ....................................................................................... 2.75g Sodium lauryl sulfate .................................................................... 0.1g L-Tryptophan ................................................................................. 0.2g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes containing an inverted Durham tube in 10.0mL volumes. Autoclave for 10 min at 10 psi pressure–115°C. Cool broth quickly to 25°C.

Use: For the detection of Escherichia coli in water samples.

LAVMm2 Medium Composition per liter: Lactalbumin hydrolysate............................................................. 10.0g Sodium acetate .............................................................................. 5.0g MgCl2·6H2O............................................................................. 20.3mg Nitrilotriacetic acid .................................................................. 19.1mg CaCl2 ........................................................................................ 11.1mg FeSO4 ..................................................................................... 0.152mg Thiamine·HCl .......................................................................... 0.05mg Cupric acetate .......................................................................... 0.04mg Biotin ....................................................................................... 0.02mg pH 8.0–8.1 at 25°C © 2010 by Taylor and Francis Group, LLC

LB Agar See: Lactobacillus bulgaricus Agar

LB Broth, Modified Composition per liter: Pancreatic digest of casein.......................................................... 10.0g NaCl.............................................................................................. 5.8g Yeast extract.................................................................................. 5.0g NaCl solution ...........................................................................16.8mL Glucose solution ......................................................................10.0mL CaCl2·2H2O solution..................................................................2.0mL MgCl2 solution...........................................................................1.6mL pH 7.0 ± 0.2 at 25°C

NaCl Solution: Composition per 100.0mL: NaCl............................................................................................ 25.0g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Glucose Solution: Composition per 100.0mL: D-Glucose .................................................................................... 40.0g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. CaCl2·2H2O Solution: Composition per 10.0mL: CaCl2·2H2O .............................................................................. 0.735g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. MgCl2 Solution: Composition per 10.0mL: MgCl2.......................................................................................... 0.95g

Preparation of MgCl2 Solution: Add MgCl2 to distilled/deion-

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

934

LB Medium

Preparation of Medium: Add components—except NaCl solution, glucose solution, CaCl2·2H2O solution, and MgCl2 solution—to distilled/deionized water and bring volume to 969.6mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 30 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 16.8mL of sterile NaCl solution, 10.0mL of sterile glucose solution, 2.0mL of sterile CaCl2·2H2O solution, and 1.6mL of sterile MgCl2 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Escherichia coli.

LB Medium (LB Broth, Miller) (ATCC Medium 1065) (ATCC Medium 1082) Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g

Source: This medium is available from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Bacillus subtilis, Corynebacterium glutamicum, Enterobacter cloacae, Erwinia uredovora, Escherichia coli, Klebsiella oxytoca, and Salmonella choleraesuis.

LB Medium (Luria-Bertani Medium) Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Bacillus subtilis, Daptobacter species, and Escherichia coli.

Ampicillin Solution: Composition per 10.0mL: Ampicillin .................................................................................. 0.1mg

Preparation of Ampicillin Solution: Add ampicillin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except ampicillin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add sterile ampicillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with Ampicillin (ATCC Medium 1364) Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Ampicillin solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Ampicillin Solution: Composition per 10.0mL: Ampicillin ................................................................................ 0.02mg

Preparation of Ampicillin Solution: Add ampicillin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except ampicillin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add sterile ampicillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with Chloramphenicol LB Medium (Luria Broth) (Lenox Broth)

Use: For the cultivation of Escherichia coli.

LB Medium with Ampicillin (ATCC Medium 1315) Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g © 2010 by Taylor and Francis Group, LLC

Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Chloramphenicol......................................................................... 0.01g pH 7.0 ± 0.2 at 25°C

LB Medium with Glucose Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Glucose ......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

LB Medium with Tetracycline and Ampicillin Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with IPTG Medium Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g IPTG solution...........................................................................10.0mL pH 7.0 ± 0.2 at 25°C

IPTG Solution: Composition per 10.0mL:

935

LB Medium with 50mg of Kanamycin Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Kanamycin.................................................................................. 0.05g pH 7.0 ± 0.2 at 25°C

LB Medium with 100mg of Kanamycin (ATCC Medium 1468)

IPTG (Isopropylthio-β-galactoside)............................................ 0.24g

Composition per liter:

Preparation of IPTG Solution: Add IPTG to distilled/deionized

NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Kanamycin.................................................................................... 0.1g pH 7.0 ± 0.2 at 25°C

water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except IPTG solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add sterile IPTG solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with Kanamycin Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl ............................................................................................ 10.0g Yeast extract................................................................................. 5.0g Kanamycin solution .................................................................50.0mL

Kanamycin Solution: Composition per 50.0mL: Kanamycin ............................................................................... 50.0mg

Preparation of Kanamycin Solution: Add kanamycin to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C.

Preparation of Medium: Add components, except kanamycin solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50–55°C. Aseptically add 50.0mL of sterile kanamycin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Escherichia coli.

LB Medium with 25mg of Kanamycin (ATCC Medium 1236)

LB Medium with Rifampicin Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Rifampicin .................................................................................... 0.1g pH 7.0 ± 0.2 at 25°C

LB Medium with Tetracycline Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Tetracycline................................................................................. 0.02g pH 7.0 ± 0.2 at 25°C

Composition per liter:

Preparation of Medium: Add components to distilled/deionized wa-

NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g Kanamycin ................................................................................ 0.025g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Escherichia coli.

ter and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

LB Medium with Tetracycline and Ampicillin (ATCC Medium 1226) Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g

936

LB Medium with Tetracycline and Ampicillin

Yeast extract.................................................................................. 5.0g Antibiotic solution ...................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Antibiotic Solution: Composition per 10.0mL: Ampicillin ................................................................................... 0.01g Tetracycline................................................................................. 0.01g

Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except antibiotic solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add sterile antibiotic solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

LB Medium with Tetracycline and Ampicillin (ATCC Medium 1235) Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g Antibiotic solution ...................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Antibiotic Solution: Composition per 10.0mL:

LB Medium with TPP (LB Medium with Thiamine Pyrophosphate) Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Thiamine pyrophosphate ....................................................... 0.046mg pH 7.0 ± 0.2 at 25°C

LB Medium for Χ1776 Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Glucose solution ......................................................................10.0mL Diaminopimelic acid solution..................................................10.0mL Thymidine solution..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 0.8g

Preparation of Glucose Solution: Add D-glucose to distilled/de-

Ampicillin ................................................................................... 0.01g Tetracycline................................................................................ 5.0mg

ionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Antibiotic Solution: Add components to distilled/

Diaminopimelic Acid Solution: Composition per 10.0mL:

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

DL-Diaminopimelic

acid ............................................................... 0.1g

Preparation of Medium: Add components, except antibiotic solu-

Preparation of Diaminopimelic Acid Solution: Add diamin-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add sterile antibiotic solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Thymidine Solution: Composition per 10.0mL:

Use: For the cultivation and maintenance of Escherichia coli.

Thymidine................................................................................... 0.02g

LB Medium with Thiamine Monophosphate See: LB Medium with TMP LB Medium with Thiamine Pyrophosphate See: LB Medium with TPP

LB Medium with TMP (LB Medium with Thiamine Monophosphate) Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g Thiamine monophosphate...................................................... 0.038mg pH 7.0 ± 0.2 at 25°C

opimelic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components—except glucose solution, diaminopimelic acid solution, and thymidine solution—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution, diaminopimelic acid solution, and thymidine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Bacillus subtilis and Escherichia coli.

LB Medium for Χ1776 with Tetracycline and Ampicillin Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g

LB Top Agar

937

Antibiotic solution ...................................................................10.0mL Glucose solution ......................................................................10.0mL Diaminopimelic acid solution ..................................................10.0mL Thymidine solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of MgCl2 Solution: Add MgCl2 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Antibiotic Solution: Composition per 10.0mL:

CaCl2 ............................................................................................. 0.5g

Ampicillin ................................................................................... 0.01g Tetracycline................................................................................. 0.01g

Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 0.8g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Diaminopimelic Acid Solution: Composition per 10.0mL: DL-Diaminopimelic

acid................................................................ 0.1g

Preparation of Diaminopimelic Acid Solution: Add diaminopimelic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thymidine Solution: Composition per 10.0mL: Thymidine ................................................................................... 0.02g

Preparation of Thymidine Solution: Add thymidine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except glucose solution, diaminopimelic acid solution, and thymidine solution—to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile antibiotic solution, glucose solution, diaminopimelic acid solution, and thymidine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Bacillus subtilis and Escherichia coli.

LB Modified Broth (ATCC Medium 1620) Composition per 1030.4mL: Tryptone ...................................................................................... 10.0g NaCl .............................................................................................. 5.8g Yeast extract.................................................................................. 5.0g

NaCl Solution: Composition per 100.0mL: NaCl ............................................................................................ 20.0g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

CaCl2 Solution: Composition per 10.0mL: Preparation of CaCl2 Solution: Add CaCl2 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Glucose Solution: Composition per 100.0mL: D-Glucose.................................................................................... 40.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components except salts and glucose solutions to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 30 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 16.8mL NaCl solution, 1.6 mL MgCl2 solution, 2.0mL CaCl2 solution, and 10.0mL glucose solution. Mix thoroughly. Use: For the cultivation of Escherichia coli (Migula) Castellani and Chalmers.

LB Streptomycin Medium Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Streptomycin solution ..............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Streptomycin Solution: Composition per 10.0mL: Streptomycin................................................................................. 0.2g

Preparation of Streptomycin Solution: Add streptomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except streptomycin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile streptomycin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Escherichia coli.

LB Top Agar Composition per liter: Pancreatic digest of casein.......................................................... 10.0g Agar .............................................................................................. 7.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Distribute into flasks in 100.0mL volumes. Reautoclave for 15 min at 15 psi pressure–121°C. Store at 25°C.

938

LBE Medium

Use: For use as a top agar for the distribution of bacteriophage or Escherichia coli.

Do not autoclave. Pour into sterile Petri dishes or distribute into sterile tubes.

LBE Medium

Use: For the selective isolation, cultivation, and enumeration of lactobacilli.

Composition per liter: NaCl ............................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract.................................................................................. 5.0g Glucose solution ......................................................................10.0mL 50X medium E ...........................................................................4.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: D-Glucose .................................................................................... 20.0g

Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

50X Medium E: Composition per liter: K2HPO4, anhydrous .................................................................. 500.0g Na(NH4)HPO4·4H2O ................................................................ 175.0g Citric acid·H2O.......................................................................... 100.0g MgSO4·7H2O .............................................................................. 10.0g

Preparation of 50X Medium E: Add components to 670.0mL of distilled/deionized water in the following order: MgSO4·7H2O, citric acid·H2O, K2HPO4, and Na(NH4)HPO4·4H2O. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water.

Preparation of Medium: Add components—except glucose solution and 50X medium E—to distilled/deionized water and bring volume to 986.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile glucose solution and 4.0mL of sterile 50X medium E. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli.

LBS™ Agar (Lactobacillus Selection Agar) Composition per liter: Sodium acetate·3H2O.................................................................. 25.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Pancreatic digest of casein .......................................................... 10.0g KH2PO4 ......................................................................................... 6.0g Yeast extract.................................................................................. 5.0g Ammonium citrate ........................................................................ 2.0g Polysorbate 80............................................................................... 1.0g MgSO4 ...................................................................................... 0.575g FeSO4 ........................................................................................ 0.034g MnSO4 ........................................................................................ 0.12g Acetic acid, glacial...................................................................1.32mL pH 5.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

LBS™ Broth (Lactobacillus Selection Broth) Composition per liter: Sodium acetate·3H2O.................................................................. 25.0g Glucose ....................................................................................... 20.0g Pancreatic digest of casein.......................................................... 10.0g KH2PO4......................................................................................... 6.0g Yeast extract.................................................................................. 6.0g Ammonium citrate ........................................................................ 2.0g Polysorbate 80 .............................................................................. 1.0g MgSO4 ...................................................................................... 0.575g FeSO4 ........................................................................................ 0.034g MnSO4 ........................................................................................ 0.12g Acetic acid, glacial...................................................................1.32mL pH 5.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the selective isolation and cultivation of lactobacilli. LBS Oxgall Agar See: Lactobacillus Selection Oxgall Agar

LC Broth Composition per liter: NaCl............................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 1.0g CaCl2·2H2O (1M solution).........................................................5.0mL MgSO4·7H2O (1M solution) ......................................................5.0mL pH 7.4 ± 0.2 at 25°C

CaCl2·2H2O Solution: Composition per 100.0mL: CaCl2·2H2O ................................................................................ 14.7g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

MgSO4·7H2O Solution: Composition per 100.0mL: MgSO4·7H2O ............................................................................ 24.65g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except CaCl2·2H2O solution and MgSO4·7H2O solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically

Lecithin Agar

939

add 5.0mL of sterile CaCl2·2H2O solution and 5.0mL of sterile MgSO4·7H2O solution. Mix thoroughly. Aseptically distibute into sterile tubes or flasks.

Vitamin K1 .................................................................................. 0.01g Fe4(P2O7)3·H2O .......................................................................... 0.01g pH 7.5 ± 0.2 at 25°C

Use: For the cultivation of Escherichia coli.

Source: This medium is available as a premixed powder from HiMe-

LD Agar See: Lombard-Dowell Agar LD Bile Agar See: Lombard-Dowell Bile Agar LD Broth See: Lombard-Dowell Broth LD Egg Yolk Agar See: Lombard-Dowell Egg Yolk Agar LD Esculin Agar See: Lombard-Dowell Esculin Agar

LD Esculin HiVeg Agar (Lombard-Dowell Esculin Agar, HiVeg) Composition per liter: Agar ............................................................................................ 20.0g Plant hydrolysate No. 1................................................................. 5.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 2.5g Esculin .......................................................................................... 1.0g Ferric citrate .................................................................................. 0.5g L-Cystine ....................................................................................... 0.4g L-Tryptophan................................................................................. 0.2g Fe4(P2O7)3·H2O........................................................................... 0.01g Vitamin K1 .................................................................................. 0.01g pH 7.5 ± 0.2 at 25°C

dia.

Use: For the cultivation and identification of a variety of obligate anaerobic bacteria. For the cultivation of Bacteroides species, Fusobacterium species, Clostridium species, and nonspore-forming Gram-positive anaerobes.

Lead Acetate Agar Composition per liter: Agar ............................................................................................ 15.0g Peptone ....................................................................................... 15.0g Proteose peptone........................................................................... 5.0g Glucose ......................................................................................... 1.0g Lead acetate .................................................................................. 0.2g Na2S2O3 ...................................................................................... 0.08g pH 6.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Allow tubes to cool in a slanted position. Use: For the cultivation and differentiation of Gram-negative coliform bacteria based on H2S production. Bacteria that produce H2S turn the medium brown.

LEB, FDA See: Listeria Enrichment Broth, FDA

Source: This medium is available as a premixed powder from HiMe-

Lecithin Agar

dia.

Use: For the cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on esculin hydrolysis, H2S production, and catalase production. Bacteria that hydrolyze esculin appear as colonies surrounded by a red-brown to dark brown zone. Bacteria that produce H2S appear as black colonies. LD Gelatin Agar See: Lombard-Dowell Gelatin Agar

LD HiVeg Agar (Lombard-Dowell Agar, HiVeg)

Composition per liter: Fraction B ..............................................................................500.0mL Fraction A ..............................................................................450.0mL Fraction C ................................................................................50.0mL pH 7.2 ± 0.2 at 25°C

Fraction A: Composition per 500.0mL: Agar ............................................................................................ 18.0g Tryptone...................................................................................... 10.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 5.0g

Preparation of Fraction A: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 43°C.

Composition per liter:

Fraction B: Composition per 450.0mL:

Agar ............................................................................................ 20.0g Plant hydrolysate........................................................................... 5.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 2.5g L-Cystine ....................................................................................... 0.4g L-Tryptophan................................................................................. 0.2g Na2SO3 ......................................................................................... 0.1g

Preparation of Fraction B: Add crude soy lethicin to distilled/deionized water and bring volume to 450.0mL. Mix thoroughly. Gently heat and bring to boiling. Swirl to form a viscous sol. Sonicate until homogeneous. Blending of unheated fraction A in a Waring blender for 2 min at high speed is also satisfactory. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 43°C.

Crude soy lecithin ....................................................................... 30.0g

940

Lecithin HiVeg Agar

Fraction C: Composition per 50.0mL: CaCl2 ............................................................................................. 0.6g

Preparation of Fraction C: Add CaCl2 to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 43°C.

Use: For the isolation, cultivation, and differentiation of histolytic clostridia from clinical specimens based on lecithinase production and lactose fermentation. It is especially useful for the differentiation of Clostridium perfringens, Clostridium sordelli, Clostridium novyi, Clostridium septicum, and Clostridium histolyticum. Bacteria that produce lecithinase appear as colonies surrounded by an opalescent zone. Bacteria that ferment lactose appear as colonies surrounded by a yellow zone.

Preparation of Medium: Combine fractions with gentle swirling. To prevent separation, immediately pour into sterile Petri plates.

Use: For the detection of microbial phospholipases.

Lecithin HiVeg Agar Composition per liter: Agar ............................................................................................ 20.5g Plant hydrolysate......................................................................... 15.0g Papaic digest of soybean meal ...................................................... 5.0g Polysorbate 80............................................................................... 5.0g NaCl .............................................................................................. 5.0g Na2S2O3 ........................................................................................ 1.0g L-Histidine..................................................................................... 1.0g Lecithin ......................................................................................... 0.7g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the detection of bacterial contamination of surfaces in unprotected and protected areas.

Lecithin Lactose Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein .......................................................... 12.7g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.5g Peptic digest of animal tissue........................................................ 5.5g Yeast extract.................................................................................. 3.9g Pancreatic digest of heart muscle.................................................. 3.3g Cornstarch ..................................................................................... 1.1g Egg lecithin ................................................................................. 0.66g L-Cysteine·HCl·H2O...................................................................... 0.5g NaN3.............................................................................................. 0.2g Neomycin sulfate ........................................................................ 0.15g CaCl2 ........................................................................................... 0.05g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Source: This medium is available as a prepared medium from BD Diagnostic Systems.

Lecithin Lipase Anaerobic Agar Composition per liter: Pancreatic digest of casein.......................................................... 40.0g Agar ............................................................................................ 25.0g Yeast extract.................................................................................. 5.0g Na2HPO4·12H2O........................................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl.............................................................................................. 2.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Egg yolk emulsion .................................................................100.0mL pH 7.6 ± 0.2 at 25°C

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Filter sterilize. Preparation of Medium: Add components, except egg yolk emulsion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile egg yolk emulsion. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation, cultivation, and differentiation of Clostridium species based on lecithinase production and lipase production. Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen.

Lecithin Tween™ Medium (LT Medium) Composition per liter: Tween™ 80................................................................................. 30.0g Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 10.0g Peptic digest of animal tissue ..................................................... 10.0g NaCl.............................................................................................. 5.0g Lecithin ......................................................................................... 5.0g Na2S2O3·5H2O .............................................................................. 5.0g Glycerol ........................................................................................ 3.0g Histidine, free base ....................................................................... 1.0g Glucose ......................................................................................... 1.0g pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Antibiotic Solution: Composition per 10.0mL:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

5–Fluorocytosine .......................................................................... 0.2g Fosfomicin .................................................................................... 0.1g Ticarcillin...................................................................................... 0.1g

Lee's Multidifferential HiVeg Agar Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except antibiotic solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile antibiotic solution. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes.

Use: For the isolation and cultivation of multiresistant lipophilic Corynebacterium species, especially Corynebacterium group JK found primarily in infections in immunocompromised hosts and patients with prosthetic valve endocarditis.

Lee’s Agar Composition per liter: Agar ............................................................................................ 18.0g Pancreatic digest of casein .......................................................... 10.0g Yeast extract................................................................................ 10.0g Lactose .......................................................................................... 5.0g Sucrose.......................................................................................... 5.0g CaCO3 ........................................................................................... 3.0g K2HPO4 ......................................................................................... 0.5g Bromcresol Purple (0.2% solution) .........................................10.0mL pH 7.0 ± 0.2 at 25°C

941

25mL volumes. Swirl flask while dispensing to evenly suspend CaCO3. Dry plates at 30°C for 18–24 hr before use.

Use: For the isolation, cultivation, and enumeration of Lactobacillus bulgaricus from yogurt.

Lee's Multidifferential Agar Composition per liter: Tomato juice, dessicated............................................................. 20.0g Peptonized milk .......................................................................... 20.0g Glucose ....................................................................................... 10.0g Yeast extract................................................................................ 10.0g Agar ............................................................................................ 15.0g CaCO3 ........................................................................................... 5.0g Calcium pantothenate ................................................................... 2.0g Citric acid...................................................................................... 1.1g Polysorbate 80 .............................................................................. 0.5g K2HPO4......................................................................................... 0.5g KH2PO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O................................................................................ 0.01g MnSO4·7H2O .............................................................................. 0.01g NaCl............................................................................................ 0.01g Bromcresol Green..................................................................... 0.022g Cycloheximide........................................................................... 7.0mg pH 5.5 ± 0.2 at 25°C

Bromcresol Purple Solution: Composition per 10.0mL:

Source: This medium is available as a premixed powder from HiMe-

Bromcresol Purple ...................................................................... 0.02g

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-

dia.

Preparation of Bromcresol Purple Solution: Add Bromcresol

mation and inhalation.

Purple to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium: Add components, except Bromcresol Purple solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile Bromcresol Purple solution. Mix thoroughly. Pour into sterile, chilled Petri dishes in 20–25mL volumes. Swirl flask while dispensing to evenly suspend CaCO3. Dry plates at 30°C for 18–24 hr before use.

Use: For the isolation, cultivation, and enumeration of Lactobacillus bulgaricus from yogurt.

Lee's HiVeg Agar Composition per liter: Agar ............................................................................................ 18.0g Plant hydrolysate......................................................................... 10.0g Yeast extract................................................................................ 10.0g Lactose .......................................................................................... 5.0g Sucrose.......................................................................................... 5.0g CaCO3 ........................................................................................... 3.0g K2HPO4 ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.02g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Autoclave for 20 min at 15 psi pressure– 121°C. Mix thoroughly. Pour into sterile, chilled Petri dishes in 20– © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–121°C. Pour into sterile Petri plates while swirling to prevent calcium carbonate from settling. The medium will have a white precipitate of calcium carbonate.

Use: For the detection of most organisms commonly encountered in a brewery. Acid producing bacteria are identified by the development of a clear zone around the colonies. Further identification is facilitated by the characteristic color reactions.

Lee's Multidifferential HiVeg Agar Composition per liter: Tomato juice, dessicated............................................................. 20.0g Plant hydrolysate No. 3............................................................... 20.0g Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Yeast extract................................................................................ 10.0g CaCO3 ........................................................................................... 5.0g Calcium pantothenate ................................................................... 2.0g Citric acid...................................................................................... 1.1g Polysorbate 80 .............................................................................. 0.5g K2HPO4......................................................................................... 0.5g KH2PO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g FeSO4·7H2O................................................................................ 0.01g MnSO4·7H2O .............................................................................. 0.01g NaCl............................................................................................ 0.01g Bromcresol Green..................................................................... 0.022g Cycloheximide........................................................................... 7.0mg pH 6.7 ± 0.2 at 25°C

942

Legionella Agar Base

Source: This medium is available as a premixed powder from HiMedia.

Agar Phase: Composition per 500.0mL:

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Agar ............................................................................................ 17.0g Charcoal, activated ....................................................................... 2.0g

Preparation of Medium: Add components to distilled/deionized

Preparation of Agar Phase: Add components to distilled/deion-

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Pour into sterile Petri plates while swirling to prevent calcium carbonate from settling. The medium will have a white precipitate of calcium carbonate.

ized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute in 20.0mL volumes into 125.0mL serum bottles with aluminum crimp seals and rubber stoppers. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 50°C. Swirl medium to put charcoal in suspension. Allow agar to solidify so that a slant with a 6.0cm height is formed.

Legionella Agar Base (Legionella Medium) (BCYEα Agar, Modified) Composition per liter: Agar ............................................................................................ 17.0g Yeast extract................................................................................ 10.0g ACES buffer (N-2-acetamido2-aminoethane sulfonic acid) ................................................. 6.0g Charcoal, activated........................................................................ 1.5g KOH.............................................................................................. 1.5g α-Ketoglutarate............................................................................. 1.0g Legionella agar enrichment .....................................................10.0mL pH 6.85–7.0 at 25°C

Source: This medium is available as a prepared medium from BD Diagnostic Systems.

Legionella Agar Enrichment: Composition per 10.0mL: L-Cysteine·HCl·H2O...................................................................... 0.4g

Fe4(P2O7)3 ................................................................................... 0.25g

Preparation of Legionella Agar Enrichment: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Legionella agar enrichment, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50° C. Add 10.0mL of sterile Legionella agar enrichment. Adjust pH to 6.9 at 50°C by adding 4.0–4.5mL of 1.0N KOH. This is a critical step. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes. Swirl medium while pouring to keep charcoal in suspension.

Use: For the preparation of Legionella agars. For the isolation and cultivation of Legionella species from clinical and nonclinical materials.

Legionella pneumophila Medium (Charcoal Yeast Extract Diphasic Blood Culture Medium) (Diphasic Blood Culture Buffered Charcoal Yeast Extract Medium) (CYE-DBCM) Composition per liter: Agar phase .............................................................................500.0mL Broth phase ............................................................................500.0mL pH 6.9–7.0 at 25°C © 2010 by Taylor and Francis Group, LLC

Broth Phase: Composition per 500.0mL: Yeast extract................................................................................ 20.0g L-Cysteine·HCl·H2O solution........................................................ 0.4g Fe(NO3)3·9H2O solution ............................................................... 0.1g

Preparation of Broth Phase: Add yeast extract to distilled/deionized water and bring volume to 480.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile L-cysteine·HCl·H2O solution and Fe(NO3)3·9H2O solution. Mix thoroughly. Adjust pH to 6.9 with 6.0mL of sterile 1N KOH. L-Cysteine·HCl·H2O Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.04g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Fe(NO3)3·9H2O Solution: Composition per 10.0mL: Fe(NO3)3·9H2O........................................................................... 0.04g

Preparation of Fe(NO3)3·9H2O Solution: Add Fe(NO3)3·9H2O

to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add 20.0mL of sterile broth phase to 125.0mL serum bottles containing 20.0mL of solidified agar phase. Seal bottles by crimping metal caps over rubber stoppers.

Use: For the isolation and cultivation of Legionella pneumophila from blood cultures.

Legionella Selective Agar Composition per liter: Agar ............................................................................................ 15.0g ACES (2-[(2-amino-2-oxoethyl)-amino]ethane sulfonic acid) buffer ......................................................... 10.0g Yeast extract................................................................................ 10.0g Charcoal, activated ....................................................................... 2.0g α-Ketoglutarate............................................................................. 1.0g L-Cysteine·HCl·H2O solution...................................................10.0mL Fe4(P2O7)3 solution ..................................................................10.0mL Antibiotic solution ...................................................................10.0mL pH 6.85–7.0 at 25°C

Source: This medium is available as a prepared medium from BD Diagnostic Systems. L-Cysteine·HCl·H2O Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O...................................................................... 0.4g

Leishmania Medium Preparation of L-Cysteine·HCl·H2O Solution: Add L-cyste-

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

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Synthetic detergent No. III ........................................................... 3.0g Ferric citrate.................................................................................. 1.0g Neutral Red................................................................................. 0.02g pH 7.5 ± 0.2 at 25°C

Fe4(P2O7)3 Solution: Composition per 10.0mL:

Source: This medium is available as a premixed powder from HiMe-

Fe4(P2O7)3 ................................................................................... 0.25g

dia.

Preparation of Fe4(P2O7)3 Solution: Add Fe4(P2O7)3 to distilled/

Preparation of Medium: Add components to distilled/deionized

Antibiotic Solution: Composition per 10.0mL:

Use: For the isolation of Salmonella and Shigella species.

Anisomycin .............................................................................. 10.0mg Colistin..................................................................................... 3.75mg Vancomycin ............................................................................... 2.0mg

Composition per liter:

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components—except L-cysteine·HCl·H2O, Fe4(P2O7)3, and antibiotic solutions—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile L-cysteine·HCl·H2O, Fe4(P2O7)3, and antibiotic solutions. Mix thoroughly. Pour into sterile Petri dishes. Swirl medium while pouring to keep charcoal in suspension. Use: Legionella selective agar is used in qualitative procedures for the isolation of Legionella species from clinical and nonclinical specimens.

Legume Extract Agar Composition per liter: Alfalfa roots ................................................................................ 35.0g Agar ............................................................................................ 20.0g Soybean meal .............................................................................. 10.0g Sucrose........................................................................................ 10.0g CaCO3 ........................................................................................... 5.0g Glucose ......................................................................................... 5.0g K2HPO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2 ............................................................................................. 0.1g NaCl .............................................................................................. 0.1g FeCl3 .......................................................................................... 1.0mg

Preparation of Medium: Wash the alfalfa roots well and cut them up. Add 10.0g of soybean meal. Add three times the volume of distilled/deionized water. Steam for 1 hr. Let stand at 25°C overnight. Bring volume to 1.0L with distilled/deionized water. Filter through paper pulp. To the filtrate, add the K2HPO4, CaCl2, MgSO4·7H2O, NaCl, FeCl3, and agar. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Add the CaCO3, sucrose, and glucose. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 20 min at 10 psi pressure– 115°C.

Use: For the cultivation of Rhizobium species.

Leifson HiVeg Agar Composition per liter: Agar ............................................................................................ 12.0g Lactose ........................................................................................ 10.0g Plant extract No. 1 ........................................................................ 6.5g Na2S2O3 ........................................................................................ 5.4g Plant peptone No. 1....................................................................... 5.0g © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Gently bring to boiling. Do not autoclave. Pour into sterile Petri plates.

Leifson Medium Agar ............................................................................................ 15.0g Pancreatic digest of casein............................................................ 2.0g MgCl2............................................................................................ 1.0g Yeast extract.................................................................................. 1.0g pH 8.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 8.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the direct isolation and routine culturing of Hyphomonas species.

Leifson's Deoxycholate HiVeg Agar, Modified (Hugh Leifson Deoxycholate HiVeg Agar, Modified) Composition per liter: Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Sodium citrate............................................................................... 5.0g Na2S2O3 ........................................................................................ 5.0g Synthetic detergent No. III ........................................................... 2.5g Ferric citrate.................................................................................. 1.0g Neutral Red............................................................................... 0.025g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Gently bring to boiling. Do not autoclave. Pour into sterile Petri plates.

Use: For the selective isolation and differentiation of Salmonella and Shigella species.

Leishmania Medium Composition per 100.0mL: Sodium citrate............................................................................... 1.2g NaCl.............................................................................................. 1.0g Rabbit blood solution...............................................................10.0mL

Rabbit Blood Solution: Composition per 10.0mL: Rabbit blood, defibrinated .........................................................5.0mL

944

Leonian’s Agar

Preparation of Rabbit Blood Solution: Add 5.0mL of whole rabbit blood to 5.0mL of sterile distilled/deionized water. Freeze and thaw twice to lyse blood cells.

Preparation of Medium: Add components, except rabbit blood solution, to distilled/deionized water and bring volume to 90.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile rabbit blood solution. Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks.

Use: For the cultivation of Leishmania donovani, Leishmania hertigi, and Leishmania tropica.

LEMB Agar See: Levine EMB Agar Lenox Broth See: LB Medium

Leonian’s Agar Composition per liter: Agar ............................................................................................ 20.0g Maltose........................................................................................ 6.25g Malt extract ................................................................................. 6.25g KH2PO4 ....................................................................................... 1.25g Peptone...................................................................................... 0.625g MgSO4·7H2O ............................................................................ 0.625g

Use: For the cultivation of Leptospira species.

Leptospira HiVeg Medium Base, Korthof, Modified with Rabbit Serum and Hemoglobin Composition per liter: NaCl.............................................................................................. 1.4g Na2HPO4 ..................................................................................... 0.88g Plant peptone ................................................................................ 0.8g KH2PO4....................................................................................... 0.24g CaCl2 ........................................................................................... 0.04g KCl.............................................................................................. 0.04g NaHCO3...................................................................................... 0.02g Rabbit serum, heat inactivated at 56°C ...................................80.0mL Hemoglobin solution .................................................................8.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without rabbit serum, is available as a premixed powder from HiMedia.

Hemoglobin Solution: Composition per 50.0mL: Bovine hemoglobin....................................................................... 1.0g

Preparation of Hemoglobin Solution: Add bovine hemoglobin to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Preparation of Medium: Add components, except rabbit serum,

and hemoglobin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Cool to 50°–56°C. Aseptically add 80.0mL of rabbit serum and 8.0mL of hemoglobin solution. Mix thoroughly. Aseptically dispense into tubes.

Use: For the cultivation and maintenance of Amorphotheca resinae, Apio-

Use: For the cultivation of Leptospira species.

Preparation of Medium: Add components to distilled/deionized

sordaria rotula, Arachnotheca glomerata, Ascotricha erinacea, Auxarthron pseudauxarthron, Coniochaeta extramundana, Coniochaetidium ostreum, Coprinus velox, Cylindrocladium couratariae, Dicranidion fragile, Dicranidion gracilis, Eupenicillium brefeldianum, Isaria sulfurea, Linderina macrospora, Microthecium retisporum, Nigrospora sacchari, Orbicula parietina, Pectinotrichum llanense, Penicillium ochrochloron, Penicillium pinophilum, Pseudogymnoascus roseus, Thielavia terricola, Triangularia batistae, Tripospermum myrti, and Zopfiella pleuropora.

Leptospira HiVeg Medium Base, Korthof, Modified with Rabbit Serum Composition per liter: NaCl .............................................................................................. 1.4g Na2HPO4 ..................................................................................... 0.88g Plant peptone................................................................................. 0.8g KH2PO4 ....................................................................................... 0.24g CaCl2 ........................................................................................... 0.04g KCl.............................................................................................. 0.04g NaHCO3 ...................................................................................... 0.02g Rabbit serum, heat inactivated at 56°C..................................100.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without rabbit serum, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components, except rabbit serum, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Cool to 50°–56°C. Aseptically add 100.0mL of rabbit serum. Mix thoroughly. © 2010 by Taylor and Francis Group, LLC

Leptospira Medium Composition per liter: (NH4)2Fe(SO4)2·6H2O .................................................................. 6.0g NaH2PO4 ..................................................................................... 0.53g L-Asparagine ................................................................................. 0.5g Glycerol ........................................................................................ 0.2g Tween™ 60................................................................................... 0.2g MgSO4·7H2O .............................................................................. 0.15g KH2PO4..................................................................................... 0.069g Tween™ 80................................................................................. 0.05g EDTA .......................................................................................... 0.01g CaCO3 ........................................................................................ 4.0mg Thiamine·HCl ............................................................................ 1.0mg Vitamin B12 ................................................................................. 1.0μg pH 7.4–7.6 at 25°C

Preparation of Medium: Add components, except thiamine·HCl, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 1.0mg of thiamine·HCl. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Leptospira species.

Leptospira Medium (DSMZ Medium 1113) Composition per liter: Agarose ......................................................................................... 1.5g Na2HPO4 ....................................................................................... 1.0g

Leptospira Protein-Free Medium

NaCl .............................................................................................. 1.0g KH2PO4 ......................................................................................... 0.3g NH4Cl ......................................................................................... 0.25g Thiamine .................................................................................... 5.0mg Leptospira enrichment EMJH (a solution of albumin, polysorbate 80 and additional growth factors available from BD Diagnostics .......................................100.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add agarose to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Boil with mixing until agarose dissolves. Add the other components, except Leptospira enrichment EMJH. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Mix thoroughly. Warm the Leptospira enrichment to approximately 25°C and add to the warm medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Leptospira spp.

Leptospira Medium, EMJH (Leptospira Medium, Ellinghausen-McCullough/ Johnson-Harris) Composition per liter: Na2HPO4 ....................................................................................... 1.0g NaCl .............................................................................................. 1.0g KH2PO4 ......................................................................................... 0.3g NH4Cl ......................................................................................... 0.25g Thiamine .................................................................................... 5.0mg Rabbit serum ..........................................................................100.0mL pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components, except rabbit serum, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile rabbit serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Leptospira species.

Leptospira Medium, Modified

945

to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 2.5mL of sterile hemin solution and 100.0mL of sterile rabbit serum. Mix thoroughly. The pH of the medium should be 7.3. Store at 4°C for 24 hr. Inactivate medium at 56°C for 60 min. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Leptospira biflexa, Leptospira borgpetersenii, Leptospira interrogans, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, and Leptospira weili.

Leptospira Protein-Free Medium (Leptospira PF Medium) Composition per liter: TES (N-Tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid) buffer ...................................................................................... 1.2g NaCl.............................................................................................. 0.9g Sodium pyruvate........................................................................... 0.2g CT-Tween™ 60........................................................................12.0mL CT-Tween™ 40..........................................................................3.0mL MgCl2-CaCl2 solution................................................................1.0mL Cyanocobalamin (0.02% solution) ............................................1.0mL Glycerol (10% solution) ............................................................1.0mL KH2PO4 (1% solution)...............................................................1.0mL MnSO4·H2O (0.1% solution) .....................................................1.0mL ZnSO4 (0.4% solution) ..............................................................0.1mL pH 7.6 ± 0.2 at 25°C

CT-Tween™ 60: Composition per 200.0mL: Charcoal, Norit A........................................................................ 40.0g Tween™ 60................................................................................. 20.0g

Preparation of CT-Tween™ 60: Add Tween™ 60 to 200.0mL of distilled/deionized water. Mix thoroughly. While stirring, add charcoal. Stir mixture for 18 hr at 25°C. Allow charcoal to settle out of suspension for 18 hr at 4°C. Carefully decant the Tween™ solution off the sediment. Centrifuge the Tween™ solution at 10,000 × g for 1 hr. Decant supernatant solution. Pass Tween™ solution through a thin-channel ultrafiltration XM 100 membrane. Store stock solution at −20°C.

CT-Tween™ 40: Composition per 200.0mL:

Composition per liter:

Charcoal, Norit A........................................................................ 40.0g Tween™ 40................................................................................. 20.0g

Agar .............................................................................................. 1.5g NaCl .............................................................................................. 0.5g Peptone.......................................................................................... 0.3g Beef extract ................................................................................... 0.2g Hemin solution...........................................................................2.5mL Sterile rabbit serum ................................................................100.0mL pH 7.3 ± 0.1 at 25°C

Preparation of CT-Tween™ 40: Add Tween™ 40 to 200.0mL of distilled/deionized water. Mix thoroughly. While stirring, add charcoal. Stir mixture for 18 hr at 25°C. Allow charcoal to settle out of suspension for 18 hr at 4°C. Carefully decant the Tween™ solution off the sediment. Centrifuge the Tween™ solution at 10,000 × g for 1 hr. Decant supernatant solution. Pass Tween™ solution through a thin-channel ultrafiltration XM 100 membrane. Store stock solution at −20°C.

Hemin Solution: Composition per 100.0mL:

MgCl2-CaCl2 Solution: Composition per 100.0mL:

Hemin.......................................................................................... 0.05g NaOH (1N solution)...................................................................1.0mL

Preparation of Hemin Solution: Add hemin to 1.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Medium: Add components, except hemin solution and rabbit serum, to distilled/deionized water and bring volume to 897.5mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH © 2010 by Taylor and Francis Group, LLC

CaCl2·2H2O .................................................................................. 1.5g MgCl2·6H2O ................................................................................. 1.5g

Preparation of MgCl2-CaCl2 Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Leptospira species.

946

Leptospirillum ferrooxidans Medium

Leptospirillum ferrooxidans Medium Composition per liter: FeSO4·7H2O................................................................................ 30.0g CaCl2·2H2O............................................................................... 0.147g (NH4)2SO4 ................................................................................... 0.13g KH2PO4 .................................................................................... 27.0mg MgCl2·6H2O............................................................................. 25.0mg Trace elements solution .............................................................1.0mL pH 2.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: CoCl2·6H2O ................................................................................ 0.12g MnCl2·4H2O.................................................................................. 0.1g Na2MoO4·2H2O ....................................................................... 85.2mg ZnCl2 ........................................................................................ 70.0mg H3BO3 ...................................................................................... 31.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add H2SO4 to 900.0mL of distilled/de-

ionized water and bring pH to 3.0. Add components. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust pH to 2.0 with H2SO4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Leptospirillum ferrooxidans.

Leptospirillum HH Medium (DSMZ Medium 882) Composition per 1001.0mL: Solution A ..............................................................................950.0mL Soultion B ................................................................................50.0mL Trace elements solution .............................................................1.0mL pH 1.8 ± 0.2 at 25°C

Preparation of Medium: Aseptically mix 950.0mL of solution A and 50.0mL solution B. Mix thoroughly. Aseptically add 1.0mL trace elements solution. Mix thoroughly. Adjust pH to 1.8.

Use: For the cultivation of Acidithiobacillus ferrooxidans=Thiobacillus ferrooxidans and Leptospirillum spp.

Leptothrix ochracea Medium Composition per liter: Agar ............................................................................................ 10.0g Manganous acetate........................................................................ 0.1g Manganese bicarbonate solution............................................100.0mL pH 7.0 ± 0.2 at 25°C

Manganese Bicarbonate Solution: Composition per 100.0mL: MnCO3 .......................................................................................... 2.0g

Preparation of Manganese Bicarbonate Solution: Add MnCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Gas with 100% CO2 for 20 min. Filter through Whatman #1 filter paper. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Leptothrix ochracea.

Leptothrix 2X PYG Medium Composition per liter:

CaCl2·2H2O............................................................................ 147.0mg (NH4)2SO4 .............................................................................. 132.0mg MgCl2·6H2O............................................................................. 53.0mg KH2PO4 .................................................................................... 27.0mg

HEPES (N-2-Hhydroxyethyl piperazine-N´2-ethanesulfonic acid) buffer............................................ 3.57g MgSO4·7H2O ................................................................................ 0.6g Glucose ......................................................................................... 0.5g Peptone ......................................................................................... 0.5g Yeast extract.................................................................................. 0.5g CaCl2·2H2O ................................................................................ 0.07g MnSO4·H2O .............................................................................. 0.017g pH 7.3 ± 0.2 at 25°C

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Adjust pH to 1.8 with 10N H2SO4. Autoclave for 30 min at 112°C. Cool to room temperature.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Solution B:

Use: For the cultivation and maintenance of Leptothrix discophora.

Solution A:

FeSO4·7H2O................................................................................ 20.0g H2SO4, 0.25N ...........................................................................50.0mL

Preparation of Solution B: Add FeSO4·7H2O to 50.0mL 0.25N H2SO4. Mix thoroughly. The pH should be 1.2. Autoclave for 30 min at 112°C. Cool to room temperature. Trace Elements Solution: ZnCl2 ........................................................................................ 68.0mg CuCl2·2H2O ............................................................................. 67.0mg CoCl2·6H2O ............................................................................. 64.0mg MnCl2·2H2O............................................................................. 62.0mg H3BO3 ...................................................................................... 31.0mg Na2MoO4.................................................................................. 10.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 1.8 with 10N H2SO4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. © 2010 by Taylor and Francis Group, LLC

Leptothrix Strains Medium Composition per liter: Agar .............................................................................................. 7.5g MnCO2 .......................................................................................... 2.0g Beef extract................................................................................... 1.0g Fe(NH4)2(SO4)2 .......................................................................... 0.15g Sodium citrate............................................................................. 0.15g Yeast extract.............................................................................. 0.075g Vitamin B12 ................................................................................. 5.0μg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Leptothrix cholodnii, Leptothrix discophora, and Sphaerotilus natans.

Letheen Agar, Modified

Leptothrix Strains Medium Composition per liter: Agar ............................................................................................ 12.0g Peptone.......................................................................................... 5.0g MgSO4·7H2O ................................................................................ 0.2g Ferric ammonium citrate............................................................. 0.15g CaCl2 ........................................................................................... 0.05g FeCl3·6H2O ................................................................................. 0.01g MnSO4·H2O ................................................................................ 0.01g pH 7.0 ± 0.2 at 25°C

Use: For the isolation and cultivation of Leptothrix species.

Leptotrichia buccalis Medium Composition per liter: Agar ............................................................................................ 15.0g Nutrient broth................................................................................ 8.0g Yeast extract.................................................................................. 2.0g Glucose solution ......................................................................10.0mL L-Cysteine solution...................................................................10.0mL Hemin solution...........................................................................4.0mL pH 7.2–7.6 at 25°C

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 2.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. L-Cysteine

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O...................................................................... 1.0g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Hemin Solution: Composition per 10.0mL: Hemin......................................................................................... 2.5mg Triethanolamine (50% solution) ..............................................10.0mL

Preparation of Hemin Solution: Add hemin to 10.0mL of triethanolamine solution. Mix thoroughly.

Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile glucose solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Leptotrichia buccalis.

Leptotrichia Medium Composition per liter: Pancreatic digest of casein .......................................................... 10.0g NaCl .............................................................................................. 5.0g Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g © 2010 by Taylor and Francis Group, LLC

947

Na2HPO4 ....................................................................................... 2.5g L-Cysteine·HCl·H2O ..................................................................... 0.5g Horse serum ...........................................................................100.0mL Tomato decoction.....................................................................50.0mL pH 7.2–7.4 at 25°C

Tomato Decoction: Composition per 100.0mL: Tomatoes..................................................................................50.0mL

Preparation of Tomato Decoction: Mince fresh tomatoes and measure 50.0mL. Add 50.0mL of tap water. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 10 min. Filter through Whatman #1 filter paper. Autoclave filtrate for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except horse serum and tomato decoction, to distilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2–7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile horse serum and tomato decoction. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Leptotrichia buccalis. LES Endo Agar See: Endo Agar, LES

Letheen Agar Composition per liter: Agar ............................................................................................ 15.0g Tween™ 80................................................................................... 7.0g Pancreatic digest of casein............................................................ 5.0g Beef extract................................................................................... 3.0g Glucose ......................................................................................... 1.0g Lecithin ......................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For determination of the antimicrobial activity of quaternary ammonium compounds.

Letheen Agar, Modified Composition per liter: Agar ............................................................................................ 20.0g Thiotone...................................................................................... 10.0g Pancreatic digest of casein.......................................................... 10.0g Tween™ 80................................................................................... 7.0g NaCl............................................................................................. 5.0g Beef extract................................................................................... 3.0g Yeast extract.................................................................................. 2.0g Glucose ......................................................................................... 1.0g Lecithin ......................................................................................... 1.0g NaHSO3 ........................................................................................ 0.1g pH 7.2 ± 0.2 at 25°C

948

Letheen Broth

Use: For determination of the antimicrobial activity of quaternary ammonium compounds.

Letheen HiVeg Agar, Modified Composition per liter:

Peptic digest of animal tissue...................................................... 10.0g Beef extract ................................................................................... 5.0g NaCl .............................................................................................. 5.0g Tween™ 80 ................................................................................... 5.0g Lecithin ......................................................................................... 0.7g pH 7.0 ± 0.2 at 25°C

Agar ............................................................................................ 20.0g Plant peptone .............................................................................. 20.0g Plant extract .................................................................................. 5.0g Plant hydrolysate .......................................................................... 5.0g NaCl.............................................................................................. 5.0g Polysorbate 80 .............................................................................. 5.0g Yeast extract.................................................................................. 2.0g Lecithin ......................................................................................... 0.7g NaHSO3 ........................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Source: This medium is available as a premixed powder from HiMe-

Letheen Broth Composition per liter:

agnostic Systems.

dia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For determination of the antimicrobial activity of quaternary ammonium compounds.

Use: For the screening of cosmetic products for microbial contamination.

Letheen Broth, Modified Composition per liter:

Letheen HiVeg Broth, AOAC

Peptic digest of animal tissue...................................................... 10.0g Thiotone peptone ........................................................................ 10.0g Beef extract ................................................................................... 5.0g NaCl .............................................................................................. 5.0g Tween™ 80 ................................................................................... 5.0g Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 2.0g Lecithin ......................................................................................... 0.7g NaHSO3 ........................................................................................ 0.1g pH 7.2 ± 0.2 at 25°C

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Plant peptone .............................................................................. 10.0g Plant extract .................................................................................. 5.0g Polysorbate 80 .............................................................................. 5.0g NaCl.............................................................................................. 5.0g Lecithin ......................................................................................... 0.7g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped bottles in 90.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the determination of the antimicrobial activity of quaternary

Use: For determination of the antimicrobial activity of quaternary

ammonium compounds.

Letheen HiVeg Agar

Letheen HiVeg Broth, Modified Composition per liter:

Agar ............................................................................................ 15.0g Polysorbate 80............................................................................... 7.0g Plant hydrolysate........................................................................... 5.0g Plant extract .................................................................................. 3.0g Glucose ......................................................................................... 1.0g Lecithin ......................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Plant peptone .............................................................................. 20.0g Plant extract .................................................................................. 5.0g Plant hydrolysate .......................................................................... 5.0g NaCl.............................................................................................. 5.0g Polysorbate 80 .............................................................................. 5.0g Yeast extract.................................................................................. 2.0g Lecithin ......................................................................................... 0.7g NaHSO3 ........................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMe-

Composition per liter:

dia.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the determination of the antimicrobial activity of quaternary

Use: For the screening of cosmetic products for microbial contamina-

ammonium compounds.

tion.

Leucothrix Medium

Leuconostoc Medium

949

Leuconostoc oenos Medium

Composition per liter:

CaCO3 ......................................................................................... 50.0g Malt extract ................................................................................. 50.0g Agar ............................................................................................ 15.0g NaCl .............................................................................................. 2.5g Beef extract ................................................................................... 1.0g Polypeptone™............................................................................... 1.0g

Glucose ....................................................................................... 10.0g Tryptone...................................................................................... 10.0g Fructose......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Diammonium citrate ..................................................................... 3.5g L-Cysteine·HCl ............................................................................. 0.5g So4Mg·7H2O................................................................................. 0.2g MnSO4·H2O................................................................................ 0.05g Tomato juice, filtered.............................................................100.0mL Tween™ 80................................................................................1.0mL pH 4.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Leuconostoc mesenteroides.

Leuconostoc oenos Medium Composition per liter: Tryptic digest of casein ............................................................... 10.0g Glucose ....................................................................................... 10.0g Fructose......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Diammonium citrate ..................................................................... 3.5g L-Cysteine·HCl.............................................................................. 0.5g MgSO4·7H2O ......................................................................... 200.0mg MnSO4·H2O ............................................................................. 50.0mg Tomato juice, filtered .............................................................100.0mL Tween™ 80 ................................................................................1.0mL pH 4.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 4.8. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Leuconostoc oenos.

Leuconostoc oenos Medium Composition per liter: Glucose ....................................................................................... 10.0g Peptone........................................................................................ 10.0g Yeast extract.................................................................................. 5.0g MnSO4·4H2O ................................................................................ 0.1g Tomato juice...........................................................................250.0mL L-Cysteine·HCl solution...........................................................10.0mL pH 4.8 ± 0.2 at 25°C L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.5g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except L-cysteine·HCl solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile Lcysteine·HCl solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Leucothrix Medium Composition per liter: Pancreatic digest of casein.......................................................... 10.0g Synthetic seawater ...............................................................1000.0mL pH 7.8 ± 0.2 at 25°C

Synthetic Seawater: Composition per liter: NaCl............................................................................................ 24.0g MgCl2·6H2O ............................................................................... 11.0g Na2SO4 .......................................................................................... 4.0g CaCl2·6H2O .................................................................................. 2.0g KCl................................................................................................ 0.7g KBr ............................................................................................... 0.1g SrCl2·6H2O ................................................................................. 0.04g H3BO3 ......................................................................................... 0.03g NaSiO3·9H2O............................................................................ 5.0mg NaF ............................................................................................ 3.0mg NH4NO3 ..................................................................................... 2.0mg FePO4·4H2O.............................................................................. 1.0mg

Preparation of Synthetic Seawater: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add 10.0g of pancreatic digest of casein to 1.0L of synthetic seawater. Mix thoroughly. Adjust pH to 7.8. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Leucothrix species.

Leucothrix Medium (ATCC Medium 429) Composition per liter: NaCl............................................................................................ 11.7g Monosodium glutamate .............................................................. 10.0g MgCl2·6H2O ............................................................................... 5.35g Na2SO4 .......................................................................................... 2.0g CaCl2·2H2O ................................................................................ 0.75g Tris(hydroxymethyl)aminomethane buffer................................... 0.5g KCl.............................................................................................. 0.35g Na2HPO4 ..................................................................................... 0.05g pH 7.6 ± 0.2 at 25°C

950

Leucothrix Medium

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Leucothrix mucor.

Leucothrix Medium (ATCC Medium 430) Composition per liter: NaCl ............................................................................................ 11.7g MgCl2·6H2O................................................................................ 5.35g Na2SO4 .......................................................................................... 2.0g CaCl2·2H2O................................................................................. 0.75g Pancreatic digest of casein ............................................................ 0.5g Yeast extract.................................................................................. 0.5g Tris(hydroxymethyl)aminomethane buffer................................... 0.5g KCl.............................................................................................. 0.35g Na2HPO4 ..................................................................................... 0.05g pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation, cultivation, and differentiation of Gram-negative enteric bacteria based on lactose fermentation. Bacteria that ferment lactose, especially the coliform bacterium Escherichia coli, appear as colonies with a green metallic sheen or blue-black to brown color. Bacteria that do not ferment lactose appear as colorless or transparent light purple colonies.

Levinthal’s Agar Composition per 105.0mL: Nutrient agar, sterile ..............................................................100.0mL Rabbit blood or human blood, sterile ........................................5.0mL pH 6.8 ± 0.2 at 25°C

Nutrient Agar: Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of gelatin........................................................... 5.0g Beef extract................................................................................... 3.0g

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Source: Nutrient agar is available as a premixed powder from BD Di-

Use: For the cultivation and maintenance of Leucothrix mucor.

Preparation of Nutrient Agar: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Leucothrix mucor Medium Composition per liter: NaCl .......................................................................................... 11.75g Monosodium glutamate .............................................................. 10.0g MgCl2·6H2O................................................................................ 5.35g Na2SO4 .......................................................................................... 2.0g Sodium lactate............................................................................... 2.0g CaCl2·6H2O................................................................................. 1.12g Tris (hydroxymethyl)aminomethane buffer.................................. 0.5g KCl.............................................................................................. 0.35g Na2HPO4 ..................................................................................... 0.05g pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Leucothrix mucor.

Levine EMB Agar (Levine Eosin Methylene Blue Agar) (Eosin Methylene Blue Agar, Levine) (LEMB Agar) Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g Peptone........................................................................................ 10.0g K2HPO4 ......................................................................................... 2.0g Eosin Y ......................................................................................... 0.4g Methylene Blue...................................................................... 0.065mg pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

agnostic Systems.

Preparation of Medium: To 100.0mL of cooled, sterile nutrient agar, aseptically add 5.0mL of human blood or rabbit blood. Mix thoroughly. Heat in a boiling water bath for 5 min. Allow the deposit to settle out of suspension. Pour the clear supernatant solution into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Haemophilus species.

Levinthal’s HiVeg Medium Base with Blood Composition per liter: Agar ............................................................................................ 20.0g Plant extract ................................................................................ 10.0g Plant peptone .............................................................................. 10.0g NaCl.............................................................................................. 5.0g Rabbit blood or human blood, sterile ......................................50.0mL pH 7.6 ± 0.2 at 25°C

Source: This medium, without blood, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components, except blood, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 50.0mL of human blood or rabbit blood. Mix thoroughly. Heat in a boiling water bath for 5 min. Allow the deposit to settle out of suspension. Pour the clear supernatant solution into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Haemophilus species.

Levinthal’s Medium Base Composition per liter: Agar ............................................................................................ 20.0g Peptic digest of animal tissue ..................................................... 10.0g Beef extract................................................................................. 10.0g

LICNR Broth

951

Source: This medium is available from HiMedia.

Yeast extract or yeast autolysate ................................................... 0.1g Pancreatic digest of casein.......................................................... 0.06g NaCl............................................................................................ 0.02g Papaic digest of soybean meal.................................................... 0.01g

Preparation of Medium: Add components, except blood, to dis-

Preparation of Solution A: Add components to distilled/deionized

NaCl .............................................................................................. 5.0g Rabbit or human blood ............................................................50.0mL pH 7.6 ± 0.2 at 25°C

tilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add blood. Mix thoroughly. Heat in boiling water bath. Allow the deposits to settle. Distribute clear supernatant into sterile tubes.

Use: For the cultivation of Haemophilus spp. LGI Medium See: Azospirillum amazonense Medium

water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 2.5–3.0 with 1N H2SO4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Solution B: Composition per 500.0mL: Agar ............................................................................................ 12.0g Glucose ......................................................................................... 1.0g

Composition per liter:

Solution A ..............................................................................500.0mL Solution B ..............................................................................500.0mL pH 2.5–3.0 at 25°C

Preparation of Medium: Aseptically combine sterile solution A and sterile solution B. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Solution A: Composition per 500.0mL:

Use: For the cultivation and maintenance of Acidiphilium angustum,

LHET2 Medium

(NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ....................................................................................... 0.51g MgSO4·7H2O ................................................................................ 0.5g KCl................................................................................................ 0.1g Pancreatic digest of casein .......................................................... 0.06g NaCl ............................................................................................ 0.02g Papaic digest of soybean meal .................................................... 0.01g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 2.5–3.0 with 1N H2SO4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Preparation of Medium: Aseptically combine sterile solution A and sterile solution B. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Acidiphilium cryptum.

LHET2 Medium with Yeast Extract or Yeast Autolysate Composition per liter: Solution A ..............................................................................500.0mL Solution B ..............................................................................500.0mL pH 2.5–3.0 at 25°C

Solution A: Composition per 500.0mL: (NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ....................................................................................... 0.51g MgSO4·7H2O ................................................................................ 0.5g KCl................................................................................................ 0.1g © 2010 by Taylor and Francis Group, LLC

Acidiphilium facilis, and Acidiphilium rubrum.

Lichen Fungi Medium Composition per liter: Agar ............................................................................................ 20.0g Malt extract................................................................................. 20.0g Yeast extract.................................................................................. 2.0g

Use: For the cultivation of Acarospora fuscata, Acarospora smaragdula, Anaptychia cilaris, Anthracothecium albescens, Arthonia cinnabarina, Bacidia incompta, Baeomyces roseus, Buellia stillingiana, Caloplaca aurantiaca, Candelariella vitellina, Cetraria islandica, numerous Cladonia species, Dermatocarpon fluviatile, Graphis tenella, Lecanora cinerea, Lecanora dispersa, Lecanora rubina, Lecidea species, Microthelia albidella, Opegrapha lichenoides, Parmelia centrifuga, Parmelia conspersa, Phisica millegrana, Phisica stellaris, Porina sandwichensis, Pyrenula nitida, Ramalina americana, Sarcogyne simplex, Stereocaulon vulcani, Umbilicaria papulosa, Usnea florida, Xanthoria parietina, and other fungi from lichen symbiotic relationships.

LICNR Broth (Lysine Iron Cystine Neutral Red Broth) Composition per 500.0mL: L-Lysine·HCl ............................................................................... 10.0g

Mannitol........................................................................................ 5.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Salicin ........................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g L-Cystine ....................................................................................... 0.1g Na2S2O3 ........................................................................................ 0.1g Neutral Red............................................................................... 0.025g Novobiocin solution.................................................................10.0mL pH 6.2 ± 0.2 at 25°C

952

Lima Bean Agar

Novobiocin Solution: Composition per 10.0mL:

lium violaceum, Pythium anandrum, Pythium irregulare, Pythium vexans, Scopulariopsis fimicola, and Sphaerostilbe repens.

Novobiocin................................................................................ 0.015g

Preparation of Novobiocin Solution: Add novobiocin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except novobiocin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 2–3 min. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 0.1mL of sterile novobiocin solution to each tube. Mix thoroughly.

Use: For the rapid, presumptive detection of Salmonella in foods, food ingredients, and feed materials.

Lima Bean Agar (ATCC Medium 322) Composition per liter:

Limnobacter Medium (DSMZ Medium 919) Composition per liter: Yeast extract.................................................................................. 0.5g Proteose peptone........................................................................... 0.5g Casamino acids ............................................................................. 0.5g Glucose ......................................................................................... 0.5g Soluble starch................................................................................ 0.5g Sodium pyruvate........................................................................... 0.3g K2HPO4......................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.05g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Lima beans, infusion from 62.5g .................................................. 8.0g Agar ............................................................................................ 15.0g pH 5.6 ± 0.2 at 25°C

Use: For the cultivation of Limnobacter thiooxidans.

Source: This medium is available as a premixed powder from BD Di-

Composition per liter:

agnostic Systems.

Yeast extract.................................................................................. 5.0g γ-Hexachlorocyclohexane (Lindane) ............................................ 0.1g pH 7.0 ± 0.2 at 25°C

Lindane Medium

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of a variety of phytopathological fungi and

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

other fungi.

Use: For the cultivation of Clostridium sphenoides.

Lima Bean Agar Composition per liter: Lima beans, solids from infusion................................................ 62.5g Agar ............................................................................................ 15.0g pH 5.6 ± 0.2 at 25°C

Use: For the cultivation of a variety of phytopathological fungi and other fungi.

Lima Bean Agar Composition per liter: Lima beans, frozen.................................................................... 250.0g Agar .............................................................................................. 5.0g pH 6.3 ± 0.3 at 25°C

Preparation of Medium: Add lima beans to distilled/deionized water and bring volume to 1.0L. Blend for 10 min. Add agar. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of numerous Colletotrichum species, Coniothyrium fuckelii, Diheterospora chlamydosporia, Glomerella cingulata, Graphium fragrans, Monochaetia mali, Penicillium crustosum, Phleospora idahoensis, Phoma eupyrena, Phoma lingam, Phyllosticta sojaecola, numerous Phytophthora species, Polysphondy© 2010 by Taylor and Francis Group, LLC

Lineola Agar Composition per liter: Mannitol...................................................................................... 25.0g Agar ............................................................................................ 15.0g Yeast extract.................................................................................. 5.0g Peptone ......................................................................................... 3.0g pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Bacillus macroides and other Bacillus species.

Lipovitellin Salt Mannitol Agar Composition per liter: NaCl............................................................................................ 75.0g Egg yolk...................................................................................... 20.0g Agar ............................................................................................ 15.0g D-Mannitol .................................................................................. 10.0g Polypeptone™ ............................................................................ 10.0g Beef extract................................................................................... 1.0g Phenol Red................................................................................ 0.025g

Listeria Enrichment Broth I, USDA FSIS Use: For the detection of Staphylococcus aureus in swimming pool water based on lipovitellin-lipase activity and mannitol fermentation. Staphylococcus aureus and other bacteria with lipovitellin-lipase activity attack the egg yolk and appear as colonies surrounded by an opaque zone. Bacteria that ferment mannitol appear as colonies surrounded by a yellow zone.

Liquoid Broth Composition per liter: Beef heart, infusion from .......................................................... 250.0g Calf brain, infusion from .......................................................... 200.0g Proteose peptone ......................................................................... 10.0g NaCl .............................................................................................. 5.0g Na2HPO4 ....................................................................................... 2.5g Glucose ......................................................................................... 2.0g Sodium polyanethol sulfonate ...................................................... 0.5g pH 7.4 ± 0.2 at 25°C

Use: For the screening of blood specimens from suspected cases of bacteremia.

953

Soybean Casein Digest Broth Yeast Extract: Composition per liter: Pancreatic digest of casein.......................................................... 17.0g Yeast extract.................................................................................. 6.0g NaCl.............................................................................................. 5.0g Papaic digest of soybean meal...................................................... 3.0g K2HPO4......................................................................................... 2.5g Glucose ......................................................................................... 2.5g

Source: This medium is available as a premixed powder from BD Diagnostic Systems. Preparation of Soybean Casein Digest Broth Yeast Extract: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Nalidixic Acid Solution: Composition per 100.0mL: Nalidixic acid................................................................................ 0.5g

Preparation of Nalidixic Acid Solution: Add nalidixic acid to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Cycloheximide Solution: Composition per 100.0mL: Cycloheximide.............................................................................. 1.5g Ethanol.....................................................................................40.0mL

Preparation of Cycloheximide Solution: Add cycloheximide to

Listeria Enrichment Broth Composition per liter: Pancreatic digest of casein .......................................................... 17.0g Yeast extract.................................................................................. 6.0g NaCl .............................................................................................. 5.0g Papaic digest of soybean meal ...................................................... 3.0g Glucose ......................................................................................... 2.5g K2HPO4 ......................................................................................... 2.5g Cycloheximide ............................................................................ 0.05g Nalidixic acid .............................................................................. 0.04g Acriflavine·HCl......................................................................... 0.015g pH 7.3 ± 0.2 at 25°C

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation and cultivation of Listeria monocytogenes according to the FDA formula.

Listeria Enrichment Broth, FDA (LEB, FDA) Composition per liter: Soybean casein digest broth yeast extract.....................................1.0L Nalidixic acid solution ...............................................................8.0mL Cycloheximide solution .............................................................5.1mL Acriflavin·HCl solution .............................................................3.0mL pH 7.3 ± 0.2 at 25°C

40.0mL of ethanol. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Acriflavin·HCl Solution: Composition per 100.0mL: Acriflavin·HCl solution ................................................................ 0.5g

Preparation of Acriflavin·HCl Solution: Add acriflavin·HCl solution to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except nalidixic acid solution, acriflavin solution, and cycloheximide solution—to distilled/ deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add 8.0mL of sterile nalidixic acid solution, 5.1mL of sterile cycloheximide solution, and 3.0mL of sterile acriflavin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and enrichment of Listeria monocytogenes from foods.

Listeria Enrichment Broth I, USDA FSIS (Listeria Primary Selective Enrichment Broth, UVM I) (University of Vermont I Listeria Primary Selective Enrichment Broth) Composition per liter: NaCl............................................................................................ 20.0g Na2HPO4 ..................................................................................... 12.0g Beef extract................................................................................... 5.0g Proteose peptone........................................................................... 5.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g

954

Listeria Enrichment Broth II, USDA FSIS

KH2PO4 ....................................................................................... 1.35g Esculin .......................................................................................... 1.0g Nalidixic acid solution ...............................................................1.0mL Acriflavine solution ...................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Nalidixic Acid Solution: Add nalidixic acid to

Source: This medium is available as a premixed powder from Oxoid

Preparation of Acriflavine Solution: Add acriflavine to distilled/

Unipath.

Nalidixic Acid Solution: Composition per 10.0mL: Nalidixic acid ................................................................................ 0.2g NaOH (0.1M solution) .............................................................10.0mL

Preparation of Nalidixic Acid Solution: Add nalidixic acid to 10.0mL of NaOH solution. Mix thoroughly. Filter sterilize. Acriflavine Solution: Composition per 10.0mL: Acriflavine .................................................................................. 0.12g

Preparation of Acriflavine Solution: Add acriflavine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use freshly prepared solution.

Preparation of Medium: Add components, except nalidixic acid solution and acriflavine solution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of sterile nalidixic acid solution. Mix thoroughly. Store at 4°C. Immediately prior to use, aseptically add 1.0mL of sterile acriflavine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the selective isolation, cultivation, and enrichment of Listeria monocytogenes from food, milk, and dairy products.

Listeria Enrichment Broth II, USDA FSIS (Listeria Primary Selective Enrichment Broth, UVM II) (University of Vermont II Listeria Primary Selective Enrichment Broth

10.0mL of NaOH solution. Mix thoroughly. Filter sterilize.

Acriflavine Solution: Composition per 10.0mL: Acriflavine .................................................................................. 0.25g deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use freshly prepared solution.

Listeria Enrichment HiVeg Agar Composition per liter: Potassium thiocyanate ................................................................ 37.5g Agar ............................................................................................ 13.0g Plant hydrolysate ........................................................................ 10.0g Plant peptone .............................................................................. 10.0g NaCl.............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Acriflavin hydrochloride (Trypaflavin) ...................................... 0.01g Thiaminium dichloride .............................................................. 5.0mg pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective isolation of Listeria monocytogenes from clinical specimens.

Composition per liter: NaCl ............................................................................................ 20.0g Na2HPO4 ..................................................................................... 12.0g Beef extract ................................................................................... 5.0g Protease peptone ........................................................................... 5.0g Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 5.0g KH2PO4 ....................................................................................... 1.35g Esculin .......................................................................................... 1.0g Nalidixic acid solution ...............................................................1.0mL Acriflavine solution ...................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Listeria Enrichment HiVeg Broth Composition per liter: Potassium thiocyanate ................................................................ 37.5g Plant hydrolysate ........................................................................ 10.0g Plant peptone .............................................................................. 10.0g NaCl.............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Acriflavin hydrochloride (Trypaflavin) ...................................... 0.01g Thiaminium dichloride .............................................................. 5.0mg pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective enrichment of Listeria monocytogenes from clinical specimens.

Listeria Identification HiVeg Agar Base with PALCAM Selective Supplement

Listeria Enrichment HiVeg Broth, Modified Composition per liter: NaCl ............................................................................................ 20.0g Plant hydrolysate No. 1............................................................... 10.0g Na2HPO4 ....................................................................................... 9.6g Yeast extract.................................................................................. 5.0g Plant extract .................................................................................. 5.0g KH2PO4 ....................................................................................... 1.35g Esculin .......................................................................................... 1.0g Nalidixic acid .............................................................................. 0.02g Acriflavin hydrochloride (Trypaflavin) .................................... 0.012g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective enrichment of Listeria species.

Listeria Enrichment HiVeg Medium Base with Acriflavine and Nalidixic Acid (UVM) Composition per liter: NaCl ............................................................................................ 20.0g Na2HPO4 ..................................................................................... 12.0g Plant extract .................................................................................. 5.0g Plant hydrolysate........................................................................... 5.0g Plant peptone No. 3....................................................................... 5.0g Yeast extract.................................................................................. 5.0g KH2PO4 ....................................................................................... 1.35g Esculin .......................................................................................... 1.0g Nalidixic acid solution ...............................................................1.0mL Acriflavine solution ...................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium, without nalidixic acid or acriflavine solutions, is available as a premixed powder from HiMedia.

Preparation of Acriflavine Solution: Add acriflavine to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use freshly prepared solution.

955

Use: For the selective isolation, cultivation, and enrichment of Listeria monocytogenes from food, milk, and dairy products. For the selective isolation and cultivation of Listeria monocytogenes from clinical specimens.

Listeria Fermentation Broth Composition per liter: Proteose peptone No. 3 ............................................................... 10.0g NaCl.............................................................................................. 5.0g Beef extract................................................................................... 1.0g Bromcresol Purple ........................................................................ 0.1g Carbohydrate solution..............................................................10.0mL

Carbohydrate Solution: Composition per 10.0mL: Carbohydrate................................................................................. 5.0g

Preparation of Carbohydrate Solution: Add carbohydrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use glucose, salicin, rhamnose, dulcitol, or raffinose. Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile carbohydrate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and differentiation of Listeria species based on the fermentation of glucose, salicin, rhamnose, dulcitol, and raffinose.

Listeria Identification HiVeg Agar Base with PALCAM Selective Supplement (PALCAM) Composition per liter: Plant peptone .............................................................................. 23.0g LiCl ............................................................................................. 15.0g Agar ............................................................................................ 13.0g Mannitol...................................................................................... 10.0g NaCl.............................................................................................. 5.0g Starch ............................................................................................ 1.0g Esculin .......................................................................................... 0.8g Ammonium ferric citrate .............................................................. 0.5g Glucose ......................................................................................... 0.5g Phenol Red.................................................................................. 0.08g PALCAM selective supplement...............................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without PALCAM selective supplement, is available as a premixed powder from HiMedia.

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately. PALCAM Selective Supplement: Composition per 10.0mL: Ceftazidime.............................................................................. 20.0mg Polymyxin B ............................................................................ 10.0mg Acriflavine·HCl ......................................................................... 5.0mg

Preparation of PALCAM Selective Supplement: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

956

Listeria Identification HiVeg Broth Base with PALCAM Selective Supplement

Preparation of Medium: Add components, except PALCAM se-

Listeria Motility Medium

lective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile PALCAM selective supplement. Mix thoroughly. Pour into sterile Petri dishes.

Composition per liter:

Use: For the selective isolation and identification of Listeria monocytogenes and other Listeria species from foods.

Source: This medium is available as a premixed powder from HiMe-

Listeria Identification HiVeg Broth Base with PALCAM Selective Supplement (PALCAM) Composition per liter: Plant peptone............................................................................... 23.0g LiCl ............................................................................................. 10.0g D-Mannitol .................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Polysorbate 80............................................................................... 2.0g Soy lecithin ................................................................................... 1.0g Esculin .......................................................................................... 0.8g Ammonium ferric citrate .............................................................. 0.5g Phenol Red .................................................................................. 0.08g PALCAM selective supplement...............................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without PALCAM selective supplement, is available as a premixed powder from HiMedia.

Casein enzymatic hydrolysate .................................................... 20.0g Peptic digest of animal tissue ....................................................... 6.1g Agar .............................................................................................. 3.5g pH 7.3 ± 0.2 at 25°C dia.

Use: For testing motility of Listeria monocytogenes.

Listeria Oxford HiVeg Medium Base with Antibiotic Inhibitor Composition per liter: Plant special peptone .................................................................. 23.0g LiCl ............................................................................................. 15.0g Agar ............................................................................................ 10.0g NaCl.............................................................................................. 5.0g Cornstarch..................................................................................... 1.0g Esculin .......................................................................................... 1.0g Ammonium ferric citrate .............................................................. 0.5g Antibiotic inhibitor ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

Source: This medium, without antibiotic inhibitor, is available as a

PALCAM Selective Supplement: Composition per 10.0mL:

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

Ceftazidime .............................................................................. 20.0mg Polymyxin B ............................................................................ 10.0mg Acriflavine·HCl.......................................................................... 5.0mg

Antibiotic Inhibitor: Composition per 10.0mL:

Preparation of PALCAM Selective Supplement: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except PALCAM selective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile PALCAM selective supplement. Mix thoroughly.

premixed powder from HiMedia.

Cycloheximide.............................................................................. 0.4g Colistin sulfate ............................................................................ 0.02g Fosfomycin ................................................................................. 0.01g Acriflavine ................................................................................. 5.0mg Cefotetan.................................................................................... 2.0mg Ethanol (50% solution) ............................................................10.0mL

Preparation of Antibiotic Inhibitor: Add antibiotics to 10.0mL of ethanol. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-

Use: For the selective cultivation of f Listeria monocytogenes and

mation and inhalation.

other Listeria species.

Preparation of Medium: Add components, except antibiotic inhib-

Listeria Motility HiVeg Medium Composition per liter: Plant hydrolysate......................................................................... 20.0g Plant peptone................................................................................. 6.1g Agar .............................................................................................. 3.5g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

itor, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile antibiotic inhibitor. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Listeria monocytogenes from specimens containing a mixed bacterial flora. For the isolation of Listeria species from pathological specimens.

Listeria Oxford Medium Base with Antibiotic Inhibitor Composition per liter: Peptone, special .......................................................................... 23.0g LiCl ............................................................................................. 15.0g

Listeria Transport Enrichment Medium

Agar ............................................................................................ 10.0g NaCl .............................................................................................. 5.0g Cornstarch ..................................................................................... 1.0g Esculin .......................................................................................... 1.0g Ammonium ferric citrate .............................................................. 0.5g Antibiotic inhibitor ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without antibiotic inhibitor, is available as a premixed powder from HiMedia.

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately. Antibiotic Inhibitor: Composition per 10.0mL: Cycloheximide .............................................................................. 0.4g Colistin sulfate ............................................................................ 0.02g Fosfomycin ................................................................................. 0.01g Acriflavine ................................................................................. 5.0mg Cefotetan .................................................................................... 2.0mg Ethanol (50% solution) ............................................................10.0mL

Preparation of Antibiotic Inhibitor: Add antibiotics to 10.0mL of ethanol. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Preparation of Medium: Add components, except antibiotic inhibitor, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile antibiotic inhibitor. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Listeria monocytogenes from specimens containing a mixed bacterial flora. For the isolation of Listeria species from pathological specimens. Listeria Primary Selective Enrichment Broth, UVM I See: Listeria Enrichment Broth I, USDA FSIS Listeria Primary Selective Enrichment Broth, UVM II See: Listeria Enrichment Broth II, USDA FSIS Listeria Selective Agar, Modified Oxford See: Oxford Agar, Modified Listeria Selective Agar, Oxford See: Oxford Agar

957

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation and cultivation of Listeria species from clinical specimens.

Listeria Selective HiVeg Broth Base with Antibiotic Inhibitor Composition per liter: Plant hydrolysate ........................................................................ 17.0g Yeast extract.................................................................................. 6.0g NaCl.............................................................................................. 5.0g Papaic digest of soybean meal...................................................... 3.0g Glucose ......................................................................................... 2.5g K2HPO4......................................................................................... 2.5g Antibiotic inhibitor ..................................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without antibiotic inhibitor, is available as a premixed powder from HiMedia.

Antibiotic Inhibitor: Composition per 10.0mL: Cycloheximide.............................................................................. 0.4g Colistin sulfate ............................................................................ 0.02g Fosfomycin ................................................................................. 0.01g Acriflavine ................................................................................. 5.0mg Cefotetan.................................................................................... 2.0mg Ethanol (50% solution) ............................................................10.0mL

Preparation of Antibiotic Inhibitor: Add antibiotics to 10.0mL of ethanol. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Use: For the cultivation of Listeria monocytogenes from specimens containing a mixed bacterial flora. For the cultivation of Listeria species from pathological specimens.

Listeria Transport Enrichment Medium Listeria Selective HiVeg Agar Composition per liter: Potassium thiocyanate................................................................. 37.5g Agar ............................................................................................ 13.0g Plant hydrolysate......................................................................... 10.0g Plant peptone............................................................................... 10.0g NaCl .............................................................................................. 5.0g Glucose ......................................................................................... 1.0g Nalidixic acid .............................................................................. 0.04g Acriflavin hydrochloride (Trypaflavin) ...................................... 0.01g Thiaminium dichloride .............................................................. 5.0mg pH 7.4 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Composition per liter: Sodium glycerophosphate........................................................... 10.0g Agar .............................................................................................. 2.0g Sodium thioglycolate .................................................................... 1.0g CaCl2 ............................................................................................. 0.1g Nalidixic acid.............................................................................. 0.04g Acridine solution .......................................................................2.0mL pH 7.4 ± 0.2 at 25°C

Acridine Solution: Composition per 10.0mL: Acridine ...................................................................................... 0.04g

958

LIT Medium

Preparation of Acridine Solution: Add acridine to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Litmus ........................................................................................... 1.0g Crystal Violet ............................................................................. 5.0mg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except acridine solu-

Preparation of Medium: Add components to distilled/deionized

tion, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2.0mL acridine solution. Mix thoroughly. Aseptically distribute into sterile tubes in 10.0mL volumes or fill bottles 4/5 full.

Use: For the maintenance—as a transport medium—and enrichment of Listeria species.

Use: For the maintenance of lactic acid bacteria and for the differentiation of several bacteria on the basis of lactose fermentation. Bacteria that ferment lactose appear as red colonies. Bacteria that do not ferment lactose appear as dark blue-purple colonies.

Litmus Lactose HiVeg Agar

LIT Medium Composition per liter:

Composition per liter:

Beef liver, infusion from........................................................... 453.0g Na2HPO4 ....................................................................................... 8.0g Pancreatic digest of casein ............................................................ 5.0g Tryptose ........................................................................................ 5.0g Glucose ......................................................................................... 1.0g NaCl .............................................................................................. 1.0g K2HPO4 ......................................................................................... 0.5g KCl............................................................................................... 0.4g Hemin....................................................................................... 10.0mg Fetal bovine serum, heat inactivated......................................100.0mL pH 7.2 ± 0.2 at 25°C

Plant peptone .............................................................................. 23.0g Lactose........................................................................................ 20.0g Agar ............................................................................................ 15.0g Plant extract .................................................................................. 5.0g NaCl.............................................................................................. 5.0g Synthetic detergent No. V............................................................. 2.0g Litmus ........................................................................................... 0.5g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Filter sterilize. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Herpetomonas mariadeanei, Trypano-

Use: For the maintenance of lactic acid bacteria and differentiation of

plasma borreli, and Trypanosoma cruzi.

Lithium Chloride Phenylethanol Moxalactam Plating Agar See: LPM Agar

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

bacteria on the basis of lactose fermentation. Bacteria that ferment lactose appear as red colonies and others as dark blue-purple colonies.

Litmus Milk Composition per liter:

Litmus Lactose Agar (LL Agar) Composition per liter: Agar ............................................................................................ 10.0g Lactose ........................................................................................ 10.0g Meat peptone................................................................................. 5.0g Beef extract ................................................................................... 3.0g Litmus ........................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Use: For the maintenance of lactic acid bacteria and differentiation of bacteria on the basis of lactose fermentation. Bacteria that ferment lactose appear as red colonies and others as dark blue-purple colonies.

Litmus Lactose Agar with Crystal Violet (LLK Agar) Composition per liter: Agar ............................................................................................ 10.0g Lactose ........................................................................................ 10.0g Meat peptone................................................................................. 5.0g Beef extract ................................................................................... 3.0g © 2010 by Taylor and Francis Group, LLC

Skim milk.................................................................................. 100.0g Azolitmin ...................................................................................... 0.5g Na2SO3 .......................................................................................... 0.5g pH 6.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems and Oxoid Unipath. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 20 min at 10 psi pressure–115°C.

Use: For the maintenance of lactic acid bacteria and for the differentiation of several bacteria, especially Clostridium species, based on their action on milk. Bacteria that do not ferment carbohydrates, such as Proteus vulgaris or Moraxella lacunata, show no change in the azolitmin litmus indicator. Bacteria that ferment lactose or glucose with the production of gas, such as Clostridium perfringens, turn the medium pink and frothy. Bacteria that proteolytically degrade milk lactalbumin turn the medium blue. Bacteria that coagulate milk casein form a curd or clot. Bacteria that peptonize casein, such as Pseudomonas aeruginosa, show a dissolution of the clot.

Littman Oxgall Agar Composition per liter: Agar ............................................................................................ 20.0g Oxgall ......................................................................................... 15.0g

Liver Broth

959

Glucose ....................................................................................... 10.0g Peptone........................................................................................ 10.0g Crystal Violet .............................................................................. 0.01g Streptomycin solution ..............................................................10.0mL pH 6.5 ± 0.2 at 25°C

Source: This medium, without streptomycin, is available as a premixed powder from HiMedia.

Source: This medium is available as a premixed powder from BD Di-

Preparation of Streptomycin Solution: Add streptomycin to dis-

agnostic Systems.

Streptomycin Solution: Composition per 10.0mL:

Streptomycin Solution: Composition per 10.0mL: Streptomycin............................................................................... 0.03g tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except streptomycin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile streptomycin solution. Mix thoroughly. Aseptically distribute into sterile tubes.

Preparation of Medium: Add components, except streptomycin so-

Use: For the selective cultivation of fungi, especially dermatophytes.

lution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile streptomycin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Allow tubes to cool in a slanted position.

Composition per liter:

Streptomycin ............................................................................... 0.03g

Preparation of Streptomycin Solution: Add streptomycin to dis-

Use: For the selective isolation and cultivation of fungi, especially dermatophytes.

Littman Oxgall HiVeg Agar Base with Streptomycin Composition per liter: Agar ............................................................................................ 20.0g Plant peptone............................................................................... 20.0g Glucose ....................................................................................... 10.0g Synthetic detergent No. II ............................................................. 5.0g Crystal Violet .............................................................................. 0.01g Streptomycin solution ..............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Liver Broth Extracted liver tissue, minced..................................................... 30.0g Infusion from fresh liver............................................................. 23.0g Peptone ....................................................................................... 10.0g K2HPO4......................................................................................... 1.0g Agar overlay solution ............................................................200.0mL pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Agar Overlay Solution: Composition per 200.0mL: Agar, Oxoid Unipath No. 3........................................................... 4.0g

Preparation of Agar Overlay Solution: Add agar to distilled/de-

Source: This medium, without streptomycin, is available as a pre-

ionized water and bring volume to 200.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C.

mixed powder from HiMedia.

Preparation of Medium: Add components, except agar, to dis-

Streptomycin Solution: Composition per 10.0mL:

tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes to a depth of 50mm. Make sure that some liver particles are transferred to each tube. Autoclave for 20 min at 10 psi pressure–115°C. After inoculation, aseptically overlay the broth with 2.0mL of sterile, cooled agar solution per tube.

Streptomycin ............................................................................... 0.03g

Preparation of Streptomycin Solution: Add streptomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the selective isolation and cultivation of fungi, especially dermatophytes.

Littman Oxgall HiVeg Broth Base with Streptomycin Composition per liter: Plant peptone............................................................................... 20.0g Glucose ....................................................................................... 10.0g Synthetic detergent No. II ............................................................. 5.0g Crystal violet............................................................................... 0.01g Streptomycin solution ..............................................................10.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Use: For the isolation and cultivation of saccharolytic or putrefactive mesophilic and thermophilic anaerobic bacteria from foods.

Liver Broth Composition per liter: Beef liver, fresh......................................................................... 453.0g Pancreatic digest of casein.......................................................... 10.0g K2HPO4......................................................................................... 1.0g Soluble starch................................................................................ 1.0g pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Remove the fat from fresh beef liver. Grind the liver. Add 1.0L of distilled/deionized water. Gently heat and bring to boiling. Continue boiling for 60 min. Adjust pH to 7.6. Filter through cheesecloth. Reserve meat. To filtrate, add pancreatic digest of casein, K2HPO4, and soluble starch. Bring volume to 1.0L with distilled/deionized water. Refilter solution. Add meat particles to test tubes to a depth of approximately 2cm. Distribute broth into tubes with meat particles in 15.0mL volumes. Autoclave for 20 min at 15 psi pressure–121°C.

960

Liver Broth with Sodium Chloride

Use: For the isolation and cultivation of anaerobic microorganisms, especially Clostridium botulinum, from foods.

Liver Broth with Sodium Chloride Composition per liter: Beef liver, fresh......................................................................... 453.0g NaCl .......................................................................................... 150.0g Pancreatic digest of casein .......................................................... 10.0g K2HPO4 ......................................................................................... 1.0g Soluble starch................................................................................ 1.0g pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Remove the fat from fresh beef liver.

Liver Infusion Broth Composition per liter: Beef liver, infusion from........................................................... 500.0g Proteose peptone......................................................................... 10.0g NaCl.............................................................................................. 5.0g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Grind the liver. Add 1.0L of distilled/deionized water. Gently heat and bring to boiling. Continue boiling for 60 min. Adjust pH to 7.6. Filter through cheesecloth. Reserve meat. To filtrate, add pancreatic digest of casein, K2HPO4, NaCl, and soluble starch. Bring volume to 1.0L with distilled/deionized water. Refilter solution. Add meat particles to test tubes to a depth of approximately 2cm. Distribute broth into tubes with meat particles in 15.0mL volumes. Autoclave for 20 min at 15 psi pressure–121°C. After inoculation, overlay each tube with sterile, melted petroleum jelly.

Use: For the cultivation of Brucella species and other fastidious pathogenic bacteria.

Use: For the cultivation of obligate halophiles from brined and dry-

Source: This medium is available as a premixed powder from HiMe-

salted vegetables.

Liver Infusion Broth, HiVeg Composition per liter: Plant infusion No. 1 .................................................................... 20.0g Plant peptone No. 3..................................................................... 10.0g NaCl.............................................................................................. 5.0g pH 6.9 ± 0.2 at 25°C dia.

Liver Infusion Agar Composition per liter: Beef liver, infusion from........................................................... 500.0g Agar ............................................................................................ 20.0g Proteose peptone ......................................................................... 10.0g NaCl .............................................................................................. 5.0g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the cultivation of Brucella species and other fastidious pathogenic bacteria.

Liver Infusion Agar, HiVeg

Use: For the cultivation of Brucella species and other fastidious pathogenic bacteria.

Liver Infusion Sake Medium Composition per liter: Liver, fresh................................................................................ 400.0g Agar ............................................................................................ 15.0g Sake........................................................................................400.0mL

Preparation of Medium: Finely mince liver and add to 600.0mL of distilled/deionized water. Gently heat and bring to boiling. Continue boiling for 5–10 min. Filter through Whatman #2 filter paper. To filtrate, add agar and sake. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 25 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Composition per liter:

Use: For the cultivation and maintenance of Lactobacillus fructiv-

Agar ............................................................................................ 20.0g Plant infusion No. 1 .................................................................... 20.0g Plant peptone No. 3..................................................................... 10.0g NaCl .............................................................................................. 5.0g pH 6.9 ± 0.2 at 25°C

orans and Lactobacillus homohiochi.

Liver Meat Infusion HiVeg Agar Composition per liter:

Plant infusion No. 2 .................................................................... 20.0g Agar ............................................................................................ 11.0g Na2SO3 ......................................................................................... 1.2g Glucose ....................................................................................... 0.75g Starch .......................................................................................... 0.75g Na2CO3 ....................................................................................... 0.67g Ferric ammonium citrate............................................................... 0.5g pH 7.6 ± 0.2 at 25°C

Use: For the cultivation of Brucella species and other fastidious patho-

Source: This medium is available as a premixed powder from HiMe-

genic bacteria.

dia.

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

Lobosphaera Medium Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Make sure that some liver and veal particles are transferred to each tube. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of a variety of anaerobic organisms.

Liver Veal Agar Composition per liter: Veal, infusion from ................................................................... 500.0g Beef liver, infusion from............................................................. 50.0g Gelatin......................................................................................... 20.0g Proteose peptone ......................................................................... 20.0g Agar ............................................................................................ 15.0g Soluble starch.............................................................................. 10.0g Glucose ......................................................................................... 5.0g NaCl .............................................................................................. 5.0g Casein............................................................................................ 2.0g NaNO3........................................................................................... 2.0g Enzymatic digest of protein .......................................................... 1.3g Pancreatic digest of casein ............................................................ 1.3g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

961

Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Preparation of Medium: To 1.0L of cooled sterile liver veal agar, aseptically add 80.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes. Dry plates at 35°C for 24 hr.

Use: For the cultivation of a variety of anaerobic organisms. LL Agar See: Litmus Lactose Agar LLK Agar See: Litmus Lactose Agar with Crystal Violet

LMX Broth Modified, Fluorocult (Fluorocult LMX Broth, Modified)

Preparation of Medium: Add components to distilled/deionized

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Make sure that some liver and veal particles are transferred to each tube. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Tryptose .......................................................................................... 5.0 NaCl................................................................................................ 5.0 Sorbitol ........................................................................................... 1.0 Tryptophan...................................................................................... 1.0 K2HPO4......................................................................................... 2.7g KH2PO4......................................................................................... 2.0g Lauryl sulfate sodium salt............................................................. 0.1g 1-Isopropyl-β-D-1-thio-galactopyranoside (IPTG) ...................... 0.1g 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-GAL) . 0.08g 4-Methylumbelliferyl-β-D-glucuronide ...................................... 0.05g pH: 6.8 ± 0.2 at 25°C

Use: For the cultivation of a variety of anaerobic organisms.

Liver Veal Egg Yolk Agar Composition per 1080.0mL: Liver veal agar ..............................................................................1.0L Egg yolk emulsion, 50% ..........................................................80.0mL pH 7.3 ± 0.2 at 25°C

Liver Veal Agar: Composition per liter: Veal, infusion from ................................................................... 500.0g Beef liver, infusion from............................................................. 50.0g Gelatin......................................................................................... 20.0g Proteose peptone ......................................................................... 20.0g Agar ............................................................................................ 15.0g Soluble starch.............................................................................. 10.0g Glucose ......................................................................................... 5.0g NaCl .............................................................................................. 5.0g Casein............................................................................................ 2.0g NaNO3........................................................................................... 2.0g Enzymatic digest of protein .......................................................... 1.3g Pancreatic digest of casein ............................................................ 1.3g

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Liver Veal Agar: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Make sure that some liver and veal particles are transferred to each tube. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. © 2010 by Taylor and Francis Group, LLC

Source: This medium is available from Merck. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into test tubes. Autoclave for 15 min at 15 psi pressure–121°C. The broth is clear and yellowish brown. Use: For the simultaneous detection of total coliforms and E. coli in water, food, and diary products by the fluorogenic procedure. A color change of the broth from yellow to blue-green indicates the presence of coliforms. A blue fluorescence under long-wave UV light permits the rapid detection of E.coli. As tryptophan is added to the broth, the indole reaction is easily done by adding KOVACS reagent. The formation of a red ring additionally confirms the presence of E. coli.

Lobosphaera Medium Composition per liter: Polypeptone™ ............................................................................ 10.0g Glycerol ........................................................................................ 7.5g Yeast extract.................................................................................. 2.0g K2HPO4......................................................................................... 1.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ............................................................................... 0.5g pH 6.5 ± 0.2 at 25°C

962

Loefer's Medium

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Filter sterilize. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Lobosphaera species.

Loefer's Medium Composition per liter: Proteose peptone ......................................................................... 15.0g Casitone ........................................................................................ 5.0g Glucose ......................................................................................... 5.0g NaCl .............................................................................................. 2.0g Na2HPO4 ....................................................................................... 1.0g KH2PO4 ......................................................................................... 1.0g Yeast extract.................................................................................. 0.5g MgCl2 ............................................................................................ 0.3g Asolectin solution ....................................................................10.0mL

Asolectin Solution: Asolectin ....................................................................................... 1.0g Ethanol ...................................................................................100.0mL

Preparation of Asolectin Solution: Add 1.0g of asolectin to ethanol and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Tetrahymena patula.

Loeffler Blood Serum Medium Composition per liter: Beef blood serum ...................................................................750.0mL Dextrose broth........................................................................250.0mL pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Dextrose Broth: Composition per liter: Enzymatic digest of protein .......................................................... 2.5g Glucose ....................................................................................... 1.25g NaCl............................................................................................ 1.25g Beef extract................................................................................ .0.75g

Preparation of Dextrose Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Combine 750.0mL of beef blood serum with 250.0mL of dextrose broth. Mix thoroughly. Distribute into screw-capped tubes. Slant tubes in the autoclave. Close the autoclave door loosely. Autoclave for 10 min at 0 psi pressure–100°C. Close the autoclave door tightly. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Corynebacterium diphtheriae. For demonstration of pigment production and proteolysis by Corynebacterium diphtheriae.

Loeffler HiVeg Medium Base with Asolectin Composition per liter: Glucose ......................................................................................... 2.5g Plant extract .................................................................................. 2.5g Plant special peptone .................................................................... 2.5g NaCl............................................................................................ 1.25g Asolectin solution ....................................................................10.0mL pH 7.6 ± 0.2 at 25°C

Source: This medium, without asolectin, is available as a premixed powder from HiMedia. Asolectin Solution: Composition per 100.0mL: Asolectin ....................................................................................... 1.0g Ethanol...................................................................................100.0mL

Preparation of Asolectin Solution: Add 1.0g of asolectin to ethanol and bring volume to 100.0mL. Mix thoroughly.

agnostic Systems.

Preparation of Medium: Add components to distilled/deionized wa-

Dextrose Broth: Composition per liter:

ter and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Tryptose ...................................................................................... 10.0g Glucose ......................................................................................... 5.0g Sodium chloride ............................................................................ 5.0g Beef extract ................................................................................... 3.0g

Use: For the cultivation of Tetrahymena patula.

Preparation of Dextrose Broth: Add components to distilled/de-

with 250.0mL of dextrose broth. Mix thoroughly. Distribute into screw-capped tubes. Slant tubes in the autoclave. Close the autoclave door loosely. Autoclave for 10 min at 0 psi pressure–100°C. Close the autoclave door tightly. Autoclave for 15 min at 15 psi pressure–121°C.

Beef serum .................................................................................. 70.0g Egg, dried...................................................................................... 7.5g Heart muscle, solids from infusion............................................. 0.72g Glucose ....................................................................................... 0.71g Peptic digest of animal tissue ..................................................... 0.71g NaCl............................................................................................ 0.36g pH 7.6 ± 0.2 at 25°C

Use: For the cultivation of Corynebacterium diphtheriae. For demon-

Source: This medium is available as a premixed powder from BD Di-

stration of pigment production and proteolysis by Corynebacterium diphtheriae.

agnostic Systems.

ionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Combine 750.0mL of beef blood serum

Loeffler Medium Composition per liter:

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped tubes. Slant tubes in the autoclave. Close the autoclave door loosely. Autoclave for 10 min at 0 psi pressure–100°C. Close the autoclave door tightly. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Corynebacterium diphtheriae. For demonstration of pigment production and proteolysis by Corynebacterium

Lombard-Dowell Broth

963

diphtheriae. For the cultivation and maintenance of Moraxella lacunata.

1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Loeffler Slant

Use: For the cultivation and identification of a variety of obligate anaerobic

Composition per liter: Tryptose ........................................................................................ 5.0g Glucose ......................................................................................... 1.0g Beef serum .............................................................................750.0mL

Use: For the cultivation of Corynebacterium diphtheriae. For demonstration of pigment production and proteolysis by Corynebacterium diphtheriae. For the cultivation and maintenance of Moraxella lacunata.

Loeffler Slant, Modified Composition per liter: Glucose ......................................................................................... 1.0g Peptone.......................................................................................... 0.5g Beef serum .............................................................................300.0mL pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add peptone and glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Add beef serum. Mix thoroughly. Adjust pH to 7.6. Distribute into screw-capped tubes in 3.0mL volumes. Slant tubes in the autoclave. Autoclave for 30 min at 0 psi pressure–100°C.

LOM Agar See: Lysine Ornithine Mannitol Agar

Lombard–Dowell Agar (LD Agar) Agar ............................................................................................ 20.0g Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 2.5g L-Cystine ....................................................................................... 0.4g L-Tryptophan ................................................................................. 0.2g Na2SO3 .......................................................................................... 0.1g Hemin....................................................................................... 10.0mg NaOH (1N).................................................................................5.0mL Vitamin K1 solution....................................................................1.0mL pH 7.5 ± 0.2 at 25°C

Vitamin K1 Solution: Composition per 100.0mL: Vitamin K1 .................................................................................... 1.0g Ethanol .....................................................................................99.0mL

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

bacteria. For the cultivation of Bacteroides species, Fusobacterium species, Clostridium species, and nonspore-forming Gram-positive anaerobes.

Lombard-Dowell Bile Agar (LD Bile Agar) Composition per liter: Agar ............................................................................................ 20.0g Oxgall ......................................................................................... 20.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 2.5g D-Glucose ...................................................................................... 1.0g L-Cystine ....................................................................................... 0.4g L-Tryptophan................................................................................. 0.2g Na2SO3 .......................................................................................... 0.1g Hemin ...................................................................................... 10.0mg Bile.........................................................................................200.0mL NaOH (1N).................................................................................5.0mL Vitamin K1 solution ...................................................................1.0mL pH 7.5 ± 0.2 at 25°C

Vitamin K1 Solution: Composition per 100.0mL: Vitamin K1 .................................................................................... 1.0g Ethanol.....................................................................................99.0mL

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Preparation of Medium: Add hemin and L-cystine to 5.0mL of NaOH. Mix thoroughly. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Pour into sterile Petri dishes. Use: For the cultivation and identification of a variety of obligate anaerobic bacteria in the presence of 20% bile.

Lombard-Dowell Broth (LD Broth) Composition per liter: Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 2.5g Agar .............................................................................................. 0.7g L-Tryptophan................................................................................. 0.2g Na2SO3 .......................................................................................... 0.1g NaOH (1N).................................................................................5.0mL Hemin solution...........................................................................1.0mL Vitamin K1 solution ...................................................................1.0mL pH 7.5 ± 0.2 at 25°C

Hemin Solution: Composition per 100.0mL: Hemin ........................................................................................... 1.0g NaOH (1N solution).................................................................20.0mL

Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water.

964

Lombard-Dowell Egg Yolk Agar

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Preparation of Medium: Add tryptophan to 5.0mL of NaOH. Mix thoroughly. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.5. Distribute into screw-capped tubes in 7.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool tubes, with caps loose, under 85% N2 + 10% H2 + 5% CO2. Tighten caps. Use: For the cultivation of a wide variety of anaerobic bacteria.

Lombard-Dowell Egg Yolk Agar (LD Egg Yolk Agar) (Egg Yolk Agar, Lombard-Dowell) Composition per 9100.0mL: Na2HPO4·12H2O........................................................................... 5.0g Glucose ......................................................................................... 2.0g LD Agar ...............................................................................9000.0mL Egg yolk emulsion .................................................................100.0mL MgSO4·7H2O (5% solution) ......................................................0.2mL pH 7.5 ± 0.2 at 25°C

LD Agar: Composition per liter: Agar ............................................................................................ 20.0g Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 5.0g NaCl .............................................................................................. 2.5g L-Cystine ....................................................................................... 0.4g L-Tryptophan ................................................................................. 0.2g Na2SO3 .......................................................................................... 0.1g Hemin....................................................................................... 10.0mg NaOH (1N NaOH) .....................................................................5.0mL Vitamin K1 solution....................................................................1.0mL

Preparation of LD Agar: Add hemin and L-cystine to 5.0mL of NaOH. Mix thoroughly. Add remaining components. Mix thoroughly. Gently heat and bring to boiling.

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Egg Yolk Emulsion: Composition: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg.

Preparation of Medium: Combine components, except egg yolk emulsion. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add 100.0mL of egg yolk emulsion. Mix thoroughly. Pour into sterile Petri dishes. © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on lecithinase production, lipase production, and proteolytic ability. Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen. Bacteria that produce proteolytic activity appear as colonies surrounded by a clear zone.

Lombard-Dowell Esculin Agar (LD Esculin Agar) (Esculin Agar, Lombard-Dowell) Composition per liter: Agar ............................................................................................ 20.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 2.5g Esculin .......................................................................................... 1.0g Ferric citrate.................................................................................. 0.5g L-Cystine ....................................................................................... 0.4g L-Tryptophan................................................................................. 0.2g Hemin ...................................................................................... 10.0mg NaOH (1N).................................................................................5.0mL Vitamin K1 solution....................................................................1.0mL pH 7.5 ± 0.2 at 25°C

Vitamin K1 Solution: Composition per 100.0mL: Vitamin K1 .................................................................................... 1.0g Ethanol.....................................................................................99.0mL

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Preparation of Medium: Add hemin and L-cystine to 5.0mL of NaOH. Mix thoroughly. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Lombard-Dowell Gelatin Agar (LD Gelatin Agar) Composition per liter: Agar ............................................................................................ 20.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g Gelatin........................................................................................... 4.0g NaCl.............................................................................................. 2.5g Glucose ......................................................................................... 1.0g L-Cystine ....................................................................................... 0.4g L-Tryptophan................................................................................. 0.2g Na2SO3 .......................................................................................... 0.1g Hemin ...................................................................................... 10.0mg NaOH (1N).................................................................................5.0mL Vitamin K1 solution....................................................................1.0mL pH 7.5 ± 0.2 at 25°C

Low Iron YC Agar

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Preparation of Medium: Add hemin and L-cystine to 5.0mL of NaOH. Mix thoroughly. Add remaining components, except agar and gelatin. Bring volume to 750.0mL with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. In a separate flask, add gelatin to 100.0mL of cold distilled/deionized water. Gently heat and bring to 70°C. Add gelatin solution to the 750.0mL of basal medium. Mix thoroughly. Add agar. Bring volume to 1.0L with distilled/deionized water. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes.

Use: For the cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on gelatinase production. After incubation of plates, gelatinase activity is determined by the addition of Frazier’s reagent. Bacteria that hydrolyze gelatin appear as colonies surrounded by a clear zone.

Lombard-Dowell Neomycin Agar (Egg Yolk Agar with Neomycin) Composition per 9100.0mL: Na2HPO4·12H2O........................................................................... 5.0g Glucose ......................................................................................... 2.0g Neomycin sulfate .......................................................................... 0.1g LD Agar ...............................................................................9000.0mL Egg yolk emulsion .................................................................100.0mL MgSO4·7H20 (5% solution) .......................................................0.2mL pH 7.5 ± 0.2 at 25°C

Preparation of LD Agar: Add hemin and L-cystine to 5.0mL of NaOH. Mix thoroughly. Add remaining components. Mix thoroughly. Gently heat and bring to boiling.

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Mix thoroughly.

Egg Yolk Emulsion: Composition: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1 © 2010 by Taylor and Francis Group, LLC

965

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Preparation of Medium: Combine components, except egg yolk emulsion and neomycin sulfate. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of egg yolk emulsion and neomycin sulfate. Mix thoroughly. Pour into sterile Petri dishes. Use: For the selective cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on lecithinase production, lipase production, and proteolytic ability. Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen. Bacteria that produce proteolytic activity appear as colonies surrounded by a clear zone.

Long-Term Preservation Medium Composition per liter: NaCl............................................................................................ 30.0g Peptone ....................................................................................... 10.0g Agar .............................................................................................. 3.0g Yeast extract.................................................................................. 3.0g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into screw-capped tubes in 4.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of a wide variety of bacteria.

Low Iron YC Agar Composition per 1033.0mL: Solution 1......................................................................................1.0L Solution 4.................................................................................30.0mL Solution 2...................................................................................2.0mL Solution 3...................................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Solution 1: Composition per liter: Yeast extract................................................................................ 20.0g Noble agar................................................................................... 10.0g Casamino acids ........................................................................... 10.0g KH2PO4......................................................................................... 5.0g CaCl2 ............................................................................................. 1.0g Tryptophan.................................................................................. 0.05g

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Gently heat and bring to boiling. Filter through #40 ashless filter paper.

Solution 2: Composition per 100.0mL: MgSO4·7H2O .............................................................................. 22.5g CuSO4·5H2O................................................................................. 0.5g ZnSO4·5H2O ................................................................................. 0.2g β-Alanine .................................................................................. 0.115g Nicotinic Acid........................................................................... 0.115g MnCl2·4H2O ............................................................................. 0.075g Pimelic acid ............................................................................... 7.5mg HCl, concentrated ......................................................................3.0mL

Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

966

Low Iron YC Broth

Solution 3: Composition per 100.0mL:

Solution 4: Composition per 100.0mL:

L-Cystine ..................................................................................... 20.0g

HCl, concentrated ....................................................................20.0mL

Maltose ....................................................................................... 50.0g CaCl2·2H2O .................................................................................. 0.5g

Preparation of Solution 3: Add components to distilled/deionized

Preparation of Solution 4: Add components to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly.

water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 10 min at 11 psi pressure–116°C. Cool to 25°C.

Solution 4: Composition per 100.0mL: Maltose........................................................................................ 50.0g CaCl2·2H2O................................................................................... 0.5g

Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 10 min at 11 psi pressure–116°C. Cool to 45°–50°C.

Preparation of Medium: To 1.0L of solution 1, add 2.0mL of solution 2 and 1.0mL of solution 3. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 30.0mL of sterile solution 4. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Medium: To 1.0L of solution 1, add 2.0mL of solution 2 and 1.0mL of solution 3. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 30.0mL of sterile solution 4. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Corynebacterium diphtheriae.

Low Phosphate Buffered Basal Medium, Modified Composition per 1030.0mL:

Solution 1 ......................................................................................1.0L Solution 4 .................................................................................30.0mL Solution 2 ...................................................................................2.0mL Solution 3 ...................................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Pectin ............................................................................................ 4.0g NH4Cl ........................................................................................... 1.0g Na2HPO4 ..................................................................................... 0.72g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.2g Reducing agent ........................................................................20.0mL Yeast extract solution...............................................................10.0mL Trace minerals..........................................................................10.0mL Wolfe’s vitamin solution............................................................5.0mL Resazurin (0.2% solution) .........................................................1.0mL FeSO4·7H2O (2.5% solution)...................................................0.03mL pH 7.3 ± 0.1 at 25°C

Solution 1: Composition per liter:

Reducing Agent: Composition per 20.0mL:

Yeast extract................................................................................ 20.0g Casamino acids ........................................................................... 10.0g KH2PO4 ......................................................................................... 5.0g CaCl2·2H2O................................................................................... 1.0g Tryptophan .................................................................................. 0.05g

Preparation of Reducing Agent: Add Na2S· 9H2O to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Gas with 100% N2 for 20 min. Cap with a rubber stopper. Autoclave for 45 min at 15 psi pressure–121°C. Use freshly prepared solution.

Preparation of Solution 1: Add components to distilled/deionized

Yeast Extract Solution: Composition per 10.0mL:

Use: For the cultivation and maintenance of Corynebacterium diphtheriae.

Low Iron YC Broth Composition per 1033.0mL:

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Gently heat and bring to boiling. Filter through #40 ashless filter paper.

Solution 2: Composition per 100.0mL: MgSO4·7H2O .............................................................................. 22.5g CuSO4·5H2O ................................................................................. 0.5g ZnSO4·5H2O ................................................................................. 0.2g β-Alanine .................................................................................. 0.115g Nicotinic acid ............................................................................ 0.115g MnCl2·4H2O.............................................................................. 0.075g Pimelic acid................................................................................ 7.5mg HCl, concentrated ......................................................................3.0mL

Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Solution 3: Composition per 100.0mL: L-Cystine ..................................................................................... 20.0g

HCl, concentrated ....................................................................20.0mL

Na2S·9H2O.................................................................................... 0.5g

Yeast extract.................................................................................. 1.0g

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C. Cool to 25°C. Trace Minerals: Composition per liter: Nitrilotriacetic acid ..................................................................... 12.8g NaCl.............................................................................................. 1.0g CoCl2·6H2O ................................................................................ 0.16g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O ................................................................................. 0.1g ZnCl2 ............................................................................................. 0.1g NiSO4·6H2O.............................................................................. 0.026g CuCl2 .......................................................................................... 0.02g Na2SeO3 ...................................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Trace Minerals: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5

Lowenstein-Jensen Medium with Sodium Chloride

with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................... 0.01g Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Cyanocobalamin ........................................................................ 0.1mg

967

Mycobacterium kansasii appears as smooth to rough photochromogenic colonies. Mycobacterium gordonae appears as smooth yellow-orange colonies. Mycobacterium avium appears as smooth, colorless colonies. Mycobacterium smegmatis appears as wrinkled, creamy white colonies.

Lowenstein-Jensen Medium Composition per 1600.0mL:

Preparation of Wolfe’s Vitamin Solution: Add components to

Potato starch................................................................................ 30.0g Asparagine .................................................................................... 3.6g KH2PO4......................................................................................... 2.4g Magnesium citrate ........................................................................ 0.6g Malachite Green............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g Homogenized whole egg ............................................................. 1.0L Glycerol ...................................................................................12.0mL

distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Source: This medium is available as a prepared medium from BD Di-

Preparation of Medium: Add components, except yeast extract so-

agnostic Systems and Oxoid Unipath.

lution and reducing agent, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Cool under 90% N2 + 10% CO2. Anaerobically distribute into tubes in 6.0mL volumes. Autoclave for 45 min at 15 psi pressure–121°C. Aseptically add 0.06mL of sterile yeast extract solution to each tube. Mix thoroughly. Immediately prior to inoculation, aseptically add 0.12mL of sterile reducing agent to each tube. Mix thoroughly.

Homogenized Whole Egg: Composition per liter:

Use: For the cultivation and maintenance of Clostridium thermosulfurogenes.

Lowenstein-Gruft Medium Composition per 1600.0mL: Potato starch................................................................................ 30.0g Asparagine .................................................................................... 3.6g KH2PO4 ......................................................................................... 2.4g Magnesium citrate......................................................................... 0.6g Malachite Green............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g Nalidixic acid ............................................................................ 0.056g Ribonucleic acid ...................................................................... 0.08mg Homogenized whole egg ............................................................. 1.0L Glycerol ...................................................................................12.0mL Penicillin ................................................................................ 80,000U

Homogenized Whole Egg: Composition per liter: Whole eggs ................................................................................ 18–24

Preparation of Homogenized Whole Egg: Use fresh eggs, less than 1 week old. Scrub the shells with soap. Let stand in a soap solution for 30 min. Rinse in running water. Soak eggs in 70% ethanol for 15 min. Break the eggs into a sterile container. Homogenize by shaking. Filter through four layers of sterile cheesecloth into a sterile graduated cylinder. Measure out 1.0L.

Preparation of Medium: Add glycerol to 600.0mL of distilled/deionized water. Mix thoroughly. Add remaining components, except fresh egg mixture. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 1.0L of homogenized whole egg. Mix thoroughly. Distribute into sterile screw-capped tubes. Place tubes in a slanted position. Inspissate at 85°C (moist heat) for 45 min.

Whole eggs ................................................................................ 18–24

Use: For the cultivation and differentiation of Mycobacterium species. Mycobacterium tuberculosis appears as granular, rough, dry colonies. Mycobacterium kansasii appears as smooth to rough photochromogenic colonies. Mycobacterium gordonae appears as smooth yellow-orange colonies. Mycobacterium avium appears as smooth, colorless colonies. Mycobacterium smegmatis appears as wrinkled, creamy white colonies. Also used for the cultivation and maintenance of Gordona species, Nocardia species, Rhodococcus species, and Tsukamurella paurometabolum.

Lowenstein-Jensen Medium with Sodium Chloride Composition per 1600.0mL: NaCl............................................................................................ 80.0g Potato starch................................................................................ 30.0g Asparagine .................................................................................... 3.6g KH2PO4......................................................................................... 2.4g Magnesium citrate ........................................................................ 0.6g Malachite Green............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g Homogenized whole egg ............................................................. 1.0L Glycerol ...................................................................................12.0mL

Use: For the cultivation and differentiation of Mycobacterium species.

Homogenized Whole Egg: Composition per liter:

Mycobacterium tuberculosis appears as granular, rough, dry colonies.

Whole eggs ................................................................................ 18–24

968

Lowenstein-Jensen Medium with Streptomycin

Lowenstein-Jensen Medium with Streptomycin Composition per 1610.0mL: Potato starch................................................................................ 30.0g Asparagine .................................................................................... 3.6g KH2PO4 ......................................................................................... 2.4g Magnesium citrate......................................................................... 0.6g Malachite Green............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g Homogenized whole egg ............................................................. 1.0L Glycerol ...................................................................................12.0mL Streptomycin solution ..............................................................10.0mL

Homogenized Whole Egg: Composition per liter: Whole eggs ................................................................................ 18–24

dona species, Nocardia species, Rhodococcus species, and Tsukamurella paurometabolum.

Lowenstein-Jensen Medium without Glycerol Composition per 1600.0mL: Potato starch................................................................................ 30.0g Asparagine .................................................................................... 3.6g KH2PO4......................................................................................... 2.4g Magnesium citrate ........................................................................ 0.6g Malachite green ............................................................................ 0.4g MgSO4·7H2O .............................................................................. 0.24g Homogenized whole egg ............................................................. 1.0L

Homogenized Whole Egg: Composition per liter: Whole eggs ................................................................................ 18–24

Preparation of Medium: Add components, except fresh egg mixture, to 600.0mL of distilled/deionized water. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 1.0L of homogenized whole egg. Mix thoroughly. Distribute into sterile screw-capped tubes. Place tubes in a slanted position. Inspissate at 85°C (moist heat) for 45 min.

Use: For the cultivation and maintenance of Mycobacterium species, especially Mycobacterium bovis and other species that are sensitive to glycerol.

LPBM Acido-Thermophile Medium Composition per liter:

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Agar ............................................................................................ 20.0g Cellulose ....................................................................................... 5.0g KH2PO4......................................................................................... 1.0g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Cellobiose ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Na2HPO4·7H2O............................................................................. 0.1g CaCl2·2H2O ................................................................................ 0.02g pH 5.2 ± 0.2 at 25°C

Preparation of Medium: Add glycerol to 600.0mL of distilled/de-

Preparation of Medium: Add components, except cellulose and

Streptomycin Solution: Composition per 10.0mL: Streptomycin .............................................................................. 0.1mg

Preparation of Streptomycin Solution: Add streptomycin to dis-

ionized water. Mix thoroughly. Add remaining components, except fresh egg mixture and streptomycin solution. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 1.0L of homogenized whole egg and 10.0mL of sterile streptomycin solution. Mix thoroughly. Distribute into sterile screw-capped tubes. Place tubes in a slanted position. Inspissate at 85°C (moist heat) for 45 min.

cellobiose, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.2 with H3PO4. Add cellulose and cellobiose. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and differentiation of Mycobacterium species.

LPM Agar (Lithium Chloride Phenylethanol Moxalactam Plating Agar)

Mycobacterium tuberculosis appears as granular, rough, dry colonies. Mycobacterium kansasii appears as smooth to rough photochromogenic colonies. Mycobacterium gordonae appears as smooth yelloworange colonies. Mycobacterium avium appears as smooth, colorless colonies. Mycobacterium smegmatis appears as wrinkled, creamy white colonies. Also used for the cultivation and maintenance of Gor© 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Acidothermus cellulolyticus.

Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g

LPM HiVeg Agar Base with Moxalactam

LiCl2.............................................................................................. 5.0g NaCl .............................................................................................. 5.0g Pancreatic digest of casein ............................................................ 5.0g Peptic digest of animal tissue........................................................ 5.0g Beef extract ................................................................................... 3.0g Phenylethyl alcohol....................................................................... 2.5g Moxalactam solution..................................................................2.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Moxalactam Solution: Composition per 10.0mL: Moxalactam .................................................................................. 0.1g

Preparation of Moxalactam Solution: Add moxalactam to dis-

969

LPM Agar with Esculin and Ferric Iron Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g LiCl ............................................................................................... 5.0g NaCl.............................................................................................. 5.0g Pancreatic digest of casein............................................................ 5.0g Peptic digest of animal tissue ....................................................... 5.0g Beef extract................................................................................... 3.0g Phenylethyl alcohol ...................................................................... 2.5g Esculin .......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Moxalactam solution .................................................................2.0mL pH 7.3 ± 0.2 at 25°C

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Moxalactam Solution: Composition per 10.0mL:

Preparation of Medium: Add components, except moxalactam so-

Moxalactam .................................................................................. 0.1g

lution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 12 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2.0mL of sterile moxalactam solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Moxalactam Solution: Add moxalactam to dis-

Use: For the isolation and cultivation of Listeria monocytogenes.

LPM Agar (Lithium Phenylethanol Moxalactam Agar) Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g Casein enzymic hydrolysate ......................................................... 5.0g Peptic digest of animal tissue........................................................ 5.0g Beef extract ................................................................................... 3.0g LiCl ............................................................................................... 5.0g NaCl .............................................................................................. 5.0g Phenylethyl alcohol....................................................................... 2.5g Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Moxalactam ............................................................................. 20.0mg

Preparation of Selective Supplement Solution: Add moxalac-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except moxalactam solution, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 12 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2.0mL of sterile moxalactam solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Listeria monocytogenes.

LPM HiVeg Agar Base with Moxalactam Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g Plant hydrolysate .......................................................................... 5.0g Plant peptone ................................................................................ 5.0g LiC ................................................................................................ 5.0g NaCl.............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Phenylethyl alcohol ...................................................................... 2.5g Moxalactam solution .................................................................2.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without moxalactam, is available as a premixed powder from HiMedia.

tam to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Moxalactam Solution: Composition per 10.0mL:

Caution: Lithium chloride is harmful. Avoid bodily contact and inha-

Moxalactam .................................................................................. 0.1g

lation of vapors. On contact with skin, wash with plenty of water immediately.

Preparation of Moxalactam Solution: Add moxalactam to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the isolation and cultivation of Listeria monocytogenes.

970

LS Differential HiVeg Medium Base with Skim Milk and Triphenyltetrazolium Chloride

Skim Milk Solution: Composition per 100.0mL:

Composition per liter:

Skim milk, antibiotic free ........................................................... 10.0g

Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Plant hydrolysate......................................................................... 10.0g Plant extract .................................................................................. 5.0g Papaic digest of soybean meal ...................................................... 5.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g L-Cysteine·HCl.............................................................................. 0.3g Skim milk solution.................................................................100.0mL Triphenyltetrazolium chloride solution....................................10.0mL pH 6.1 ± 0.2 at 25°C

Preparation of Skim Milk Solution: Add skim milk to distilled/

Source: This medium, without skim milk and triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia.

Skim Milk Solution: Composition per 100.0mL: Skim milk, antibiotic free ........................................................... 10.0g

Preparation of Skim Milk Solution: Add skim milk to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 5 min at 15 psi pressure–121°C. Cool to 50°C.

Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.2g

Preparation of Triphenyltetrazolium Chloride Solution: Add triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C.

Preparation of Medium: Add components, except skim milk solution and triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile skim milk solution and sterile triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the differentiation and enumeration of lactobacilli and streptococci in yogurt. Lactobacillus species appear as irregular, red colonies surrounded by a white, opaque zone. Streptococcus species appear as round, red colonies surrounded by a clear zone.

LS Differential Medium (Lactobacillus Streptococcus Differential Medium) Composition per liter: Glucose ....................................................................................... 20.0g Agar ............................................................................................ 13.0g Pancreatic digest of casein .......................................................... 10.0g Beef extract ................................................................................... 5.0g NaCl .............................................................................................. 5.0g Papaic digest of soybean meal ...................................................... 5.0g Yeast extract.................................................................................. 5.0g L-Cysteine·HC1·H2O..................................................................... 0.3g Skim milk solution.................................................................100.0mL Triphenyltetrazolium chloride solution....................................10.0mL pH 6.1 ± 0.2 at 25°C

deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 5 min at 15 psi pressure–121°C. Cool to 50°C.

Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride ............................................. 0.2g

Preparation of Triphenyltetrazolium Chloride Solution: Add triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Warm to 50°C.

LST-MUG Broth Composition per liter: Tryptose ...................................................................................... 20.0g Lactose.......................................................................................... 5.0g K2HPO4 ...................................................................................... 2.75g KH2PO4 ...................................................................................... 2.75g NaCl.............................................................................................. 5.0g L-Tryptophan ................................................................................ 1.0g Sodium lauryl sulfate.................................................................... 0.1g 4-Methylumbelliferyl-β-D-glucuronide ........................................ 0.1g pH 6.8 ± 0.2 at 37°C

Source: This medium is available from Fluka, Sigma-Aldrich. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into test tubes that contain an inverted Durham tube in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the detection of E. coli by the fluorogenic method. The presence of E. coli results in fluorescence in the UV. A positive indole test provides confirmation. β-D-glucoronidase, which is produced by E. coli, cleaves 4-methylumbelliferyl-β-D-glucuronide to 4-methylumbelliferone and glucuronide. The fluorogen 4-methylumbelliferone can be detected under a long wavelength UV lamp. LT Medium See: Lecithin Tween™ Medium

LTH Medium for Thiothrix Composition per liter: HEPES (N-[2-hydroxyethyl]piperazineN´-2-ethanesulfonic acid) buffer ........................................ .2.38.g Sodium lactate .............................................................................. 1.0g Na2S2O3·5H2O .............................................................................. 0.5g (NH4)2SO4 .................................................................................... 0.5g

LUP

K2HPO4 ....................................................................................... 0.11g MgSO4·7H2O ................................................................................ 0.1g CaCl2·2H2O................................................................................. 0.05g KH2PO4 .................................................................................... 85.0mg EDTA ......................................................................................... 3.0mg FeCl3·6H2O ................................................................................ 2.0mg Wolfe’s vitamin solution ..........................................................10.0mL pH 7.3 ± 0.2 at 25°C

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Calcium DL-pantothenate........................................................... 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Wolfe’s vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.3 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL of sterile Wolfe’s vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Thiothrix species.

971

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except Wolfe’s vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.3 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile Wolfe’s vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of unidentified bacterium ATCC 49750.

Luedemann Medium (DSMZ Medium 877) Composition per 100.0mL: Agar .............................................................................................. 1.5g Malt extract broth ......................................................................... 1.5g Soluble starch................................................................................ 1.0g Glucose ......................................................................................... 1.0g Yeast extract.................................................................................. 0.5g NaCl.............................................................................................. 0.5g CaCO3 ........................................................................................... 0.2g pH 8.6 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Blastococcus saxobsidens and Blastococcus sp.

Luminous Medium Composition per liter:

LTH Medium for Thiothrix with Casitone Composition per liter: HEPES (N-[2-hydroxyethyl]piperazine-N´-2ethanesulfonic acid) buffer ............................................... 2.38g Casitone ........................................................................................ 1.0g Sodium lactate............................................................................... 1.0g Na2S2O3·5H2O .............................................................................. 0.5g (NH4)2SO4 ..................................................................................... 0.5g K2HPO4 ....................................................................................... 0.11g MgSO4·7H2O ................................................................................ 0.1g CaCl2·2H2O................................................................................. 0.05g KH2PO4 .................................................................................... 85.0mg EDTA ......................................................................................... 3.0mg FeCl3·6H2O ................................................................................ 2.0mg Wolfe’s vitamin solution ..........................................................10.0mL

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Calcium DL-pantothenate........................................................... 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg © 2010 by Taylor and Francis Group, LLC

NaCl............................................................................................ 30.0g Agar ............................................................................................ 20.0g NH4Cl ........................................................................................... 5.0g Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g K2HPO4......................................................................................... 3.9g KH2PO4......................................................................................... 2.1g CaCO3 ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g KCl.............................................................................................. 0.75g Tris buffer (1M solution, pH 7.5).............................................50.0mL Glycerol .....................................................................................3.0mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Alteromonas hanedai, Photobacterium species, Shewanella hanedai, and Vibrio species.

LUP (Lupine Medium) Composition per liter: Agar ............................................................................................ 15.0g Lupine stems........................................................................... variable

Preparation of Medium: Add agar to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boil-

972

LuPhet1 Medium

ing. Distribute 6.0mL volumes into tubes. Cut lupine stems into 8.0cmlong pieces. Add 2–3 lupine stems per tube. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position.

Use: For the cultivation of Botryosphaeria berengeriana and Mycosphaerella ligulicola.

LuPhet1 Medium (DSMZ Medium 298e) Composition per liter: NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................10.0mL Sodium lactate solution............................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Yeast extract solution ...............................................................10.0mL Seven vitamin solution...............................................................1.0mL Selenite-tungstate solution .........................................................1.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Selenite-Tungstate Solution Composition per liter:

Na2S·9H2O Solution: Composition per 10.0mL:

NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Na2S·9H2O .................................................................................. 0.36g

Preparation of Selenite-Tungstate Solution: Add components

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Sodium Lactate Solution: Composition per 10.0mL: Sodium lactate............................................................................... 1.5g

Preparation of Sodium Lactate Solution: Add sodium lactate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except NaHCO3 solution, sodium lactate solution, Na2S·9H2O solution, seven vitamin solution, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 957.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL NaHCO3 solution, 10.0mL Na2S·9H2O solution, 10.0mL sodium lactate solution, 1.0mL seven vitamin solution, 1.0mL selenite- tungstate solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% N2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Acetobacterium sp. Luria Agar See: L Agar

Yeast Extract Solution: Composition per 10.0mL: Yeast extract.................................................................................. 1.0g

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg © 2010 by Taylor and Francis Group, LLC

Luria Agar Base, Miller Composition per liter: Agar ............................................................................................ 15.0g Tryptone...................................................................................... 10.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 0.5g pH 7.0 ± 0.2 at 25°C

LuTria3 Medium Use: For the cultivation and maintenance of bacteria for genetic and molecular studies.

Luria Bertani Agar, Miller (LB Agar, Miller) Composition per liter: Agar ............................................................................................ 15.0g Tryptone ...................................................................................... 10.0g NaCl ............................................................................................ 10.0g Yeast extract.................................................................................. 5.0g pH 7.5 ± 0.2 at 25°C

Luria Broth, HiVeg Composition per liter: Plant hydrolysate ........................................................................ 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Escherichia coli.

Composition per liter:

Use: For the cultivation and maintenance of bacteria for genetic and molecular studies.

Luria Bertani HiVeg Agar, Miller Composition per liter: Agar ............................................................................................ 15.0g Plant hydrolysate......................................................................... 10.0g NaCl ............................................................................................ 10.0g Yeast extract.................................................................................. 5.0g pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation and maintenance of bacteria for genetic and molecular studies. For the cultivation and maintenance of recombinant strains of Escherichia coli for genetic studies.

Luria Bertani HiVeg Broth, Miller

973

Luria HiVeg Agar Agar ............................................................................................ 15.0g Plant hydrolysate ........................................................................ 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the routine cultivation and estimation of not particularly fastidious microorganisms.

Luria HiVeg Agar with Glucose Composition per liter: Agar ............................................................................................ 15.0g Plant hydrolysate ........................................................................ 10.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Glucose solution ......................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Composition per liter:

Source: This medium, without glucose solution, is available as a pre-

Plant hydrolysate......................................................................... 10.0g NaCl ............................................................................................ 10.0g Yeast extract.................................................................................. 5.0g pH 7.5 ± 0.2 at 25°C

mixed powder from HiMedia.

Glucose Solution: Composition per 100.0mL:

Source: This medium is available as a premixed powder from HiMe-

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

dia.

Use: For the cultivation of bacteria for genetic and molecular studies. For the cultivation and maintenance of recombinant strains of Escherichia coli for genetic studies.

Glucose ....................................................................................... 10.0g

Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Bring pH to 7.0. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile glucose solution. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Escherichia coli.

LuTria3 Medium (DSMZ Medium 298d) Composition per liter: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g

974

LY Agar for Filobasidium

MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................10.0mL Butanediol solution ..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Seven vitamin solution.............................................................10.0mL Triethanolamine hydrochloride solution..................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.36g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Butanediol Solution: Composition per 10.0mL: 2,3 butanediol................................................................................ 0.9g

Preparation of Butanediol Solution: Add butanediol to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

oughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Triethanolamine Hydrochloride Solution: Composition per 10.0mL: Triethanolamine hydrochloride..................................................... 1.4g

Preparation of Triethanolamine Hydrochloride Solution: Add triethanolamine hydrochloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Adjust pH to 7.0. Sparge with 100% N2. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except NaHCO3 solution, butanediol solution, Na2S·9H2O solution, seven vitamin solution, triethanolamine hydrochloride solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 949.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL NaHCO3 solution, 10.0mL butanediol solution, 10.0mL Na2S·9H2O solution, 10.0mL seven vitamin solution, 10.0mL triethanolamine hydrochloride solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% N2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Acetobacterium sp.

LY Agar for Filobasidium Composition per liter: Agar ............................................................................................ 15.0g Lactose.......................................................................................... 1.0g Yeast extract.................................................................................. 0.5g

Use: For the cultivation and maintenance of Filobasidium floriforme. Lyngby Iron Agar See: Iron Agar, Lyngby

Lysine Agar, Selective Composition per liter: Agar ............................................................................................ 15.0g L-Lysine....................................................................................... 10.0g Peptone ......................................................................................... 5.0g Glucose ......................................................................................... 3.5g Yeast extract.................................................................................. 3.0g Bile salts mixture .......................................................................... 1.5g Sulfapyridine................................................................................. 0.3g Bromcresol Purple ...................................................................... 0.03g Crystal Violet ............................................................................ 0.001g pH 6.8 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Lysine Decarboxylase Broth, Taylor Modification Use: For the selective isolation and cultivation of Salmonella species from food by the hydrophobic grid membrane filter method.

Lysine Arginine Iron Agar Composition per liter: Agar ............................................................................................ 15.0g L-Arginine ................................................................................... 10.0g L-Lysine....................................................................................... 10.0g

Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Sodium thiosulfate ...................................................................... 0.04g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.8. Distribute into screw-capped tubes in 5.0mL volumes. Autoclave for 12 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position. Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate lysine, decarboxylate arginine, and produce H2S. Bacteria that decarboxylate lysine or arginine turn the medium purple. Bacteria that produce H2S appear as black colonies.

975

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH so that is will be 6.5 ± 0.2 after sterilization. Distribute into 16 × 150mm screw-capped tubes in 5.0mL volumes. Autoclave medium with loosely capped tubes for 10 min at 15 psi pressure–121°C. Screw the caps on tightly for storage and after inoculation.

Use: For the cultivation and differentiation of Vibrio spp. based on their ability to decarboxylate the amino acid lysine. Bacteria that decarboxylate lysine turn the medium turbid purple.

Lysine Decarboxylase Broth, Falkow Composition per liter: Peptone ......................................................................................... 5.0g L-Lysine......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.5–6.8 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.5–6.8. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria, especially Salmonella, based on their ability to decarboxylate lysine. Bacteria that decarboxylate lysine turn the medium turbid purple.

Lysine Arginine Iron HiVeg Agar Composition per liter: Agar ............................................................................................ 15.0g L-Arginine ................................................................................... 10.0g L-Lysine....................................................................................... 10.0g Plant peptone................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ...................................................................................... 0.04g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Lysine Decarboxylase Broth, Taylor Modification Composition per liter: L-Lysine......................................................................................... 5.0g

Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.1. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria, especially Salmonella, based on their ability to decarboxylate lysine. Bacteria that decarboxylate lysine turn the medium turbid purple.

Lysine Decarboxylase Broth, Taylor Modification (Lysine Decarboxylase Broth) Composition per liter: L-Lysine......................................................................................... 5.0g

Lysine Broth Falkow with Sodium Chloride (BAM M44) Composition per liter: L-Lysine......................................................................................... 5.0g Peptone or gelysate ....................................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.5 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Peptone ......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring

976

Lysine Decarboxylase HiVeg Broth

to boiling. Adjust pH to 6.1. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria, especially Salmonella, based on their ability to decarboxylate lysine. Bacteria that decarboxylate lysine turn the medium turbid purple.

Lysine Decarboxylase HiVeg Broth Composition per liter: Plant peptone................................................................................. 5.0g L-Lysine hydrochloride ................................................................. 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically distribute into sterile tubes in 1.0mL volumes.

Use: For the cultivation and differentiation of members of the Enterobacteriaceae based on their ability to decarboxylate lysine and to form H2S. Bacteria that decarboxylate lysine turn the medium purple. Bacteria that produce H2S appear as black colonies.

Lysine Iron Cystine HiVeg Broth Base with Novobiocin Composition per liter: L-Lysine

hydrochloride ............................................................... 10.0g Plant hydrolysate .......................................................................... 5.0g Mannitol........................................................................................ 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Salicin ........................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ........................................................................................ 0.1g L-Cystine....................................................................................... 0.1g Neutral Red............................................................................... 0.025g Novobiocin solution.................................................................10.0mL pH 6.2 ± 0.2 at 25°C

Source: This medium, without novobiocin solution, is available as a

Use: For the cultivation and differentiation of Gram-negative, nonfer-

premixed powder from HiMedia.

mentative bacteria based on their ability to decarboxylate lysine. Bacteria that decarboxylate lysine turn the medium purple.

Novobiocin Solution: Composition per 10.0mL: Novobiocin ............................................................................... 0.015g

Lysine Decarboxylase Medium Composition per liter: Glucose ......................................................................................... 0.5g KH2PO4 ......................................................................................... 0.5g L-Lysine·HCl ................................................................................. 0.5g pH 4.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Preparation of Novobiocin Solution: Add novobiocin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 4.6. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically distribute into sterile tubes in 1.0mL volumes.

Use: For the rapid, presumptive detection of Salmonella in foods, food

Use: For the cultivation and differentiation of Gram-negative, nonfer-

ingredients, and feed materials.

mentative bacteria based on their ability to decarboxylate lysine. Bacteria that decarboxylate lysine turn the medium turbid purple.

Lysine Iron Cystine Neutral Red Broth See: LICNR Broth

Lysine Iron Agar Composition per liter:

Lysine Iron HiVeg Agar Composition per liter:

Agar ............................................................................................ 13.5g L-Lysine....................................................................................... 10.0g Pancreatic digest of gelatin ........................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3·5H2O ............................................................................ 0.04g Bromcresol Purple ...................................................................... 0.02g pH 6.7 ± 0.2 at 25°C

Agar ............................................................................................ 15.0g L-Lysine ...................................................................................... 10.0g Plant peptone ................................................................................ 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Na2S2O3 ...................................................................................... 0.04g Bromcresol Purple ...................................................................... 0.02g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Source: This medium is available as a premixed powder from HiMe-

agnostic Systems and Oxoid Unipath.

dia.

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes in 10.0mL volumes. Autoclave for 12 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position.

Lysobacter deserti Medium Use: For the cultivation and differentiation of members of the Enterobacteriaceae based on their ability to decarboxylate lysine and to form H2S. Bacteria that decarboxylate lysine turn the medium purple. Bacteria that produce H2S appear as black colonies. For the differentiation of enteric organisms, especially Salmonella serotype Arizona.

Lysine Lactose HiVeg Broth Composition per liter: Lactose ........................................................................................ 10.0g Plant peptone No. 2....................................................................... 5.0g L-Lysine......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically distribute into sterile tubes in 1.0mL volumes.

Use: For the determination of lysine decarboxylase activity of lactose nonfermenting members of Enterobacteriaceae, especially Salmonella species.

Lysine Medium Composition per liter: Glucose ....................................................................................... 44.5g Agar ............................................................................................ 17.8g KH2PO4 ....................................................................................... 1.78g Lysine............................................................................................ 1.0g MgSO4·7H2O .............................................................................. 0.89g CaCl2·2H2O............................................................................... 0.178g NaCl .......................................................................................... 0.089g Inositol ........................................................................................ 0.02g Calcium pantothenate ................................................................ 2.0mg Adenine .................................................................................... 1.78mg DL-Methionine.......................................................................... 0.89mg L-Histidine................................................................................ 0.89mg DL-Tryptophan.......................................................................... 0.89mg Thiamine·HCl ............................................................................ 0.4mg Pyridoxine .................................................................................. 0.4mg Nicotinic acid ............................................................................. 0.4mg FeSO4·7H2O............................................................................. 0.22mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg MnSO4·H2O ........................................................................... 0.035mg ZnSO4·7H2O .......................................................................... 0.035mg (NH4)2MoO4·4H2O ................................................................ 0.018mg H3BO3 .........................................................................................8.9μg Biotin ..........................................................................................2.0μg Folic acid.....................................................................................1.0μg Potassium lactate (50% solution).............................................10.0mL Lactic acid..................................................................................1.0mL pH 4.8 ± 0.2 at 25°C

977

maining components, except lactic acid. Mix thoroughly. Gently heat while stirring and bring to boiling. Do not autoclave. Cool to 50°C. Adjust pH to 4.8 by adding 1.0mL of lactic acid. Pour into sterile Petri dishes. Dry the surface of the plates by incubation at 37°C for 24 hr.

Use: For the isolation, cultivation, and enumeration of wild yeasts encountered in brewing.

Lysine Ornithine Mannitol Agar (LOM Agar) Composition per liter: Agar ............................................................................................ 13.5g L–Ornithine·HCl ........................................................................... 6.5g D–Mannitol ................................................................................. 5.25g L–Lysine·HCl ................................................................................ 5.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Bromthymol Blue ......................................................................... 0.3g Vancomycin solution ...............................................................10.0mL pH 6.5 ± 0.2 at 25°C

Vancomycin Solution: Composition per 10.0mL: Vancomycin·HCl......................................................................... 0.03g

Preparation of Vancomycin Solution: Add vancomycin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vancomycin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile vancomycin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and differentiation of Gram-negative bacilli based on their ability to decarboxylate lysine or ornithine and mannitol fermentation. Especially useful for the identification of Enterobacter agglomerans. Bacteria that ferment mannitol appear as dark yellow colonies. Bacteria that decarboxylate lysine or ornithine appear as green-yellow colonies.

Lysobacter deserti Medium (DSMZ Medium 1060) Composition per liter: Solution A..............................................................................700.0mL Solution B ..............................................................................300.0mL pH 8.1 ± 0.2 at 25°C

Solution A: Composition per 700.0mL:

Source: This medium is available as a premixed powder from Oxoid

Agar ............................................................................................ 15.0g Glucose ...................................................................................... 10.0g Peptone ......................................................................................... 5.0g Yeast extract.................................................................................. 5.0g K2HPO4 ........................................................................................ 1.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 40.0g

Unipath.

Preparation of Solution A: Add components to distilled/deionized

water and bring volume to 700.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

978

Lysozyme Broth

Solution B: Composition per 300.0mL:

into bottles in 99.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

NaCl ............................................................................................ 20.0g Na2CO3 ......................................................................................... 1.0g

Lysozyme Solution: Composition per 100.0mL:

Preparation of Solution B: Add components to distilled/deionized

Lysozyme...................................................................................... 0.1g

water and bring volume to 300.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Preparation of Lysozyme Solution: Add lysozyme to distilled/

Preparation of Medium: Aseptically combine 700.0mL sterile solution A and 300.0mL sterile solution B. Mix thoroughly. Adjust pH to 8.1. Pour into Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Lysobacter deserti.

Lysozyme Broth Composition per 1005.0mL: Basal glycerol broth ......................................................................1.0L Lysozyme solution .....................................................................5.0mL

Basal Glycerol Broth: Composition per liter: Peptone.......................................................................................... 5.0g Beef extract ................................................................................... 3.0g Glycerol ...................................................................................70.0mL

Preparation of Basal Glycerol Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute 500.0mL of the broth into screw-capped tubes in 5.0mL volumes. Autoclave the tubes and the flask with the remaining broth for 15 min at 15 psi pressure–121°C. Cool to 25°C.

deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add 1.0mL of sterile lysozyme solution to 99.0mL of cooled, sterile nutrient broth. Mix thoroughly. Aseptically distribute into sterile tubes in 2.5mL volumes. Use: For the cultivation and differentiation of Bacillus cereus in foods. Bacillus cereus is resistant to lysozyme and will grow in this medium.

M Broth Composition per liter: Pancreatic digest of casein.......................................................... 12.5g K2HPO4......................................................................................... 5.0g NaCl.............................................................................................. 5.0g Sodium citrate............................................................................... 5.0g Yeast extract.................................................................................. 5.0g Mannose........................................................................................ 2.0g MgSO4·7H2O ................................................................................ 0.8g Polysorbate 80 ............................................................................ 0.75g FeSO4 .......................................................................................... 0.04g pH 7.0 ± 0.22 at 25°C

Source: Available as a premixed powder from BD Diagnostic Sys-

Lysozyme Solution: Composition per 100.0mL:

tems.

Lysozyme ...................................................................................... 0.1g HCl (0.01N solution)..............................................................100.0mL

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Lysozyme Solution: Add lysozyme to 100.0mL

Use: For the detection of Salmonella in dried foods and feeds.

Preparation of Medium: Add components to distilled/deionized

of HCl solution. Mix thoroughly. Filter sterilize. Store for up to 1 week at 4°C.

Preparation of Medium: Add 5.0mL of the sterile lysozyme solution to 95.0mL of the cooled, sterile basal glycerol broth. Mix thoroughly. Aseptically distribute into sterile screw-capped tubes in 5.0mL volumes.

Use: For the cultivation and differentiation of Nocardia asteroides, Streptomyces griseus, and Actinomadura madurae based on sensitivity to lysozyme. Nocardia asteroides grows well in both the basal glycerol broth and the lysozyme broth. Actinomadura madurae and Streptomyces griseus grow well in the basal glycerol broth but not in the lysozyme broth.

Lysozyme Broth

M Medium Composition per liter: Beef............................................................................................... 5.0g Neopeptone ................................................................................... 4.0g NaCl.............................................................................................. 1.6g Glucose ......................................................................................... 0.5g CaCl2 ........................................................................................... 0.06g KH2PO4....................................................................................... 0.06g KCl.............................................................................................. 0.04g Rabbit blood solution.............................................................200.0mL

Rabbit Blood Solution: Composition per liter:

Composition per 1010.0mL:

Rabbit blood...........................................................................500.0mL

Nutrient broth................................................................................1.0L Lysozyme solution ...................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Preparation of Rabbit Blood Solution: Add 500.0mL of whole

Nutrient Broth: Composition per liter:

Preparation of Medium: Trim beef to remove fat. Add 5.0g of lean beef to 200.0mL of distilled/deionized water. Gently heat and bring to boiling. Boil for 2–3 min. Filter through Whatman #2 filter paper. Add other components, except rabbit blood solution. Bring volume to 800.0mL with distilled/deionized water. Mix thoroughly. Adjust pH to 7.2 with 1N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 200.0mL of lysed rabbit blood solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Pancreatic digest of gelatin ........................................................... 5.0g Beef extract ................................................................................... 3.0g

Source: Nutrient broth is available as a premixed powder from BD Diagnostic Systems. Preparation of Nutrient Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute © 2010 by Taylor and Francis Group, LLC

rabbit blood to 500.0mL of sterile distilled/deionized water. Freeze and thaw twice to lyse blood cells.

M1A Medium Use: For the cultivation of Herpetomonas megaseliae.

M1 Medium Composition per liter: L-Leucine....................................................................................... 2.0g L-Alanine....................................................................................... 1.0g L-Isoleucine ................................................................................... 1.0g L-Phenylalanine............................................................................. 1.0g L-Proline ........................................................................................ 1.0g L-Tryptophane ............................................................................... 1.0g L-Asparagine ................................................................................. 0.5g L-Lysine......................................................................................... 0.5g L-Methionine ................................................................................. 0.5g L-Tyrosine...................................................................................... 0.4g L-Valine ......................................................................................... 0.2g L-Serine ......................................................................................... 0.2g

MgSO4·7H2O ................................................................................ 0.2g NaCl .............................................................................................. 0.2g KH2PO4 ....................................................................................... 0.14g L-Arginine ..................................................................................... 0.1g L-Cysteine...................................................................................... 0.1g L-Glycine....................................................................................... 0.1g L-Histidine..................................................................................... 0.1g L-Threonine ................................................................................... 0.1g CaCl2 .......................................................................................... 2.0mg FeCl3·6H2O ................................................................................ 2.0mg Tris(hydroxymethyl)aminomethane buffer (0.01M solution, pH 7.6).............................................1.0L pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add solid components to 1.0L of Tris buffer. Mix thoroughly. Filter sterilize. Aseptically distribute into tubes or flasks.

Use: For the cultivation of Myxococcus xanthus.

M1-Nocardiopsis arabia Medium (DSMZ Medium 1065) Composition per liter: NaCl ........................................................................................... 20.0g Agar .......................................................................................... 18.0 g Starch ......................................................................................... 10.0g Yeast extract ................................................................................. 4.0g Peptone ......................................................................................... 2.0g Seawater .......................................................................................1.0L pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to seawater and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Pour into Petri dishes or leave in tubes.

Use: For the cultivation of Nocardiopsis arabia.

M1A Medium Composition per 1001.0mL: Sorbitol........................................................................................ 23.3g Peptone.......................................................................................... 6.0g Sucrose.......................................................................................... 3.3g Pancreatic digest of casein ............................................................ 3.3g Beef heart infusion........................................................................ 2.0g Glucose ......................................................................................... 1.3g © 2010 by Taylor and Francis Group, LLC

979

Yeast extract.................................................................................. 1.0g Fructose......................................................................................... 0.3g Phenol Red............................................................................... 20.0mg Schneider’s Drosophila medium ...........................................533.0mL Fetal calf serum, heat inactivated ..........................................167.0mL Fresh yeast extract solution .....................................................33.0mL Penicillin solution ......................................................................8.0mL

Schneider’s Drosophila Medium: Composition per liter: MgSO4·7H2O ................................................................................ 3.7g NaCl.............................................................................................. 2.1g Yeast extract.................................................................................. 2.0g Trehalose....................................................................................... 2.0g D-Glucose ...................................................................................... 2.0g L-Glutamine .................................................................................. 1.8g L-Lysine·HCl ................................................................................. 1.7g L-Proline........................................................................................ 1.7g KCl................................................................................................ 1.6g Na2HPO4·7H2O............................................................................. 1.3g L-Glutamic acid............................................................................. 0.8g L-Methionine ................................................................................. 0.8g CaCl2, anhydrous .......................................................................... 0.6g KH2PO4......................................................................................... 0.5g β-Alanine ...................................................................................... 0.5g L-Tyrosine ..................................................................................... 0.5g L-Arginine ..................................................................................... 0.4g L-Aspartic acid .............................................................................. 0.4g L-Histidine..................................................................................... 0.4g L-Threonine ................................................................................... 0.4g NaHCO3 ........................................................................................ 0.4g Glycine.......................................................................................... 0.3g L-Serine ......................................................................................... 0.3g L-Valine ......................................................................................... 0.3g L-Isoleucine ................................................................................... 0.2g L-Leucine ...................................................................................... 0.2g L-Phenylalanine............................................................................. 0.2g α-Ketoglutaric acid....................................................................... 0.2g Fumaric acid ................................................................................. 0.1g Malic acid ..................................................................................... 0.1g Succinic acid................................................................................. 0.1g L-Cystine ....................................................................................... 0.1g L-Tryptophan................................................................................. 0.1g L-Cysteine ................................................................................... 0.06g

Preparation of Schneider’s Drosophila Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Penicillin Solution: Composition per 10.0mL: Penicillin ........................................................................... 2,500,000U

Preparation of Penicillin Solution: Add penicillin to distilled/deionized water and bring volume to 10.0mL. Filter sterilize.

Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

980

M3 Agar

Preparation of Medium: Add components—except Schneider’s Drosophila medium, fetal calf serum, fresh yeast extract solution, and penicillin solution— to distilled/deionized water and bring volume to 260.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 533.0mL of sterile Schneider’s Drosophila medium, 167.0mL of sterile fetal calf serum, 33.0mL of sterile fresh yeast extract solution, and 8.0mL of sterile penicillin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and cultivation of Spiroplasma species that cause corn stunt.

M3 Agar Composition per 1020.0mL: Agar ............................................................................................ 18.0g Na2HPO4 ................................................................................... 0.732g KH2PO4 ..................................................................................... 0.466g NaCl ............................................................................................ 0.29g Sodium propionate ........................................................................ 0.2g MgSO4·7H2O ................................................................................ 0.1g CaCO3 ......................................................................................... 0.02g KNO3 .......................................................................................... 0.01g FeSO4·7H2O............................................................................... 0.2mg ZnSO4·7H2O ............................................................................ 0.18mg MnSO4·4H2O ........................................................................... 0.02mg Cycloheximide solution ...........................................................10.0mL Thiamine·HCl solution.............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Cycloheximide Solution: Composition per 10.0mL: Cycloheximide ............................................................................ 0.05g

Preparation of Cycloheximide Solution: Add cycloheximide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation. Thiamine·HCl Solution: Composition per 10.0mL: Thiamine·HCl ............................................................................ 4.0mg

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except cycloheximide solution and thiamine·HCl solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add 10.0mL of sterile cycloheximide solution and 10.0mL of thiamine·HCl solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Sodium propionate........................................................................ 0.2g KNO3 ............................................................................................ 0.1g MgSO4·7H2O ................................................................................ 0.1g CaCO3 ......................................................................................... 0.02g Thiamine·HCl ............................................................................ 4.0mg FeSO4·7H2O............................................................................... 0.2mg ZnSO4·7H2O ............................................................................ 0.18mg MnSO4·4H2O ........................................................................... 0.02mg Cycloheximide solution ...........................................................10.0mL Thiamine·HCl solution ............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Cycloheximide Solution: Composition per 10.0mL: Cycloheximide............................................................................ 0.04g

Preparation of Cycloheximide Solution: Add cycloheximide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Thiamine·HCl Solution: Composition per 10.0mL: Thiamine·HCl ............................................................................. 0.04g

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except cycloheximide solution and thiamine·HCl solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add sterile cycloheximide solution and thiamine·HCl solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Micromonospora species.

M3 Chytrid Agar Composition per liter: Agar ............................................................................................ 15.0g Soluble starch................................................................................ 5.0g Glucose ......................................................................................... 5.0g Corn meal, solids from infusion ................................................... 2.0g Peptone ......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g

Use: For the cultivation and maintenance of Rhyzophydium species.

M7 Medium

Use: For the selective isolation and cultivation of Nocardia species

Composition per liter:

and Rhodococcus species.

Yeast extract solution.............................................................200.0mL Dialyzed fetal bovine serum ..................................................100.0mL L-Methionine solution..............................................................30.0mL Buffer solution .........................................................................20.0mL Glucose solution ......................................................................20.0mL

M3 Agar Medium Composition per liter: Agar ............................................................................................ 18.0g Na2HPO4 ................................................................................... 0.732g KH2PO4 ..................................................................................... 0.466g NaCl ............................................................................................ 0.29g © 2010 by Taylor and Francis Group, LLC

Yeast Extract Solution: Composition per liter: Yeast extract................................................................................ 25.0g

M9 Medium with Arginine

981

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

MgSO4·7H2O Solution: Composition per liter:

L-Methionine

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Solution: Composition per liter:

L-Methionine................................................................................. 1.5g

Preparation of L-Methionine Solution: Add L-methionine to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Glucose Solution: Composition per liter: Glucose ..................................................................................... 270.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

MgSO4·7H2O ............................................................................ 246.5g

Thiamine·HCl Solution: Composition per 10.0mL: Thiamine·HCl .......................................................................... 10.0mg

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. CaCl2 Solution: Composition per liter: CaCl2 solution............................................................................. 14.7g

Preparation of CaCl2 Solution: Add CaCl2 solution to distilled/

Buffer Solution: Composition per liter: Na2HPO4 .................................................................................... 25.0g KH2PO4 ....................................................................................... 18.1g

Preparation of Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Dialyzed Fetal Bovine Serum: Composition per 100.0mL: Fetal bovine serum, heat inactivated......................................100.0mL

Preparation of Dialyzed Fetal Bovine Serum: Dialyze the heatinactivated serum at 0°–4°C against 10 volumes of water. Clean the dialysis tubing before use by boiling in a solution of 0.37g/L of EDTA and rinsing with water. Change the water four times at 8–16 hr intervals. Centrifuge the dialyzed serum for 30 min at 35,000 x g and filter sterilize.

Preparation of Medium: Aseptically combine the sterile solutions. Mix thoroughly. Bring volume to 1.0L with sterile distilled/deionized water.

Use: For the cultivation of Naegleria fowleri, Naegleria gruberi, and Nuclearia species.

M9 Medium Composition per liter:

deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except MgSO4·7H2O

solution, glucose solution, thiamine·HCl solution, and CaCl2 solution, to distilled/deionized water and bring volume to 987.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Aseptically add sterile MgSO4·7H2O solution, sterile glucose solution, sterile thiamine·HCl solution, and sterile CaCl2 solution. Mix thoroughly. Distribute into tubes or flasks.

Use: For the cultivation and maintenance of Escherichia coli and a variety of other bacteria.

M9 Medium with Arginine Composition per 1013.0mL: L-Proline

.................................................................................. 20.0mg

L-Arginine................................................................................ 20.0mg

10X M9 salts..........................................................................100.0mL Glucose solution ......................................................................10.0mL CaCl2·2H2O solution..................................................................1.0mL MgSO4 solution..........................................................................1.0mL Thiamine·HCl solution ..............................................................1.0mL pH 7.4 ± 0.2 at 25°C

10X M9 Salts: Composition per liter:

Na2HPO4 ....................................................................................... 6.0g KH2PO4 ......................................................................................... 3.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.5g Glucose solution ......................................................................10.0mL MgSO4·7H2O solution ...............................................................1.0mL Thiamine·HCl solution...............................................................1.0mL CaCl2 solution ............................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL:

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

D-Glucose .................................................................................... 20.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC

Preparation of 10X M9 Salts: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 20.0g

CaCl2·2H2O Solution: Composition per 100.0mL: CaCl2·2H2O ................................................................................ 1.47g

982

M9 Medium with Arginine

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis-

Preparation of Proline Solution: Add proline to distilled/deion-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

MgSO4 Solution: Composition per 100.0mL:

Calcium Chloride Solution: Composition per 10.0mL:

MgSO4 ...................................................................................... 12.04g

CaCl2 ............................................................................................ 0.1g

Preparation of MgSO4 Solution: Add MgSO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Calcium Chloride Solution: Add CaCl2 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thiamine·HCl Solution: Composition per 10.0mL:

Magnesium Sulfate Solution: Composition per 10.0mL:

Thiamine·HCl ............................................................................. 3.37g

MgSO4 .......................................................................................... 1.2g

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to

Preparation of Magnesium Sulfate Solution: Add MgSO4 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add L-proline, L-arginine, and 10X M9 salts to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile glucose solution, 1.0mL of sterile CaCl2·2H2O solution, 1.0mL of sterile MgSO4 solution, and 1.0mL of sterile thiamine·HCl solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

10X M9 Salts Solution: Composition per liter:

Use: For the cultivation of Escherichia coli.

Preparation of 10X M9 Salts Solution: Add components to dis-

M9 Medium with Arginine (DSMZ Medium 450) Composition per liter: 10X M9 salts ..........................................................................100.0mL Glucose solution ......................................................................10.0mL Calcium chloride solution ........................................................10.0mL Magnesium sulfate solution .....................................................10.0mL Thiamine solution ......................................................................1.0mL Proline solution ..........................................................................1.0mL Arginine solution .......................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 2.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Na2HPO4 ..................................................................................... 60.0g KH2PO4....................................................................................... 30.0g NH4Cl ......................................................................................... 10.0g NaCl.............................................................................................. 5.0g MgSO4 .......................................................................................... 1.2g tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine sterile component solutions. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Escherichia coli arginine auxotrophs.

M9 Medium with Casamino Acids Composition per liter: Na2HPO4 ....................................................................................... 6.0g Casamino acids ............................................................................. 5.0g KH2PO4......................................................................................... 3.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.5g Glucose solution ......................................................................10.0mL MgSO4·7H2O solution ...............................................................1.0mL Thiamine·HCl solution ..............................................................1.0mL CaCl2 solution............................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Thiamine Solution: Composition per 10.0mL: Thiamine-HCl·2H2O.................................................................... 3.7g

Glucose solution: Composition per 100.0mL:

Preparation of Thiamine Solution: Add thiamine-HCl·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Arginine Solution: Composition per 10.0mL:

MgSO4·7H2O Solution: Composition per liter:

Arginine ........................................................................................ 0.2g

MgSO4·7H2O ............................................................................ 246.5g

Preparation of Arginine Solution: Add arginine to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Proline Solution: Composition per 10.0mL:

Thiamine·HCl Solution: Composition per 10.0mL:

Proline ........................................................................................... 0.2g

Thiamine·HCl .......................................................................... 10.0mg

D-Glucose .................................................................................... 20.0g

M9 Medium with Tryptophan Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

CaCl2 Solution: Composition per liter:

983

Thiamine·HCl Solution: Composition per 10.0mL: Thiamine·HCl ............................................................................. 3.37g

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to

CaCl2 solution ............................................................................. 14.7g

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of CaCl2 Solution: Add CaCl2 solution to distilled/

Preparation of Medium: Add L-proline, L-tryptophan, and 10X

deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except MgSO4·7H2O

Use: For the cultivation and maintenance of Flavobacterium meningosepticum.

M9 Medium with Tryptophan Composition per 1013.0mL: L-Proline................................................................................... 20.0mg L-Tryptophan............................................................................ 20.0mg 10X M9 salts..........................................................................100.0mL Glucose solution ......................................................................10.0mL CaCl2·2H2O solution..................................................................1.0mL MgSO4 solution..........................................................................1.0mL Thiamine·HCl solution...............................................................1.0mL pH 7.4 ± 0.2 at 25°C

10X M9 Salts: Composition per liter:

M9 salts to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile glucose solution, 1.0mL of sterile CaCl2·2H2O solution, 1.0mL of sterile MgSO4 solution, and 1.0mL of sterile thiamine·HCl solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Escherichia coli.

M9 Medium with Tryptophan (DSMZ Medium 451) Composition per liter: 10X M9 salts..........................................................................100.0mL Glucose solution ......................................................................10.0mL Calcium chloride solution........................................................10.0mL Magnesium sulfate solution.....................................................10.0mL Thiamine solution ......................................................................1.0mL Proline solution..........................................................................1.0mL Tryptophan solution...................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Glucose Solution: Composition per 10.0mL: D-Glucose ...................................................................................... 2.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thiamine Solution: Composition per 10.0mL: Thiamine-HCl·2H2O ................................................................... 3.7g

Preparation of 10X M9 Salts: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4.

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Glucose Solution: Composition per 100.0mL:

Tryptophan Solution: Composition per 10.0mL:

Glucose ....................................................................................... 20.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Thiamine Solution: Add thiamine-HCl·2H2O to

Tryptophan................................................................................... .0.2g

Preparation of Tryptophan Solution: Add tryptophan to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

CaCl2·2H2O Solution: Composition per 100.0mL:

Proline Solution: Composition per 10.0mL:

CaCl2·2H2O................................................................................. 1.47g

Proline.......................................................................................... .0.2g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis-

Preparation of Proline Solution: Add proline to distilled/deion-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

MgSO4 Solution: Composition per 100.0mL:

Calcium Chloride Solution: Composition per 10.0mL:

MgSO4 ...................................................................................... 12.04g

CaCl2 ........................................................................................... .0.1g

Preparation of MgSO4 Solution: Add MgSO4 to distilled/deion-

Preparation of Calcium Chloride Solution: Add CaCl2 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

984

M10 Broth

Magnesium Sulfate Solution: Composition per 10.0mL: MgSO4 .......................................................................................... 1.2g

Preparation of Magnesium Sulfate Solution: Add MgSO4 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Hemin Solution: Add components to 100.0mL of distilled/deionized water. Mix thoroughly.

10X M9 Salts Solution: Composition per liter:

L-Cysteine·HCl

Na2HPO4 ..................................................................................... 60.0g KH2PO4 ....................................................................................... 30.0g NH4Cl ......................................................................................... 10.0g NaCl .............................................................................................. 5.0g MgSO4 .......................................................................................... 1.2g

L-Cysteine·HCl ........................................................................... 0.25g

Preparation of 10X M9 Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically combine sterile component solutions. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Escherichia coli tryptophan auxotrophs.

M10 Broth Composition per liter: Pancreatic digest of casein ............................................................ 2.0g Yeast extract.................................................................................. 2.0g Cellobiose ..................................................................................... 1.0g Glucose ......................................................................................... 1.0g Maltose.......................................................................................... 1.0g Starch ............................................................................................ 1.0g Resazurin ................................................................................... 1.0mg Mineral solution I...................................................................100.0mL Mineral solution II .................................................................100.0mL Na2CO3 solution......................................................................50.0mL Hemin solution.........................................................................10.0mL L-Cysteine·HCl solution...........................................................10.0mL Volatile fatty acid mixture..........................................................3.1mL pH 6.8 ± 0.2 at 25°C

Mineral Solution I: Composition per 100.0mL: K2HPO4 ......................................................................................... 0.2g

Preparation of Mineral Solution I: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Mineral Solution II: Composition per 100.0mL: NaCl .............................................................................................. 0.4g (NH4)2SO4 ..................................................................................... 0.4g KH2PO4 ......................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.09g CaCl2 ........................................................................................... 0.05g

Preparation of Mineral Solution II: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Na2CO3 Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 8.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deion-

ized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution: Composition per 10.0mL:

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Volatile Fatty Acid Mixture: Composition per 31.0mL: Acetic acid ...............................................................................17.0mL Propionic acid ............................................................................6.0mL Butyric acid................................................................................4.0mL DL-α-Methylbutyric acid ...........................................................1.0mL Isobutyric acid ...........................................................................1.0mL Isovaleric acid............................................................................1.0mL n-Valeric acid .............................................................................1.0mL

Preparation of Volatile Fatty Acid Mixture: Combine components. Mix thoroughly. Store under 100% N2.

Preparation of Medium: Prepare and dispense medium under 100% CO2. Add components, except L-cysteine·HCl solution and Na2CO3 solution, to distilled/deionized water and bring volume to 930.0mL. Mix thoroughly. Sparge with 100% CO2. Adjust pH to 6.8 with 1N NaOH. Distribute anaerobically in 9.3mL volumes into Hungate tubes. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 0.2mL of sterile L-cysteine·HCl solution and 0.5mL of sterile Na2CO3 solution. Check that final pH is 6.8.

Use: For the cultivation of Enterococcus species, Lactobacillus species, Streptococcus species, and Vagococcus fluvialis.

M13 Verrucomicrobium Medium Composition per liter: Glucose ....................................................................................... 0.25g Peptone ....................................................................................... 0.25g Yeast extract................................................................................ 0.25g Distilled water........................................................................670.0mL Artificial seawater..................................................................250.0mL Tris-HCl buffer, (0.1M solution, pH 7.5).................................50.0mL Modified Huntner’s basal salts ................................................20.0mL Vitamin solution.......................................................................10.0mL pH 7.5 ± 0.2 at 25°C

Artificial Seawater: Composition per liter: NaCl.......................................................................................... 23.48g MgCl2.......................................................................................... 4.98g Na2SO4 ........................................................................................ 3.92g CaCl2 ............................................................................................. 1.1g KCl.............................................................................................. 0.66g NaHCO3 ...................................................................................... 0.19g H3BO3 ....................................................................................... 0.026g SrCl2.......................................................................................... 0.024g KBr ............................................................................................ 6.0mg NaF ............................................................................................ 3.0mg

M16 Agar Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O................................................................................. 3.34g FeSO4·7H2O............................................................................. 99.0mg (NH4)2MoO4 ............................................................................ 9.25mg Metals “44” ..............................................................................50.0mL

Metals “44”: Composition per 100.0mL: ZnSO4·7H2O ................................................................................. 1.1g FeSO4·7H2O.................................................................................. 0.5g EDTA .......................................................................................... 0.25g MnSO4·7H2O ............................................................................ 0.154g CuSO4·5H2O ............................................................................... 0.04g Co(NO3)2·6H2O ........................................................................ 0.025g Na2B4O7·10H2O........................................................................ 0.018g

Preparation of Metals “44”: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Vitamin Solution: Composition per liter: D-Calcium pantothenate ............................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except modified Hutner’s basal salts, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Aseptically add 20.0mL of sterile modified Hutner’s basal salts. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Verrucomicrobium spinosum.

M14 Medium Composition per liter: Yeast extract.................................................................................. 1.0g D-Glucose ...................................................................................... 1.0g Tris(hydroxymethyl)aminomethane.......................................... 0.753g Artificial seawater..................................................................250.0mL Modified Hutner’s basal salts ..................................................20.0mL pH 7.5 ± 0.2 at 25°C

985

CaCl2 ............................................................................................. 1.1g KCl.............................................................................................. 0.66g NaHCO3 ...................................................................................... 0.19g H3BO3 ....................................................................................... 0.026g SrCl2.......................................................................................... 0.024g KBr ............................................................................................ 6.0mg NaF ............................................................................................ 3.0mg

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O ................................................................................ 3.34g FeSO4·7H2O............................................................................. 99.0mg (NH4)2MoO4 ............................................................................ 9.25mg Metals “44”..............................................................................50.0mL

Metals “44”: Composition per 100.0mL: ZnSO4·7H2O ................................................................................. 1.1g FeSO4·7H2O.................................................................................. 0.5g EDTA .......................................................................................... 0.25g MnSO4·7H2O ............................................................................ 0.154g CuSO4·5H2O............................................................................... 0.04g Co(NO3)2·6H2O ........................................................................ 0.025g Na2B4O7·10H2O........................................................................ 0.018g

Preparation of Metals “44”: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components, except modified Hutner’s basal salts, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 20.0mL of sterile modified Hutner’s basal salts. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Pirellula marina.

M16 Agar Composition per liter: Agar ............................................................................................ 10.0g Beef extract................................................................................... 5.0g Pancreatic digest of soybean meal................................................ 5.0g Polypeptone™ ............................................................................... 5.0g Sodium acetate·3H2O.................................................................... 3.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g Carbohydrate solution..............................................................50.0mL pH 7.2 ± 0.2 at 25°C

Carbohydrate Solution: Composition per 50.0mL: Lactose or glucose ........................................................................ 5.0g

Preparation of Carbohydrate Solution: Add lactose or glucose to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize.

986

M17 Agar

Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2 with 2N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of sterile carbohydrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of lactobacilli from cheddar cheese.

M17 Agar (LMG Medium 261) Composition per liter: Disodium β-glycerophosphate .................................................... 19.0g Agar ............................................................................................ 11.0g Polypeptone™............................................................................... 5.0g Beef extract ................................................................................... 5.0g Papaic digest of soybean meal ...................................................... 5.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g Lactose solution .......................................................................50.0mL MgSO4·7H2O (1M solution) ......................................................1.0mL pH 6.9 ± 0.2 at 25°C

Lactose Solution: Composition per 100.0mL: Lactose ........................................................................................ 10.0g

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except lactose solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of sterile lactose solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of streptococci and their bacteriophages. Also used for the cultivation and maintenance of starter cultures for cheese and yogurt manufacture as well as detecting streptococcal mutants which are unable to ferment lactose.

M17 Broth Composition per liter: Disodium β-glycerophosphate.................................................... 19.0g Beef extract................................................................................... 5.0g Lactose.......................................................................................... 5.0g Papaic digest of soybean meal...................................................... 5.0g Pancreatic digest of casein............................................................ 2.5g Peptic digest of animal tissue ....................................................... 2.5g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g MgSO4·7H2O .............................................................................. 0.25g pH 7.15 ± 0.05 at 25°C

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Source: This medium is available as a premixed powder from BD Di-

Preparation of Medium: Add components, except lactose solu-

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

tion, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of sterile lactose solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

agnostic Systems and Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation and maintenance of streptococci and their bacteriophages. Also used for the cultivation and maintenance of starter cultures for cheese and yogurt manufacture as well as detecting streptococcal mutants that are unable to ferment lactose.

Use: For the cultivation of Streptococcus thermophilus and for the cultivation and maintenance of streptococci and their bacteriophages. Also used for the cultivation and maintenance of starter cultures for cheese and yogurt manufacture as well as detecting streptococcal mutants that are unable to ferment lactose

M17 Agar Composition per liter: Disodium β-glycerophosphate .................................................... 19.0g Agar ............................................................................................ 11.0g Beef extract ................................................................................... 5.0g Papaic digest of soybean meal ...................................................... 5.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g MgSO4·7H2O .............................................................................. 0.25g Lactose solution .......................................................................50.0mL pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

M17 HiVeg Agar Base with Disodium-β-glycerophosphate Composition per liter: Disodium-β-glycerophosphate ................................................... 19.0g Agar ............................................................................................ 10.0g Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Lactose.......................................................................................... 5.0g Papaic digest of soybean meal...................................................... 5.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g MgSO4 ........................................................................................ 0.25g pH 7.1 ± 0.2 at 25°C

Source: This medium, without disodium-β-glycerophosphate, is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Lactose Solution: Composition per 100.0mL:

Use: For the cultivation and maintenance of streptococci and their bac-

Lactose ........................................................................................ 10.0g

teriophages. Also used for the cultivation and maintenance of starter

M17 Medium, Modified

987

cultures for cheese and yogurt manufacture as well as detecting streptococcal mutants which are unable to ferment lactose.

Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

M17 Medium for Filomicrobium fusiforme (DSMZ Medium 768)

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Composition per liter: Na-acetate ..................................................................................... 1.0g KNO3 ............................................................................................ 1.0g Artificial seawater, concentrated............................................500.0mL Hutner's salts solution ..............................................................20.0mL Vitamin solution.......................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Hutner’s Salts Solution: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O............................................................................... 3.335g FeSO4·7H2O............................................................................. 99.0mg (NH4)6MoO7O24·4H2O ............................................................ 9.25mg “Metals 44” ..............................................................................50.0mL

“Metals 44”: Composition per 100.0mL: ZnSO4·7H2O ............................................................................. 1.095g FeSO4·7H2O.................................................................................. 0.5g Sodium EDTA............................................................................. 0.25g MnSO4·H2O.............................................................................. 0.154g CuSO4·5H2O ............................................................................ 39.2mg Co(NO3)2·6H2O ....................................................................... 24.8mg Na2B4O7·10H2O....................................................................... 17.7mg

Preparation of Medium: Add components, except artificial sea water and vitamin solution, to distilled/deionized water and bring volume to 490.0mL. Mix thoroughly. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 500.0mL artificial sea water and 10.0mL vitamin solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Filomicrobium fusiforme.

M17 Medium for Lactic Streptococci (DSMZ Medium 449) Composition per liter: Na2-ß-glycerophosphate ............................................................. 19.0g Peptone from casein...................................................................... 5.0g Soy peptone .................................................................................. 5.0g Peptone bacteriological................................................................. 5.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g MgSO4·7H2O .............................................................................. 0.25g Lactose solution .......................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Lactose Solution: Composition per 10.0mL:

Preparation of “Metals 44”: Add sodium EDTA to distilled/de-

Lactose.......................................................................................... 5.0g

ionized water and bring volume to 90.0mL. Mix thoroughly. Add a few drops of concentrated H2SO4 to retard precipitation of heavy metal ions. Add remaining components. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water.

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Hutner’s Salts Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Artificial Seawater, Concentrated: Composition per liter: NaCl .......................................................................................... 70.43g MgCl2·6H2O.............................................................................. 31.86g Na2SO4 ...................................................................................... 11.75g CaCl2·2H2O................................................................................. 4.35g NaHCO3 ...................................................................................... 2.88g KCl.............................................................................................. 1.99g KBr.............................................................................................. 0.29g H3BO3 ......................................................................................... 0.08g

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Vitamin Solution: Composition per liter: Riboflavin .................................................................................. 5.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Ca-pantothenate ......................................................................... 5.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Add components, except lactose solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL sterile lactose solution. Aseptically distribute to sterile tubes or flasks. Use: For the cultivation and maintenance of Lactococcus lactis subsp. lactis=Streptococcus lactis.

M17 Medium, Modified Composition per 1001.2mL: Disodium-ß-glycerophosphate...................................................... 9.5g Pancreatic digest of casein............................................................ 5.0g Meat peptone ................................................................................ 5.0g Papaic digest of soybean meal...................................................... 5.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g MgSO4·7H2O .............................................................................. 0.25g Lactose solution .......................................................................50.0mL CaCl2 solution............................................................................1.2mL pH 7.15 ± 0.05 at 25°C

Lactose Solution: Composition per 50.0mL: Lactose.......................................................................................... 8.0g

988

M40 Y Agar

Preparation of Lactose Solution: Add lactose to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize.

FeSO4·7H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg pH 7.0 ± 0.2 at 25°C

CaCl2 Solution: Composition per 100.0mL:

Preparation of Medium: Add components to distilled/deionized

CaCl2·2H2O................................................................................. 14.7g

Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except lactose solution and CaCl2 solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 50.0mL of sterile lactose solution and 1.2mL of sterile CaCl2 solution. Mix thoroughly. Aseptically adjust pH to 7.15 ± 0.05. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Streptococcus thermophilus. M40 Y See: Medium for Osmophilic Fungi

M40 Y Agar (Harrold’s Agar) Composition per liter: Sucrose...................................................................................... 400.0g Agar ............................................................................................ 20.0g Malt extract ................................................................................. 20.0g Yeast extract.................................................................................. 5.0g

Preparation of Medium: Add components, except sucrose, to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. In a separate flask, add sucrose to distilled/deionized water and bring volume to 600.0mL. Mix thoroughly. Autoclave both solutions separately for 15 min at 15 psi pressure–121°C. Cool to 50°C. Combine the sterile solutions. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Ascosphaera osmophila, Aspergillus halophilicus, Aspergillus penicilloides, Aspergillus restictus, Aspergillus tonophilus, Eremascus albus, Eremascus fertilis, Eurotium halophilicum, Eurotium herbariorum, Geomyces pulvereus, Monascus bisporus, Monascus eremophilus, Oidiodendron sindenia, Penicillium isariiforme, Penicillium ochro-chloron, Penicillium pinophilum, Physalospora tucumanensis, Polypaecilum pisce, Saccharomyces cerevisiae, Trichophaea abundans, Trichophaea contradicta, Wallemia sebi, Wardomyces dimerus, Xeromyces bisporus, and Zygosaccharomyces rouxii.

M56 Agar Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 8.7g KH2PO4 ......................................................................................... 5.3g D-Glucose ...................................................................................... 4.0g (NH4)2SO4 ..................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.1g L-Histidine................................................................................... 0.05g L-Leucine..................................................................................... 0.05g Uracil .......................................................................................... 0.03g Ca(NO3)2·4H2O.......................................................................... 5.0mg © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Escherichia coli.

M56 Medium Composition per liter: Na2HPO4 ....................................................................................... 8.7g KH2PO4......................................................................................... 5.3g D-Glucose ...................................................................................... 4.0g (NH4)2SO4 .................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.1g L-Histidine................................................................................... 0.05g L-Leucine .................................................................................... 0.05g Uracil .......................................................................................... 0.03g Ca(NO3)2·4H2O ......................................................................... 5.0mg FeSO4·7H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Escherichia coli.

M63 Medium, 5X Composition per liter: KH2PO4 ...................................................................................... 68.0g (NH4)2SO4 .................................................................................. 10.0g FeSO4·7H2O .............................................................................. 2.5mg Carbohydrate solution..............................................................10.0mL MgSO4·7H2O solution ...............................................................1.0mL pH 7.0 ± 0.2 at 25°C

Carbohydrate Solution: Composition per 100.0mL: Carbohydrate............................................................................... 20.0g

Preparation of Carbohydrate Solution: Add carbohydrate to distilled/deionized water and bring volume to 100.0mL. Glucose or glycerol may be used. Mix thoroughly. Filter sterilize.

MgSO4·7H2O Solution: Composition per 100.0mL: MgSO4·7H2O ............................................................................ 24.65g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except carbohydrate solution and MgSO4·7H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. To prepare medium for use (1×), aseptically dilute 200.0mL of 5× stock solution with 789.0mL of sterile distilled/deionized water. Aseptically add 10.0mL of sterile carbohydrate solution and 1.0mL of sterile MgSO4·7H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

MAB1 Medium Use: For the cultivation of Escherichia coli.

MA Medium Composition per 1002.0mL: Peptone........................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g Ribonucleic acid from Torula yeast .............................................. 1.0g Asolectin ....................................................................................... 0.2g Artificial seawater..................................................................500.0mL Vitamin solution.........................................................................2.0mL

Preparation of Medium: Emulsify asolectin in warm, distilled water before adding remaining powdered ingredients. Adjust pH to 7.2. Add vitamin mix and artificial seawater; readjust to pH 7.2, if necessary. Dispense 5.0mL per 16 x 125mm screw-capped test tube and autoclave at 121°C for 15 min.

Artificial Seawater: Composition per 500.0mL: Aqua-Marin sea salts .................................................................. 20.8g

Source: Aqua-Marin sea salts are available from Aquatrol, Inc., Anaheim, CA. Preparation of Artificial Seawater: Add Aqua-Marin sea salts to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly.

Vitamin Solution: Composition per 110.0mL: Thiamine·HCl ) ...................................................................... 150.0mg Calcium D-(+)-pantothenate................................................... 100.0mg Folic acid.................................................................................. 50.0mg Nicotinamide............................................................................ 50.0mg Pyridoxal·HCl .......................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg DL-6-Thioctic acid...................................................................... 1.0mg Biotin solution..........................................................................10.0mL

Biotin Solution: Composition per 10.0mL: Biotin ....................................................................................... 0.01mg

Preparation of Biotin Solution: Add biotin to 10.0mL of absolute ethanol. Mix thoroughly.

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. For long-term storage, preserve under nitrogen at −20°C. Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 2.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Anophryoides soldoi, Metanophrys diminuta, Mesanophrys chesapeakensis, Miamiensis avidus, Parauronema acutum, and Paranophrys species.

989

MA4 See: Malt Agar 4% MA4 with Lupine Stems See: Malt Agar 4% with Lupine Stems MA8 See: Malt Agar 8%

MAB1 Medium Composition per 1003.0mL: NaCl............................................................................................ 20.0g MgCl2·6H2O ................................................................................. 3.0g Na2SO4 .......................................................................................... 3.0g KCl................................................................................................ 0.5g NH4Cl ......................................................................................... 0.25g Yeast extract.................................................................................. 0.2g KH2PO4......................................................................................... 0.2g Sodium benzoate......................................................................... 0.15g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 1.0mg Wolfe’s vitamin solution..........................................................20.0mL NaHCO3 solution .....................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Na2SeO3/Na2WO4 solution........................................................1.0mL Sodium dithionite solution.........................................................1.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

MA1 See: Malt Agar 1%

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

MA2 See: Malt Agar 2%

Na2SeO3/Na2WO4 Solution: Composition per liter:

990

MACA with Maltose

Preparation of Na2SeO3/Na2WO4 Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Sodium Dithionite Solution: Composition per 10.0mL: Sodium dithioninium .................................................................... 0.2g

Preparation of Sodium Dithionite Solution: Add sodium dithioninium to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

MacConkey Agar Composition per liter: Pancreatic digest of gelatin......................................................... 17.0g Agar ............................................................................................ 13.5g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Bile salts........................................................................................ 1.5g Pancreatic digest of casein............................................................ 1.5g Peptic digest of animal tissue ....................................................... 1.5g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation, cultivation, and differentiation of coliforms and enteric pathogens based on the ability to ferment lactose. Lactose-fermenting organisms appear as red to pink colonies. Lactosenonfermenting organisms appear as colorless or transparent colonies.

Preparation of Trace Elements Solution SL-10: Prepare and dispense under 80% N2 + 20% CO2. Add FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare medium and dispense under 80% N2 + 20% CO2. Add components, except Wolfe’s vitamin solution, NaHCO3 solution, sodium dithionite solution, Na2S·9H2O solution, Na2SeO3/Na2WO4 solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of Wolfe’s vitamin solution, 10.0mL of sterile NaHCO3 solution, 10.0mL of sterile Na2S·9H2O solution, 1.0mL Na2SeO3/Na2WO4 solution, 1.0mL of sterile sodium dithionite solution, and 1.0mL of sterile trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of Desulfotomaculum species.

MACA with Maltose Composition per liter: Yeast extract................................................................................ 20.0g Agar ............................................................................................ 10.0g Maltose........................................................................................ 10.0g Glucose ....................................................................................... 10.0g Proteose peptone No. 3 ................................................................. 5.0g KH2PO4 ......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 0.1g pH 6.7 ± 0.2 at 25°C

MacConkey Agar Composition per liter: Peptone ....................................................................................... 20.0g Agar ............................................................................................ 12.0g Lactose........................................................................................ 10.0g Bile salts........................................................................................ 5.0g NaCl.............................................................................................. 5.0g Neutral Red............................................................................... 0.075g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

MacConkey Agar Base, HiVeg Composition per liter: Plant peptone .............................................................................. 17.0g Agar ............................................................................................ 13.5g NaCl.............................................................................................. 5.0g Plant peptone No. 3....................................................................... 3.0g Synthetic detergent ....................................................................... 1.5g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation and maintenance of Lactobacillus sanfran-

Preparation of Medium: Add components to distilled/deionized

cisco.

water and bring volume to 1.0L. Mix thoroughly. Gently heat while

MacConkey Agar without Crystal Violet with Sodium Chloride and 0.5% Sodium Taurocholate, HiVeg

stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and differentiation of lactose-fermenting and nonfermenting Gram-negative bacteria. Lactose-fermenting organisms appear as red to pink colonies. Lactose-nonfermenting organisms appear as colorless or transparent colonies.

MacConkey Agar with 0.15% Bile Salts, Crystal Violet, and Sodium Chloride, HiVeg

991

Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Peptone from meat........................................................................ 3.0g Bile salt mixture............................................................................ 1.5g 4-Methylumbelliferyl-β-D-glucuronide ........................................ 0.1g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................ 0.001g pH 7.1 ± 0.2 at 25°C

Source: This medium is available from Merck.

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Plant peptone No. 2..................................................................... 17.0g Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Plant hydrolysate........................................................................... 1.5g Plant peptone................................................................................. 1.5g Synthetic detergent........................................................................ 1.5g Neutral Red ................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Pour into sterile Petri dishes. The plates are clear and red to red-brown.

Use: For the isolation of Salmonella, Shigella, and coliform bacteria, in particular E. coli, from various materials. The bile salts and Crystal Violet largely inhibit the growth of Gram-positive microbial flora. Lactose together with the pH indicator Neutral Red are used to detect lactose-positive colonies and E. coli can be seen among these because of fluorescence under UV light.

MacConkey Agar with Sorbitol See: Sorbitol MacConkey Agar

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective isolation, cultivation, and differentiation of enteric pathogens, especially enterococci, in clinical specimens and in materials of sanitary importance.

MacConkey Agar, CS Composition per liter: Peptone........................................................................................ 17.0g Agar ............................................................................................ 13.5g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Proteose peptone ........................................................................... 3.0g Bile salts........................................................................................ 1.5g Neutral Red ................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a prepared medium from BD Diagnostic Systems.

Use: For the cultivation and differentiation of lactose-fermenting and lactose-nonfermenting Gram-negative bacteria while also controlling the swarming of Proteus species, if present. Lactose-fermenting organisms appear as red to pink colonies. Lactose-nonfermenting organisms appear as colorless or transparent colonies.

MacConkey Agar, Fluorocult (Fluorocult MacConkey Agar)

MacConkey Agar without Crystal Violet Composition per liter: Agar ............................................................................................ 12.0g Lactose........................................................................................ 10.0g Pancreatic digest of casein.......................................................... 10.0g Peptic digest of animal tissue ..................................................... 10.0g Bile salts........................................................................................ 5.0g NaCl.............................................................................................. 5.0g Neutral Red................................................................................. 0.05g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the detection of members of the Enterobacteriaceae and enterococci as well as some staphylococci. For the isolation and detection of coliforms and enteric pathogens from water and wastewater.

MacConkey Agar without Crystal Violet with Sodium Chloride and 0.5% Sodium Taurocholate, HiVeg Composition per liter: Agar ............................................................................................ 20.0g Plant peptone .............................................................................. 20.0g Lactose........................................................................................ 10.0g Synthetic detergent No. V............................................................. 5.0g NaCl.............................................................................................. 5.0g Neutral Red................................................................................. 0.04g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Composition per liter:

Media.

Peptone from casein.................................................................... 17.0g Agar ............................................................................................ 13.5g

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat while

992

MacConkey Agar without Crystal Violet and Sodium Chloride with 0.5% Sodium Taurocholate, HiVeg

stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation, cultivation, and differentiation of Vibrio spp. in clinical specimens and in materials of sanitary importance.

MacConkey Agar without Crystal Violet and Sodium Chloride with 0.5% Sodium Taurocholate, HiVeg Composition per liter: Agar ............................................................................................ 20.0g Plant peptone............................................................................... 20.0g Lactose ........................................................................................ 10.0g Synthetic detergent No. V............................................................. 5.0g Neutral Red ................................................................................. 0.04g pH 7.2 ± 0.2 at 25°C

Source: This medium, without NaCl, is available as a premixed powder from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation, cultivation, and differentiation of enteric pathogens, especially enterococci, in clinical specimens and in materials of sanitary importance.

MacConkey Agar without Salt Composition per liter: Peptone........................................................................................ 20.0g Agar ............................................................................................ 12.0g Lactose ........................................................................................ 10.0g Bile salts........................................................................................ 5.0g Neutral Red ............................................................................... 0.075g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems and Oxoid Unipath.

Use: For the isolation and detection of coliforms and enteric pathogens from urine. Provides a low electrolyte medium on which most Proteus species will not swarm and therefore avoids overgrowth of the plate.

MacConkey Agar No. 2 (MacConkey II Agar) Composition per liter:

Use: For the selective isolation, cultivation, and differentiation of enteric pathogens, especially enterococci, in clinical specimens and in materials of sanitary importance.

MacConkey Agar No. 3 Composition per liter: Peptone ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Bile salts No. 3.............................................................................. 1.5g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................ 0.001g pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Use: For the selective isolation, cultivation, and differentiation of enteric pathogens, especially Salmonella and Shigella, in clinical specimens and in foods.

MacConkey Broth Composition per liter: Pancreatic digest of gelatin......................................................... 20.0g Lactose........................................................................................ 10.0g Oxgall ........................................................................................... 5.0g Bromcresol Purple ...................................................................... 0.02g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. If testing 10.0mL samples, prepare medium double strength. Mix thoroughly. Gently heat while stirring until boiling. Distribute into test tubes containing inverted Durham tubes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the selective isolation and cultivation of coliforms in milk and water.

MacConkey Broth Composition per liter:

Peptone........................................................................................ 20.0g Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Bile salts No. 2.............................................................................. 1.5g Neutral Red ................................................................................. 0.05g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Peptone ....................................................................................... 20.0g Lactose........................................................................................ 10.0g Bile salts........................................................................................ 5.0g NaCl.............................................................................................. 5.0g Neutral Red............................................................................... 0.075g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Preparation of Medium: Add components to distilled/deionized

agnostic Systems and Oxoid Unipath.

water and bring volume to 1.0L. If testing 10.0mL samples, prepare

Source: This medium is available as a premixed powder from Oxoid Unipath.

MacConkey HiVeg Agar without Crystal Violet and Sodium Chloride

993

medium double strength. Mix thoroughly. Gently heat while stirring until boiling. Distribute into test tubes containing inverted Durham tubes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the selective isolation, cultivation, and differentiation of

Use: For the selective isolation and cultivation of coliforms in milk

MacConkey HiVeg Agar with Crystal Violet and Sodium Chloride

enteric bacteria.

and water.

MacConkey Broth, Purple

Composition per liter:

Peptone........................................................................................ 20.0g Lactose ........................................................................................ 10.0g Bile salts........................................................................................ 5.0g NaCl .............................................................................................. 5.0g Bromcresol Purple ...................................................................... 0.01g pH 7.4 ± 0.2 at 25°C

Plant peptone .............................................................................. 20.0g Agar ............................................................................................ 15.0g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Synthetic detergent ....................................................................... 1.5g Neutral Red................................................................................. 0.05g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder or tablets

Source: This medium is available as a premixed powder from Hi-

Composition per liter:

from Oxoid Unipath.

Use: For the selective isolation and cultivation of coliforms in milk and water.

MacConkey Broth, Purple, with Bromcresol Purple, HiVeg

Media.

Use: For the selective isolation, cultivation, and differentiation of enteric bacteria. For the identification of Enterobacteriaceae in the presence of coliforms and lactose nonfermenters from water and sewage.

MacConkey HiVeg Agar with 1.35% Agar, Crystal Violet, and Sodium Chloride

Composition per liter: Plant special peptone .................................................................. 23.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Synthetic detergent No. V............................................................. 2.0g Bromcresol Purple ...................................................................... 0.01g pH 7.2 ± 0.2 at 25°C

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Plant peptone No. 2..................................................................... 17.0g Agar ............................................................................................ 13.5g Lactose........................................................................................ 10.0g NaCl.............................................................................................. 5.0g Plant hydrolysate .......................................................................... 1.5g Plant peptone ................................................................................ 1.5g Sodium acetate (anhydrous) ......................................................... 1.5g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Use: For the selective isolation, cultivation, and differentiation of

Source: This medium is available as a premixed powder from Hi-

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

enteric bacteria, especially coliforms.

MacConkey HiVeg Agar with Bromthymol Blue Composition per liter: Plant peptone............................................................................... 17.0g Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Plant peptone No. 3....................................................................... 3.0g Synthetic detergent........................................................................ 1.5g Bromthymol Blue ....................................................................... 0.03g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Media.

Use: For the selective isolation and differentiation of lactose-fermenting and lactose-nonfermenting enteric bacteria.

MacConkey HiVeg Agar without Crystal Violet and Sodium Chloride Composition per liter: Plant peptone .............................................................................. 23.0g Agar ............................................................................................ 12.0g Lactose........................................................................................ 10.0g Synthetic detergent ....................................................................... 2.0g Neutral Red............................................................................... 0.075g pH 7.2 ± 0.2 at 25°C

994

MacConkey HiVeg Agar, Modified

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components to distilled/deionized

Media.

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the presumptive identification of coliforms from water.

MacConkey Sorbitol HiVeg Agar (Sorbitol HiVeg Agar)

Use: For the cultivation and differentiation of enteric bacteria, restricting swarming of Proteus species.

MacConkey HiVeg Agar, Modified Composition per liter: Plant peptone............................................................................... 17.0g Agar ............................................................................................ 13.5g Inositol ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Plant peptone No. 3....................................................................... 3.0g Synthetic detergent........................................................................ 1.5g Neutral Red ................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the isolation of Klebsiella species from water samples.

MacConkey HiVeg Broth (Double Strength) with Neutral Red

Composition per liter: Plant peptone .............................................................................. 17.0g Agar ............................................................................................ 13.5g D-Sorbitol.................................................................................... 10.0g NaCl.............................................................................................. 5.0g Plant peptone No. 5....................................................................... 3.0g Synthetic detergent No. I .............................................................. 1.5g Neutral Red................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the isolation and cultivation of pathogenic Escherichia coli.

M-Aeromonas Selective Agar Base, Havelaar Composition per liter:

Media.

Agar ............................................................................................ 13.0g Dextrin ........................................................................................ 11.4g Tryptose ........................................................................................ 5.0g NaCl.............................................................................................. 3.0g KCl................................................................................................ 2.0g Yeast extract.................................................................................. 2.0g MgSO4·7H2O ................................................................................ 0.1g Sodium deoxycholate.................................................................... 0.1g Bromthymol Blue ....................................................................... 0.08g FeCl3·6H2O ................................................................................. 0.06g pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available from HiMedia.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or leave in flasks. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized

Composition per liter: Plant peptone............................................................................... 46.0g Lactose ........................................................................................ 20.0g NaCl ............................................................................................ 10.0g Synthetic detergent........................................................................ 4.0g Neutral Red ................................................................................. 0.15g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Use: For the primary isolation of coliforms from large samples such as water or wastewater.

MacConkey HiVeg Broth Purple with Bromo Cresol Purple Composition per liter: Plant special peptone .................................................................. 23.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Synthetic detergent No. V............................................................. 2.0g Bromcresol Purple ...................................................................... 0.01g pH 7.2 ± 0.2 at 25°C

Use: For the detection of Aeromonas species in water and other liquid samples by the membrane filter technique.

Magnesium Oxalate Agar (MOX Agar) Composition per liter: Pancreatic digest of casein.......................................................... 15.0g Agar ............................................................................................ 15.0g Papaic digest of soybean meal...................................................... 5.0g NaCl.............................................................................................. 5.0g MgCl2·6H2O ................................................................................. 4.1g Sodium oxalate ........................................................................... 2.68g pH 7.4–7.6 at 25°C

Magnetospirillum Medium Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Yersinia enterocolitica from foods.

Magnetic Spirillum Growth Medium, Revised (MSGM, Revised) Composition per liter: Agar .............................................................................................. 1.3g KH2PO4 ....................................................................................... 0.68g Tartaric acid ................................................................................ 0.37g Succinic acid ............................................................................... 0.37g NaNO3......................................................................................... 0.12g Sodium acetate ............................................................................ 0.05g Ascorbic acid ............................................................................ 0.035g Wolfe’s vitamin solution ..........................................................10.0mL Wolfe’s mineral solution ............................................................5.0mL Ferric quinate solution ...............................................................2.0mL Resazurin (0.1% solution)........................................................0.45mL pH 6.75 ± 0.2 at 25°C

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Cyanocobalamin .....................................................................100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitriloacetic acid........................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Ferric Quinate Solution: Composition per 100.0mL: FeCl3 ........................................................................................... 0.27g Quinic acid.................................................................................. 0.19g © 2010 by Taylor and Francis Group, LLC

995

Preparation of Ferric Quinate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: To 1.0L of distilled/deionized water add components in the following order: Wolfe’s vitamin solution, Wolfe’s mineral solution, ferric quinate solution, resazurin, KH2PO4, NaNO3, ascorbic acid, tartaric acid, succinic acid, sodium acetate, and agar. Mix thoroughly after each addition. Adjust pH to 6.75 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically distribute into sterile screw-capped tubes. Fill tubes to capacity with medium. Use a heavy inoculum in each tube and do not introduce a headspace of air. Screw down caps tightly. Use: For the cultivation and maintenance of Aquaspirillum magnetotacticum.

Magnetospirillum Medium (DSMZ Medium 380) Composition per liter: KH2PO4....................................................................................... 0.68g L(+)-Tartaric acid ........................................................................ 0.37g Succinic acid............................................................................... 0.37g NaNO3 ........................................................................................ 0.12g Na-thioglycolate ......................................................................... 0.05g Na-acetate ................................................................................... 0.05g Resazurin ................................................................................... 0.5mg Vitamin solution.......................................................................10.0mL Trace elements solution .............................................................5.0mL Ferric quinate solution ...............................................................2.0mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

996

Magnetospirillum 2 Medium

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Ferric Quinate Solution: Composition per 100.0mL: FeCl3·6H2O ................................................................................. 0.45g Quinic acid .................................................................................. 0.19g

Preparation of Ferric Quinate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Magnetospirillum 2 Medium Composition per liter: Sodium acetate .............................................................................. 1.0g K2HPO4 ......................................................................................... 0.5g Sodium thioglycolate .................................................................... 0.5g NH4Cl ........................................................................................... 0.1g Yeast extract.................................................................................. 0.1g Ferric citrate ..............................................................................20.0μg pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Allow medium to stand upright at room temperature for 2 to 3 days before inoculation. Do not shake.

Use: For the cultivation and maintenance of Magnetospirillum gryphiswaldense.

Magnetospirillum Semi-solid Medium (DSMZ Medium 380) Composition per liter: Agar .............................................................................................. 1.3g KH2PO4 ....................................................................................... 0.68g L(+)-Tartaric acid ........................................................................ 0.37g Succinic acid ............................................................................... 0.37g NaNO3 ........................................................................................ 0.12g Na-thioglycolate.......................................................................... 0.05g Na-acetate ................................................................................... 0.05g Resazurin ................................................................................... 0.5mg Vitamin solution.......................................................................10.0mL Trace elements solution .............................................................5.0mL Ferric quinate solution ...............................................................2.0mL pH 6.8 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Ferric Quinate Solution: Composition per 100.0mL: FeCl3·6H2O ................................................................................. 0.45g Quinic acid.................................................................................. 0.19g

Preparation of Ferric Quinate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C.

Preparation of Medium: Add components, except vitamin solution and ferric quinate solution, to distilled/deionized water and bring volume to 988.0mL. Purge medium with N2 gas for 10 min. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptically and anerobically add 10.0mL vitamin solution and 2.0mL ferric quinate solution. Mix thoroughly. Purge medium with N2 gas for 10 min. Dispense 12mL of medium per 16 x 150mm anaerobe screwcap tube under N2 gas. Prior to inoculation, remove caps briefly under air, tighten the caps again, and wait several hours to establish oxygen gradients. The medium should be slightly pink in color. Strongly reduced conditions will not support growth of the organism. During growth O2 will be consumed, resazurin decolorized, and the pH increased. Feed oxygen (by adding air) and succinic acid from sterile 0.05M solution (to maintain pH below 7.0). If higher densities of magnetic cell are wanted, ferric quinate also can be fed. For transfer use cell material which has been concentrated at the glass wall of the culture vessel by means of a magnetic rod attached outside.

Maleate Medium for Pseudomonas fluorescens with Glucose and Phenol Red Use: For the cultivation of Magnetospirillum magnetotacticum (Aquaspirillum magnetotacticum) and Magnetospirillum gryphiswaldense.

Maintenance HiVeg Medium for B. subtilis ATCC 6633

997

Malachite Green Broth Composition per liter: Peptone ....................................................................................... 15.0g Beef extract................................................................................... 9.0g Malachite Green....................................................................... 0.01mg pH 7.3 ± 0.2 at 25°C

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Agar ............................................................................................ 15.0g Plant peptone................................................................................. 6.0g Plant hydrolysate........................................................................... 4.0g Yeast extract.................................................................................. 3.0g Plant extract .................................................................................. 1.5g Glucose ......................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of Bacillus subtilis.

Maintenance of L Antigen in Neisseria Composition per liter: Proteose peptone No. 3 ............................................................... 20.0g Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 5.0g NaCl .............................................................................................. 5.0g Glucose ......................................................................................... 0.5g Rabbit blood, defibrinated .....................................................100.0mL pH 7.4–7.6 at 25°C

Preparation of Medium: Add components, except rabbit blood, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 60°C. Aseptically add 100.0mL of sterile, defibrinated rabbit blood. Maintain at 75°C while shaking for 30 min. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Neisseria gonorrhoeae. Maintenance SCY Medium See: SCY Medium

Maintenance (SCY) HiVeg Medium Composition per liter: Agar ............................................................................................ 10.0g Sucrose.......................................................................................... 1.0g Plant hydrolysate......................................................................... 0.91g Yeast extract................................................................................ 0.25g NaCl ............................................................................................ 0.05g Papaic digest of soybean meal .................................................... 0.03g K2HPO4 ....................................................................................... 0.02g Thiamine .................................................................................... 0.4mg pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Use: For the cultivation of Pseudomonas aeruginosa.

Maleate Medium for Pseudomonas fluorescens Composition per liter: Agar ............................................................................................ 15.0g K2HPO4....................................................................................... 1.13g NH4NO3 ........................................................................................ 1.0g KH2PO4....................................................................................... 0.48g MgSO4·7H2O ................................................................................ 0.2g Potassium maleate solution......................................................8.61mL pH 7.0 ± 0.2 at 25°C

Potassium Maleate Solution: Composition per liter: Maleic acid.............................................................................. 116.07g KOH (10N solution) ..............................................................200.0mL

Preparation of Potassium Maleate Solution: Add maleic acid to distilled/deionized water and bring volume to 600.0mL. Slowly add KOH solution (generates heat). Bring volume to 1.0L with distilled/deionized water. Adjust pH to 7.0. Filter sterilize. Preparation of Medium: Add components, except potassium maleate solution, to distilled/deionized water and bring volume to 991.4mL. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 8.61mL of the potassium maleate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Pseudomonas fluorescens and Mycoplasma pneumoniae.

Maleate Medium for Pseudomonas fluorescens with Glucose and Phenol Red Composition per liter: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g K2HPO4....................................................................................... 1.13g NH4NO3 ........................................................................................ 1.0g KH2PO4....................................................................................... 0.48g MgSO4·7H2O ................................................................................ 0.2g Phenol Red.................................................................................. 0.04g Potassium maleate solution......................................................8.61mL pH 7.0 ± 0.2 at 25°C

998

Malonate Broth

Preparation of Medium: Add components, except potassium

Source: This medium is available as a premixed powder from BD Di-

maleate solution, to distilled/deionized water and bring volume to 991.4mL. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 8.61mL of the potassium maleate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

agnostic Systems.

Use: For the cultivation and maintenance of Pseudomonas fluorescens and Mycoplasma pneumoniae.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–118°C. Do not overheat or agar will not harden. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of yeasts and molds.

Malonate Broth Composition per liter: Sodium malonate .......................................................................... 3.0g NaCl .............................................................................................. 2.0g (NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ......................................................................................... 0.6g KH2PO4 ......................................................................................... 0.4g Bromthymol Blue ..................................................................... 0.025g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Malt Agar Composition per liter: Agar ............................................................................................ 20.0g Malt extract................................................................................. 12.5g

Use: For the cultivation and maintenance of fungi.

agnostic Systems.

Malt Agar, 1/3 Strength (ATCC Medium 2365)

Use: For the cultivation and differentiation of coliforms and other enteric organisms, particularly Enterobacter and Escherichia, based on their ability to utilize malonate as the sole carbon source and ammonium sulfate as the sole nitrogen source. Malonate-utilizing organisms turn the medium blue.

Malonate Broth, Ewing Modified Composition per liter: Sodium malonate .......................................................................... 3.0g NaCl .............................................................................................. 2.0g (NH4)2SO4 ..................................................................................... 2.0g Yeast extract.................................................................................. 1.0g Glucose ....................................................................................... 0.25g K2HPO4 ......................................................................................... 0.6g KH2PO4 ......................................................................................... 0.4g Bromthymol Blue ..................................................................... 0.025g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of coliforms and other enteric organisms, particularly Enterobacter and Escherichia, based on their ability to utilize malonate as a carbon source and ammonium sulfate as a nitrogen source. The small amount of yeast extract and glucose encourages the growth of some organisms that may be distressed or fail to respond. Malonate-utilizing organisms turn the medium blue.

Malt Agar Composition per liter: Malt extract ................................................................................. 30.0g Agar ............................................................................................ 15.0g pH 5.5 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Composition per liter: Agar ............................................................................................ 15.0g Malt extract................................................................................. 10.0g pH 5.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the cultivation of yeasts and molds.

Malt Agar, Blakeslee Composition per liter: Glucose ....................................................................................... 20.0g Malt extract................................................................................. 20.0g Agar ............................................................................................ 16.0g Mycological peptone .................................................................... 1.0g pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Acremonium chrysogenum, Agaricus bisporus, Agrocybe aegerita, Armillaria mellea, Aspergillus species, Aureobasidium pullulans, Basidiomycetes species, Candida species, Ceratocystis adiposa, Citeromyces matritensis, Cladosporium cucumerinum, Cochliobolus miyaheanus, Colletotrichum destructivum, Colletotrichum lindemuthianum, Cryptococcus albidus, numerous other Cryptococcus species, Debaryomyces polymorphus, Diaporthe magnusii, Diaporthephaseolorum, Drechslera spicifera, Fusarium graminearum, Geotrichum lactis, other Geotrichum species, Hanseniaspora uvarum, Hanseniaspora valbyensis, Hansenula subpelliculosa, Humicola species, Kloeckera apiculata, Kloeckera corticis, Kluyveromyces lodderi, Kluyveromyces marxianus, Metschnikowia pulcherrima, Monilinia fructigena,

Malt Agar 4% with Lupine Stems

Myrothecium verrucaria, Myxozyma melibiosi, Nadsonia fulvescens, Neurospora crassa, Octosporomyces octosporus, Pachysolen tannophilus, Paecilomyces variotii, Penicillium aurantiogriseum, many other Penicillium species, Pithoascus schumacheri, Rhizoctonia crocorum, Rhizomucor miehei, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula rubra, Saccharomyces cerevisiae, Saccharomyces exiguus, Saccharomyces ludwigii, Saccharomyces capsularis, Saccharomyces fibuligera, Schizosaccharomyces pombe, Sporobolomyces pararoseus, Sporobolomyces salmonicolor, Sporopachydermia lactativora, Stachybotrys species, Talaromyces emersonii, Taphrina deformans, Torulaspora delbrueckii, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon beigelii, Wickerhamia fluorescens, Yarrowia lipolytica, and Zygosaccharomyces veronae.

999

Malt Agar 8% (MA8) Composition per liter: Malt extract................................................................................. 80.0g Agar ............................................................................................ 15.0g

Use: For the cultivation of Moniliella pollinis.

Malt Agar with 0.5% CaCO3

Composition per liter:

Malt extract................................................................................. 20.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g CaCO3 ........................................................................................... 5.0g Mycological peptone .................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Malt Agar 1% (MA1) Composition per liter:

Use: For the cultivation of Acrodontium griseum, Acrodontium simplex, Acrophialophora fusispora, Acrothecium tenebrosum, Acrothecium capsic, Agaricus bisporus, Chaetocladium jonesii, Chaetomium globosum, Chaetomium cupreum, Chaetomium irregulare, Farlowiella carmichaeliana, and many other filamentous fungi.

Use: For the cultivation and maintenance of Brettanomyces anomalus,

Malt Agar 2% (MA2) Composition per liter: Malt extract ................................................................................. 20.0g Agar ............................................................................................ 15.0g

Use: For the cultivation of Acrophialophora fusispora, Agaricus augustus, Ceratocystis penicillata, Chaetocladium brefeldii, and many other fungi.

Malt Agar 4% (MA4) Composition per liter: Malt extract ................................................................................. 40.0g Agar ............................................................................................ 15.0g

Use: For the cultivation of Chaetomium globosum, Chaetomium indicum, Chaetomium funicola, Chaetomium megalocarpum, Chaetomium murorum, Chaetomium seminudum, Chaetomium pachypodioides, Chaetomium perlucidum, Chaetomium quadrangulatum, Chaetomium reflexum, Chaetomium subaffine, Chaetomium subspirilliferum, Chaetomium succineum, Chaetomium luknowense, Daedalea quercina, Mycosphaerella ligulicola, Polypaecilum pisce, and many other fungi. © 2010 by Taylor and Francis Group, LLC

Brettanomyces bruxellensis, Brettanomyces claussenii, Brettanomyces lambicus, Dekkera bruxellensis, and Dekkera intermedia.

Malt Agar with 2% Malt Composition per liter: Agar ............................................................................................ 20.0g Malt extract................................................................................. 20.0g pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of a variety of yeasts and other fungi.

Malt Agar 2% with Lupine Stems (MA2 with Lupine Stems) Composition per liter: Malt extract................................................................................. 20.0g Agar ............................................................................................ 15.0g Lupine stems........................................................................... variable

Preparation of Medium: Add components, except lupine stems, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute 6.0mL volumes into tubes. Cut lupine stems into 8.0cm-long pieces. Add 2–3 lupine stems per tube. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position.

Use: For the cultivation of Chaetomium bostrychodes.

Malt Agar 4% with Lupine Stems (MA4 with Lupine Stems) Composition per liter: Malt extract................................................................................. 40.0g Agar ............................................................................................ 15.0g Lupine stems........................................................................... variable

1000

Malt Dextrose Peptone Agar

Use: For the cultivation of Chaetomium indicum and Chaetospermum chaetosporum.

Malt 4% Dextrose Yeast Agar (MDYA4) Composition per liter: Malt extract................................................................................. 40.0g Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g Yeast extract.................................................................................. 1.0g

Preparation of Medium: Add components to distilled/deionized

Malt Dextrose Peptone Agar (MDPA) Composition per liter: Agar ............................................................................................ 25.0g Malt extract ................................................................................. 20.0g Glucose ....................................................................................... 20.0g Peptone.......................................................................................... 1.0g

Use: For the cultivation of Aspergillus aeneus, Aspergillus candidus, Odontia uda, Oidiodendron chlamydosporicum, Oidiodendron cerealis, Oidiodendron flavum, Oidiodendron griseum, Oidiodendron periconioides, Oidiodendron tenuissimum, Penicillium spinulosum, and other fungi.

Use: For the cultivation of Torulaspora delbrueckii.

Malt Extract Agar Composition per liter: Malt extract................................................................................. 30.0g Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Xanthomonas species.

Malt Dextrose 40% Peptone Agar (MDPA 40)

Malt Extract Agar (MEA)

Composition per liter:

Glucose ..................................................................................... 400.0g Agar ............................................................................................ 25.0g Malt extract ................................................................................. 20.0g Peptone.......................................................................................... 1.0g

Use: For the cultivation of Aspergillus penicilloides.

Malt 4% Dextrose Peptone Yeast Agar (MDPYA4) Composition per liter: Malt extract ................................................................................. 40.0g Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g Peptone.......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g

Use: For the cultivation of Candida glabrata, Candida haemulonii, Candida lactis-condensi, Candida magnoliae, Candida nemodendra, Metschnikowia pulcherrima, Metschnikowia reukaufii, Phoma glomerata, Saccharomyces exiguus, and Trigonopsis variabilis. © 2010 by Taylor and Francis Group, LLC

Use: For the isolation, cultivation, and identification of heat-resistant filamentous fungi (molds) from foods.

Malt Extract Agar Composition per liter: Malt extract................................................................................. 30.0g Agar ............................................................................................ 15.0g Mycological peptone .................................................................... 5.0g pH 5.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring until boiling. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–115°C. Do not overheat or agar will not harden. If a lower pH (3.5) is desired, cool medium to 55°C and aseptically add 100.0mL of sterile lactic acid. Pour into sterile Petri dishes or distribute into sterile tubes.

Malt Extract Agar, Half Strength Use: For the detection, isolation, and enumeration of yeasts and molds. The addition of lactic acid suppresses bacterial growth.

Malt Extract Agar Composition per liter: Malt extract ................................................................................. 30.0g Agar ............................................................................................ 20.0g Chlortetracycline solution........................................................10.0mL pH 5.5 ± 0.2 at 25°C

1001

Malt Extract Agar (MEA) Composition per liter: Glucose ....................................................................................... 20.0g Malt extract................................................................................. 20.0g Agar ............................................................................................ 15.0g Peptone ......................................................................................... 1.0g

Preparation of Medium: Add components to distilled/deionized

Chlortetracycline Solution: Composition per 10.0mL:

Chlortetracycline...................................................................... 0.04mg

Use: For the cultivation of Arthrinium phaeospermum, Aspergillus

Preparation of Chlortetracycline Solution: Add chlortetracy-

fumigatus, Aspergillus clavatus, Penicillium verruculosum, and Penicillium spinulosum.

cline to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except chlortetracy-

Malt Extract Agar (BAM M93)

cline solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile chlortetracycline solution. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes.

Malt extract................................................................................. 30.0g Agar ............................................................................................ 20.0g pH 5.5 ± 0.2 at 25°C

Use: For the cultivation of yeasts and filamentous fungi (molds) from

Preparation of Medium: Add components to distilled/deionized

cosmetics.

Malt Extract Agar Composition per liter: Malt extract ................................................................................. 20.0g Agar ............................................................................................ 15.0g Peptone.......................................................................................... 5.0g

Use: For the cultivation and maintenance of Candida melibiosica,

Composition per liter:

Use: For the isolation, cultivation, and identification of heat-resistant filamentous fungi (molds) from foods.

Malt Extract Agar, Blakeslee’s Composition per liter: Malt extract................................................................................. 20.0g Glucose ....................................................................................... 20.0g Agar ............................................................................................ 20.0g Peptone ......................................................................................... 1.0g

Cryptococcus curvatus, Kluyveromyces species, Metschnikowia hawaiiensis, Rhodosporidium paludigenum, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Stephanoascus species, and Trichosporon nigrescens.

Preparation of Medium: Add components to distilled/deionized

Malt Extract Agar (ATCC Medium 109)

Use: For the cultivation and maintenance of a variety of yeasts, includ-

Composition per liter: Agar ............................................................................................ 15.0g Maltose...................................................................................... 12.75g Dextrin ........................................................................................ 2.75g Glycerol ...................................................................................... 2.35g Pancreatic digest of gelatin ......................................................... 0.78g pH 4.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. ing Candida versatilis, Cryptococcus elinovii, Kluyveromyces marxianus, Pichia species, Reniforma strues, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Sporobolomyces roseus, Trichosporon nigrescens, Yarrowia lipolytica, Zygosaccharomyces rouxii, and numerous filamentous fungi.

Malt Extract Agar, Half Strength (ATCC Medium 2418) Composition per liter: Agar ............................................................................................ 15.0g Malt extract................................................................................. 10.0g Peptone ......................................................................................... 2.5g pH 5.5 ± 0.2 at 25°C

1002

Malt Extract Agar for Yeasts and Molds

Use: For the cultivation of yeasts and molds.

Malt Extract Agar for Yeasts and Molds (MEAYM) (BAM M182)

or flasks. Autoclave for 15 min at 15 psi pressure–118°C. Do not overheat.

Use: For the cultivation of yeasts and molds.

Malt Extract Charcoal Medium (DSMZ Medium 801)

Use: For the isolation, cultivation, and identification of heat-resistant

Composition per liter: Malt extract................................................................................. 30.0g Agar ............................................................................................ 15.0g Charcoal........................................................................................ 3.0g pH 6.5 ± 0.2 at 25°C

filamentous fungi (molds) from foods. Recommended for identification of Aspergillus spp. and Penicillium spp.

Use: For the cultivation and maintenance of Boletus edulis, Xerocomus badius DSM 4436, Antrodia serialis, and other filamentous fungi.

Malt Extract Broth

Malt Extract Glucose Agar (DSMZ Medium 735)

Composition per liter: Malt extract ................................................................................... 6.0g Glucose ......................................................................................... 6.0g Maltose.......................................................................................... 1.8g Yeast extract.................................................................................. 1.2g pH 4.7 ± 0.2 at 25°C

Composition per liter:

Source: This medium is available as a premixed powder from BD Di-

Preparation of Medium: Add components to distilled/deionized

agnostic Systems.

Use: For the isolation, cultivation, and enumeration of yeast and fila-

Agar ............................................................................................ 15.0g Malt extract................................................................................. 10.0g Glucose ......................................................................................... 5.0g pH 7.1 ± 0.2 at 25°C water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Morchella esculenta spp., Verpa digitaliformis, Disciotis venosa, Mitrophora semilibera, Gyromitra spp., and Mitrophora semilibera.

mentous fungi (mold).

Malt Extract Broth Composition per liter:

Malt Extract HiVeg Agar Base Composition per liter:

Malt extract ................................................................................. 17.0g Mycological peptone..................................................................... 3.0g pH 5.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid

Source: This medium is available as a premixed powder from Hi-

Unipath.

Media.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–115°C.

Use: For the cultivation of molds and yeasts, especially for sterility testing.

Malt Extract Broth Composition per liter: Malt extract, purified solids ........................................................ 15.0g pH 4.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.4. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of fungi, especially yeasts.

Malt Extract HiVeg Agar Base, Modified Composition per liter: Agar ............................................................................................ 15.0g Maltose ..................................................................................... 12.75g Dextrin ........................................................................................ 2.75g Plant peptone .............................................................................. 0.78g pH 6.0 ± 0.2 at 25°C

Malt Yeast Extract 50% Glucose Agar (MY50G) Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of yeasts and molds.

Malt Extract HiVeg Broth Base Composition per liter: Malt extract ................................................................................. 17.0g Plant peptone No. 4....................................................................... 3.0g pH 5.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of yeasts and molds.

Malt Extract Peptone Agar Composition per liter: Malt extract ................................................................................. 30.0g Agar ............................................................................................ 15.0g Soy peptone................................................................................... 3.0g pH 5.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.6. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Basidiobolus ranarum and Sclerophoma pityophila.

Malt Extract Peptone Agar Composition per liter: Malt extract ................................................................................. 30.0g Agar ............................................................................................ 15.0g Papaic digest of soybean meal ...................................................... 3.0g pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Ascobolus immersus, Aspergillus amstelodami, and Aspergillus clavatus.

Malt Extract Yeast Extract 40% Glucose Agar (MY40G) Composition per liter: Glucose ..................................................................................... 400.0g Agar ............................................................................................ 12.0g © 2010 by Taylor and Francis Group, LLC

1003

Malt extract powder.................................................................... 12.0g Yeast extract.................................................................................. 3.0g pH 5.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except glucose, to 550.0mL of distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Bring volume to 600.0mL with distilled/deionized water. While the solution is still hot, add the glucose all at once while stirring to avoid formation of lumps. Autoclave for 30 min at 0 psi pressure–100°C.

Use: For the isolation and cultivation of osmotolerant microorganisms from foods.

Malt and Peptone Medium Composition per liter: Agar ............................................................................................ 15.0g Malt extract................................................................................. 10.0g Peptone ......................................................................................... 5.0g NaCl.............................................................................................. 1.0g pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Adjust pH to 6.5. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation and maintenance of Flavobacterium species. Malt Peptone Yeast Extract Agar See: MPY Agar

Malt Yeast Agar Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 10.0g Peptone ......................................................................................... 5.0g Malt extract................................................................................... 3.0g Yeast extract.................................................................................. 3.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Arthrobacter viscosus.

Malt Yeast Extract 50% Glucose Agar (MY50G) (ATCC Medium 2093) Composition per liter: Glucose ..................................................................................... 500.0g Agar ............................................................................................ 10.0g Malt extract................................................................................. 10.0g Yeast extract.................................................................................. 2.5g pH 5.5 ± 0.2 at 25°C

Preparation of Medium: Add agar, yeast extract, and malt extract to distilled/deionized water and bring volume to 500mL. Mix thoroughly. Gently heat while stirring until boiling. Slowly add glucose while stirring to avoid lumps. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Do not overheat or agar will not harden. Pour into sterile Petri dishes or leave in tubes. Note: This agar hardens very slowly.

Use: For the cultivation of yeasts and molds.

1004

Malt 2% Yeast Extract Agar

Malt 2% Yeast Extract Agar (MYA2) Composition per liter: Agar ............................................................................................ 20.0g Malt extract ................................................................................. 20.0g Yeast extract.................................................................................. 1.0g

Use: For the cultivation of Thielavia hyalocarpa.

Maltea

Use: For the cultivation of Actinomucor elegans, Actinospora mega-

Composition per liter: Maltea ......................................................................................... 40.0g Agar ............................................................................................ 22.0g Yeast extract.................................................................................. 3.0g

lospora, Agaricus bisporus, Ceratocystis perfecta, Ceratocystis cana, Ceratocystis seticollis, Chaetomium trilaterale, Chaetomium indicum, Chaetomium seminudum, Chaetomium piluliferum, Cirrenalia macrocephala, Kluyveromyces species, Lepista inversa, Torula dematia, Trichoderma pseudokoningii, and other fungi.

Preparation of Medium: Add components to distilled/deionized

Malt 4% Yeast Extract Agar (MYA4)

Maltose Peptone Yeast Extract Agar See: MPY Agar

Composition per liter: Malt extract ................................................................................. 40.0g Agar ............................................................................................ 20.0g Yeast extract.................................................................................. 1.0g

Use: For the cultivation of Arthrobotrys arthrobotryoides, Ascosphaera apis, Bettsia alvei, Ceratocystiopsis minuta, Chaetomium spinosum, Chaetomium piluliferum, Ciboriopsis simulata, Dactylella minuta, Dactylella rhombospora, Dactylella lysipaga, Eriopeziza caesia, Europhium clavigerum, Issatchenkia orientalis, Moniliella suaveolens, and other fungi.

Malt 4% Yeast Extract Agar with Lupine Stems (MYA4 with Lupine Stems) Composition per liter: Malt extract ................................................................................. 40.0g Agar ............................................................................................ 20.0g Yeast extract.................................................................................. 1.0g Lupine stems ........................................................................... variable

Use: For the cultivation of Ceratocystiopsis minuta-bicolor, Ceratocystiopsis retusi, Ceratocystis pilifera, Ceratocystis olivaceapini, Ceratocystis multiannulata, Ceratocystis nigra, Ceratocystis olivacea, Ceratocystis tremuloaurea, Chaetomium indicum, and Chaetospermum chaetosporum.

Malt 8% Yeast Extract Agar (MYA8) Composition per liter: Malt extract ................................................................................. 80.0g Agar ............................................................................................ 20.0g Yeast extract.................................................................................. 1.0g © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation of Leucogyrophana mollusca.

Maltose Peptone Yeast Extract Broth See: MPY Broth Maltose Peptone Yeast Extract Medium See: MPY Agar

Manganese Acetate Agar Composition per liter: Agar, highly purified................................................................... 10.0g Manganous acetate........................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add manganous acetate to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Add agar. Steam the medium to dissolve agar. Distribute into screw-capped tubes or bottles. Autoclave for 15 min at 15 psi pressure– 121°C. Allow tubes or bottles to cool in a slanted position.

Use: For the cultivation of manganese-oxidizing bacteria.

Manganese Agar No. 1 (Mn Agar No. 1) Composition per liter: Agar ............................................................................................ 10.0g MnCO3 .......................................................................................... 2.0g Beef extract................................................................................... 1.0g Fe(NH4)2(SO4)2 .......................................................................... 0.15g Sodium citrate............................................................................. 0.15g Yeast extract.............................................................................. 0.075g Cyanocobalamin solution ........................................................10.0mL

Cyanocobalamin Solution: Composition per 10.0mL: Cyanocobalamin .................................................................... 0.005mg

Preparation of Cyanocobalamin Solution: Add cyanocobalamin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except cyanocobalamin, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of the sterile cyanocobalamin solu-

Mannitol Agar with Prilion

tion. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

1005

Use: For the isolation and cultivation of iron and sulfur bacteria. Also

Solution A..............................................................................100.0mL Solution B ................................................................................10.0mL pH 2.6 ± 0.1 at 25°C

used to differentiate Leptothrix (Sphaerotilus) discophorus from Sphaerotilus natans.

Solution A: Composition per 100.0mL: FeSO4·7H2O................................................................................ 33.4g

Manganese Agar No. 2 (Mn Agar No. 2)

Preparation of Solution A: Add FeSO4·7H2O to distilled/deion-

Composition per liter:

ized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Agar ............................................................................................ 15.0g MnSO4·H2O ............................................................................. 10.0mg

Solution B: Composition per 10.0mL:

Preparation of Medium: Add components to distilled/deionized

Yeast extract.................................................................................. 0.2g

Preparation of Solution B: Add yeast extract to distilled/deionized

Use: For the enumeration, enrichment, and isolation of iron and sulfur bacteria. For the isolation and cultivation of Leptothrix species from water.

Manganese Medium for Pseudomonas species Composition per liter: Noble agar................................................................................... 10.0g MnCO3 .......................................................................................... 1.0g Fe(NH4)2(SO4)2·6H2O ................................................................ 0.15g Sodium citrate ............................................................................. 0.15g Yeast extract.............................................................................. 0.075g Na4P2O7·10H2O .......................................................................... 0.05g pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Pseudomonas putida and other Pseudomonas species.

Manganese Nutrient Agar

water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except solutions A and B, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Adjust pH to 2.6. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add solutions A and B. Adjust pH to 2.5–2.7 with 0.1N H2SO4. Use: For the cultivation of iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage.

Mannitol Agar Composition per liter: Mannitol...................................................................................... 25.0g Agar ............................................................................................ 15.0g Yeast extract.................................................................................. 5.0g Peptone ......................................................................................... 3.0g

Use: For the cultivation and maintenance of Acetobacter aceti, Acetobacter hansenii, Acetobacter pasteurianus, Frateuria aurantia, Gluconobacter asaii, Gluconobacter cerinus, Gluconobacter oxydans, and other bacteria that can utilize mannitol as a carbon source.

Composition per liter: Agar ............................................................................................ 15.0g Peptone.......................................................................................... 5.0g Meat extract .................................................................................. 3.0g MnSO4·H2O ............................................................................... 5.0mg

Use: For the cultivation and obtaining the sporulation of Bacillus species.

Manning Medium (DSMZ Medium 1023)

Mannitol Agar with Prilion Composition per liter: D-Mannitol .................................................................................. 15.0g Agar ............................................................................................ 13.0g Meat peptone .............................................................................. 10.0g Meat extract .................................................................................. 7.0g NaCl.............................................................................................. 3.0g Na2H2PO4 ..................................................................................... 2.0g Prilion ........................................................................................... 2.0g Metachrome Yellow.................................................................. 1.875g Water Blue ................................................................................ 0.625g pH 7.2 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

(NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 1.0g KCl................................................................................................ 0.2g K2HPO4 ........................................................................................ 0.2g Ca(NO3)2 .................................................................................... 0.02g

Use: For the isolation and differentiation of Salmonella spp. from Proteus species.

1006

Mannitol Egg Yolk Polymyxin Agar

Mannitol Egg Yolk Polymyxin Agar Composition per liter:

Preparation of Dye Stock Solution, 1000X: Add Bromthymol Blue and Cresol Red to 100.0mL of ethanol. Mix thoroughly.

Agar ............................................................................................ 15.0g D-Mannitol................................................................................... 10.0g Peptone........................................................................................ 10.0g NaCl ............................................................................................ 10.0g Beef extract ................................................................................... 1.0g Phenol Red ................................................................................ 0.025g Egg yolk emulsion, 50% ..........................................................50.0mL Polymyxin B solution ..............................................................10.0mL pH 7.1 ± 0.1 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: Available as a prepared medium from BD Diagnostic Sys-

Composition per liter:

tems.

Preparation of Polymyxin B Solution: Add polymyxin B to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

water and bring volume to 1.0L. Mix thoroughly. Adjust to pH 7.8. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Vibrio species from foods.

Mannitol Motility Test HiVeg Medium Plant peptone .............................................................................. 20.0g Agar .............................................................................................. 3.0g Mannitol........................................................................................ 2.0g Potassium nitrate........................................................................... 1.0g Phenol red ................................................................................... 0.04g pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective isolation, cultivation, and enumeration of staphylococci from clinical and nonclinical specimens. Mannitol-utilizing organisms turn the medium yellow.

Mannitol Salt Agar

Preparation of Medium: Add components—except egg yolk

Composition per liter:

emulsion, 50%, and polymyxin B solution—to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL of sterile egg yolk emulsion, 50%, and 10.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes.

NaCl............................................................................................ 75.0g Agar ............................................................................................ 15.0g D-Mannitol .................................................................................. 10.0g Pancreatic digest of casein............................................................ 5.0g Peptic digest of animal tissue ....................................................... 5.0g Beef extract................................................................................... 1.0g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Use: For the cultivation and enumeration of Bacillus cereus from foods.

Mannitol Lysine Crystal Violet Brilliant Green Agar See: MLCB Agar

Source: This medium is available as a premixed powder from BD Di-

Mannitol Maltose Agar

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Composition per liter: NaCl ............................................................................................ 20.0g Agar ............................................................................................ 13.0g D-Mannitol................................................................................... 10.0g Maltose........................................................................................ 10.0g Beef extract ................................................................................... 5.0g Papaic digest of soybean meal ...................................................... 5.0g Polypeptone™............................................................................... 5.0g Dye stock solution, 1000X.........................................................1.0mL pH 7.8 ± 0.2 at 25°C

Dye Stock Solution, 1000X: Composition per 100.0mL: Bromthymol Blue ......................................................................... 4.0g Cresol Red..................................................................................... 4.0g Ethanol, 95%..........................................................................100.0mL © 2010 by Taylor and Francis Group, LLC

agnostic Systems and Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized

Use: For the selective isolation, cultivation, and enumeration of staphylococci from clinical and nonclinical specimens. Mannitol-utilizing organisms turn the medium yellow.

Mannitol Salt Agar (BAM M97) Composition per liter: NaCl............................................................................................ 75.0g Agar ............................................................................................ 15.0g D-Mannitol .................................................................................. 10.0g Polypeptone ................................................................................ 10.0g

Mannitol Selenite Broth

Beef extract ................................................................................... 1.0g Phenol Red ................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Use: For the selective isolation, cultivation, and enumeration of staphylococci. Mannitol-utilizing organisms turn the medium yellow.

Mannitol Salt Agar with Egg Yolk Emulsion Composition per liter: NaCl ............................................................................................ 75.0g Agar ............................................................................................ 15.0g D-Mannitol .................................................................................. 10.0g Proteose peptone ......................................................................... 10.0g Beef extract ................................................................................... 1.0g Phenol Red ................................................................................ 0.025g Egg yolk emulsion .................................................................100.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Egg Yolk Emulsion Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C.

Preparation of Medium: Add components, except egg yolk emulsion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add egg yolk emulsion. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes or flasks.

Use: For the selective isolation of pathogenic staphylococci.

Mannitol Salt Broth Composition per liter:

1007

Mannitol Salt Broth Composition per liter: NaCl.......................................................................................... 100.0g Pancreatic digest of casein.......................................................... 17.0g Papaic digest of soybean meal...................................................... 3.0g K2HPO4......................................................................................... 2.5g D-Mannitol .................................................................................... 2.5g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the selective isolation and cultivation of staphylococci from foods and nonclinical specimens. Mannitol-utilizing organisms turn the medium yellow.

Mannitol Salt HiVeg Agar Base Composition per liter: NaCl............................................................................................ 75.0g Agar ............................................................................................ 15.0g D-Mannitol .................................................................................. 10.0g Plant peptone No. 3..................................................................... 10.0g Plant extract .................................................................................. 1.0g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective isolation of pathogenic staphylococci.

Mannitol Salt HiVeg Broth Composition per liter: NaCl............................................................................................ 75.0g D-Mannitol .................................................................................. 10.0g Plant peptone No. 3..................................................................... 10.0g Plant extract .................................................................................. 1.0g Phenol Red................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

NaCl ............................................................................................ 75.0g Proteose peptone ......................................................................... 10.0g D-Mannitol .................................................................................. 10.0g Beef extract ................................................................................... 1.0g Phenol Red ................................................................................ 0.025g pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Source: This medium is available from HiMedia.

Use: For the selective isolation of presumptive pathogenic staphylo-

Media.

Mannitol Selenite Broth (Selenite Mannitol Broth) Composition per liter: NaH2PO4 ..................................................................................... 10.0g Peptic digest of animal tissue ....................................................... 5.0g

1008

Mannitol Selenite Broth with Brilliant Green

Mannitol........................................................................................ 4.0g NaHSeO3 ...................................................................................... 4.0g pH 7.1 ± 0.2 at 25°C

FeCl3·6H2O Solution: Composition per 10.0mL:

Source: This medium is available from HiMedia.

Preparation of FeCl3·6H2O Solution: Add FeCl3·6H2O to dis-

FeCl3·6H2O .............................................................................. 0.66mg

Caution: Sodium hydrogen selenite (sodium biselenite) is a very tox-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

ic, corrosive agent and causes teratogenicity; it should be handled with great care. If there is contact, wash immediately with lots of water.

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium: Add NaHSeO3 to distilled/deionized wa-

ter and bring volume to 1.0L. Mix thoroughly. Add remaining components. Mix thoroughly. Distribute into tubes or flasks. Gently heat and bring to boiling. Sterilize in a boiling water bath or free flowing steam for 10 min. Do not autoclave. Discard the prepared medium if a large amount of selenite is reduced (indicated by red precipitate at the bottom of the tube).

Use: For the selective enrichment of Salmonella spp. from clinical materials.

Use: For the cultivation and maintenance of Rhizobium species and Bradyrhizobium species.

Mannitol-Yeast Extract-Peptone (MYP) (DSMZ Medium 1087) Composition per liter:

Mannitol Selenite Broth with Brilliant Green Composition per liter: Meat peptone................................................................................. 5.0g Yeast extract.................................................................................. 5.0g Mannitol........................................................................................ 5.0g K2HPO4 ....................................................................................... 4.35g KH2PO4 ......................................................................................... 3.4g Sodium taurocholate ..................................................................... 1.0g Brilliant Green .......................................................................... 0.005g Na2SeO3·5H2O.............................................................................. 1.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Caution: Sodium hydrogen selenite (sodium biselenite) is a very toxic, corrosive agent and causes teratogenicity; iit should be handled with great care. If there is contact, wash immediately with lots of water.

Preparation of Medium: Add NaHSeO3 to distilled/deionized wa-

Use: For the enrichment of Salmonella spp. from feces, foodstuffs, and other materials.

Mannitol Yeast Extract Medium (LMG 135) Composition per liter: Agar ............................................................................................ 20.0g Mannitol...................................................................................... 10.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g NaCl .............................................................................................. 0.1g CaCl2·2H2O solution..................................................................1.0mL FeCl3·6H2O solution ..................................................................1.0mL

CaCl2·2H2O Solution: Composition per 10.0mL: CaCl2·2H2O.............................................................................. 5.28mg

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

D-Mannitol ................................................................................. 25.0g Agar ........................................................................................... 15.0g Yeast extract ................................................................................. 5.0g Peptone ........................................................................................ 3.0g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation of Acetobacter fabarum.

Mannitol Yolk Polymyxin Agar (MYP Agar) Composition per 110.0mL: Agar .............................................................................................. 1.5g NaCl.............................................................................................. 1.0g Peptone ......................................................................................... 1.0g D-Mannitol .................................................................................... 1.0g (NH4)2PO4 .................................................................................... 0.1g Meat extract .................................................................................. 0.1g Phenol Red................................................................................. 2.5mg Egg yolk emulsion, 20%..........................................................10.0mL Polymyxin B solution ................................................................1.0mL pH 7.1 ± 0.2 at 25°C

Egg Yolk Emulsion, 20%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................80.0mL

Preparation of Egg Yolk Emulsion, 20%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Measure 20.0mL of egg yolk emulsion and add to 80.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Polymyxin B Solution: Composition per 1.0mL: Polymyxin B .............................................................................. 1.0mg

Preparation of Polymyxin B Solution: Add polymyxin B to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize.

Marine Agar 2216 Preparation of Medium: Add components—except egg yolk emulsion, 20%, and polymyxin B solution—to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile egg yolk emulsion, 20%, and 1.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes. Use: For the cultivation and maintenance of Bacillus cereus.

Maricaulis Medium (DSMZ Medium 1025)

1009

Na2SO4 .......................................................................................... 4.0g CaCl2·6H2O .................................................................................. 2.0g KCl................................................................................................ 0.7g KBr ............................................................................................... 0.1g SrCl2·6H2O ................................................................................. 0.04g H3BO3 ......................................................................................... 0.03g NaSiO3·9H2O............................................................................. 5.0mg NaF ............................................................................................ 3.0mg NH4NO3 ..................................................................................... 2.0mg Fe3PO4·4H2O ............................................................................. 1.0mg

Preparation of Synthetic Seawater: Add components to distilled

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly.

Sea salts, Sigma .......................................................................... 30.0g NH4Cl ........................................................................................... 0.5g Peptone yeast extract solution..................................................20.0mL Glucose solution ........................................................................2.0mL Riboflavin solution ....................................................................5.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add agar, tryptone, peptone, and yeast

Glucose Solution: Composition per 10.0mL: Glucose ......................................................................................... 5.0g

extract to synthetic seawater and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.8. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Halobacillus halophilus, Halomonas spp., Vibrio harveyi, Cobetia marina, and Ruegeria atlantica.

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Riboflavin Solution: Composition per 10.0mL: Riboflavin .................................................................................. 2.0mg

Preparation of Riboflavin Solution: Add riboflavin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Peptone Yeast Extract Solution: Composition per 100.0mL: Peptone........................................................................................ 10.0g Yeast extract.................................................................................. 5.0g

Preparation of Peptone Yeast Extract Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except riboflavin, glucose, and peptone yeast extract solutions, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 7.2. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add riboflavin, glucose, and peptone yeast extract solutions.

Use: For the cultivation of Maricaulis spp.

Marine Agar (DSMZ Medium 123) Composition per liter: Agar ............................................................................................ 15.0g Tryptone ...................................................................................... 10.0g Peptone.......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g Synthetic seawater ........................................................................1.0L pH 7.8 ± 0.2 at 25°C

Marine Agar 2216 (DSMZ Medium 604) Composition per liter: NaCl.......................................................................................... 19.45g Agar ............................................................................................ 15.0g MgCl2............................................................................................ 8.8g Peptone ......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Use: For the cultivation and maintenance of Hyphomonas spp., Oceanospirillum spp., Hyphomicrobium indicum, Psychroflexus gondwanensis=Flavobacterium gondwanense, Salegentibacter salegens=Flavobacterium salegens, Psychromonas antarctica, Sulfitobacter mediterraneus, Thalassomonas viridans, Vibrio spp., Marinospirillum minutulum=Oceanospirillum minutulum, Terasakiella pusilla=Oceanospirillum pusillum, Pseudoalteromonas atlantica=Alteromonas atlantica, Pseudomonas atlantica, Roseobacter spp., Erythrobacter longus, Pseudospirillum japonicum=Oceanospirillum japonicum, Marinobacter hydrocarbonoclasticus (Pseudomonas nautica), Psychrobacter

1010

Marine Agar with Biphenyl

spp., and Moritella japonica. For the isolation, cultivation, and maintenance of a wide variety of heterotrophic marine bacteria.

Solution C: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.3g

Marine Agar with Biphenyl Composition per liter: NaCl .......................................................................................... 19.45g Agar ............................................................................................ 15.0g MgCl2 ............................................................................................ 8.8g Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg Biphenyl..................................................................................... 1.0mg pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components, except biphenyl, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. After agar solidifies, aseptically add a few crystals of biphenyl to each plate.

Use: For the cultivation and maintenance of biphenyl-utilizing marine bacteria, such as Cycloclasticus pugetii.

Marine Agar with Lambda Carrageenan Composition per 1070.0mL: Solution A .....................................................................................1.0L Solution B ................................................................................60.0mL Solution C ................................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Solution A: Composition per liter: NaCl ............................................................................................ 25.0g Agar ............................................................................................ 15.0g MgSO4·7H2O ................................................................................ 5.0g Casamino acids ............................................................................. 2.5g Lambda-carrageenan..................................................................... 2.5g NaNO3........................................................................................... 2.0g CaCl2·2H2O................................................................................... 0.2g KCl................................................................................................ 0.1g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL:

Preparation of Solution C: Add FeSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of ATCC strain 43554.

Marine Agar with Kappa and Lambda Carrageenan Composition per 1070.0mL: Solution A.....................................................................................1.0L Solution B ................................................................................60.0mL Solution C ................................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Solution A: Composition per liter: NaCl............................................................................................ 25.0g Agar ............................................................................................ 15.0g MgSO4·7H2O ................................................................................ 5.0g Casamino acids ............................................................................. 2.5g NaNO3 .......................................................................................... 2.0g κ-Carrageenan............................................................................. 1.25g λ-Carrageenan............................................................................. 1.25g CaCl2·2H2O .................................................................................. 0.2g KCl................................................................................................ 0.1g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Solution B: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 3.56g

Preparation of Solution B: Add Na2HPO4·2H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.3g

Use: For the cultivation and maintenance of Pseudomonas carrageenovora.

Marine Agar with Naphthalene

Na2HPO4·2H2O........................................................................... 3.56g

Composition per liter:

Preparation of Solution B: Add Na2HPO4·2H2O to distilled/de-

NaCl.......................................................................................... 19.45g Agar ............................................................................................ 15.0g MgCl2............................................................................................ 8.8g

Marine Broth with Biphenyl

Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg Naphthalene .................................................................................. 1mg pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components, except naphthalene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. After agar solidifies, aseptically add a few crystals of naphthalene to each plate.

Use: For the cultivation and maintenance of naphthalene-utilizing

Marine Ameba Medium Composition per liter: Agar ............................................................................................ 10.0g Malt extract................................................................................... 0.1g Yeast extract.................................................................................. 0.1g Artificial seawater.........................................................................1.0L

Artificial Seawater: Composition per liter: NaCl............................................................................................ 27.5g MgSO4·7H2O .............................................................................. 6.78g MgCl2·6H2O ............................................................................... 5.38g KCl.............................................................................................. 0.72g NaHCO3 ....................................................................................... 0.2g CaCL2·2H2O ................................................................................. 1.4g

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to artificial seawater and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Cochliopodium clarum, Heteramoeba clara, Lingulamoeba leei, Paramoeba pemaquidensis, and Vannella species.

marine bacteria

Marine Agar with Sulfur (ATCC Medium 1922) Composition per liter: NaCl .......................................................................................... 19.45g Sulfur .......................................................................................... 10.0g MgCl2 ............................................................................................ 8.8g Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg pH 7.6 ± 0.2 at 25°C

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Marine Broth 2216 (LMG Medium 164) Composition per liter: NaCl.......................................................................................... 19.45g MgCl2............................................................................................ 8.8g Peptone ......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized

100°C on 3 successive days.

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare anaerobically under a gas phase

Use: For the cultivation of Vibrio liquefaciens and for the isolation,

of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50–55°C. Aseptically add 10.0g of sterile sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes.

cultivation, and maintenance of a wide variety of heterotrophic marine bacteria.

Preparation of Sulfur: Autoclave sulfur for 15 min at 0 psi pressure–

Marine Broth with Biphenyl Composition per liter: NaCl.......................................................................................... 19.45g MgCl2............................................................................................ 8.8g

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Marine Broth with Lambda Carrageenan

Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg Biphenyl..................................................................................... 1.0mg pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components, except biphenyl, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add a few crytals of biphenyl to each tube or flask. Use: For the cultivation of biphenyl-utilizing marine bacteria.

Marine Broth with Lambda Carrageenan Composition per 1070.0mL: Solution A .....................................................................................1.0L Solution B ................................................................................60.0mL Solution C ................................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Solution A: Composition per liter: NaCl ............................................................................................ 25.0g MgSO4·7H2O ................................................................................ 5.0g Casamino acids ............................................................................. 2.5g λ-Carrageenan............................................................................... 2.5g NaNO3........................................................................................... 2.0g CaCl2·2H2O................................................................................... 0.2g KCl................................................................................................ 0.1g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Solution B: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 3.56g

Preparation of Solution B: Add Na2HPO4·2H2O to distilled/de-

ionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Solution C: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.3g

Marine Broth with Kappa and Lambda Carrageenan Composition per 1070.0mL: Solution A.....................................................................................1.0L Solution B ................................................................................60.0mL Solution C ................................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Solution A: Composition per liter: NaCl............................................................................................ 25.0g MgSO4·7H2O ................................................................................ 5.0g Casamino acids ............................................................................. 2.5g NaNO3 .......................................................................................... 2.0g κ-Carrageenan............................................................................. 1.25g λ-Carrageenan............................................................................. 1.25g CaCl2·2H2O .................................................................................. 0.2g KCl................................................................................................ 0.1g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C.

Solution B: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 3.56g

Use: For the cultivation and maintenance of Pseudomonas carrageenovora.

Marine Broth with Naphthalene Composition per liter: NaCl.......................................................................................... 19.45g MgCl2............................................................................................ 8.8g Peptone ......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg Naphthalene .................................................................................. 1mg pH 7.6 ± 0.2 at 25°C

Marine Chlorobiaceae Medium 2 Preparation of Medium: Add components, except naphthalene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add a few crytals of naphthalene to each tube or flask.

Use: For the cultivation of naphthalene-utilizing marine bacteria.

Marine Broth with Sulfur Composition per liter: NaCl .......................................................................................... 19.45g Sulfur .......................................................................................... 10.0g MgCl2 ............................................................................................ 8.8g Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg pH 7.6 ± 0.2 at 25°C

Preparation of Sulfur: Autoclave for 15 min at 0 psi pressure–100°C on three successive days.

Preparation of Medium: Prepare anaerobically under a gas phase of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0g of sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes.

Use: For the cultivation of Thermococcus litoralis.

Marine Caulobacter Medium Composition per liter: Proteose peptone ......................................................................... 10.0g Yeast extract.................................................................................. 3.0g Artificial seawater.........................................................................1.0L pH 7.2–7.4 at 25°C

Artificial Seawater: Composition per liter: Commercially available marine aquarium salts mixture ..................................................... variable

Preparation of Artificial Seawater: Add commercially available marine aquarium salts mixture to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Combine components. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

1013

Marine Chlorobiaceae Medium 2 Composition per 1051.0mL: Solution 1...............................................................................950.0mL Na2S·9H2O solution .................................................................60.0mL NaHCO3 solution.....................................................................40.0mL Vitamin B12 solution ..................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Solution 1: Composition per 950.0mL: NaCl............................................................................................ 20.0g MgSO4·7H2O ................................................................................ 3.0g KH2PO4......................................................................................... 1.0g NH4Cl ........................................................................................... 0.5g CaCl2·2H2O ................................................................................ 0.05g Trace elements solution SL-8 ....................................................1.0mL

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Trace Elements Solution SL-8: Composition per liter: Disodium EDTA ........................................................................... 5.2g FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.19g MnCl2·4H2O ................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g H3BO3 ......................................................................................... 0.06g NaMoO4·2H2O............................................................................ 0.04g CuCl2·2H2O ................................................................................ 0.02g NiCl2·6H2O................................................................................. 0.02g

Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................... 5.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ................................................................................ 2.0mg

Preparation of Vitamin B12 Solution: Add vitamin B12 to dis-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S·9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 6.8 with sterile H2SO4 or Na2CO3 . Aseptically distribute into sterile 50.0mL or 100.0mL bottles with metal screw caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble.

1014

Marine Chromatiaceae Medium 2

Use: For the isolation and cultivation of marine members of the Chlorobiaceae.

Marine Chromatiaceae Medium 2

ly distribute into sterile 50.0mL or 100.0mL bottles with metal screw caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble.

Use: For the isolation and cultivation of marine members of the Chro-

Composition per 1051.0mL:

matiaceae.

Solution 1 ...............................................................................950.0mL Na2S·9H2O solution .................................................................60.0mL NaHCO3 solution .....................................................................40.0mL Vitamin B12 solution ..................................................................1.0mL pH 7.3 ± 0.2 at 25°C

Composition per liter:

Solution 1: Composition per 950.0mL: NaCl ............................................................................................ 20.0g MgSO4·7H2O ................................................................................ 3.0g KH2PO4 ......................................................................................... 1.0g NH4Cl ........................................................................................... 0.5g CaCl2·2H2O................................................................................. 0.05g Trace elements solution SL-8 ....................................................1.0mL

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Trace Elements Solution SL-8: Composition per liter: Disodium EDTA ........................................................................... 5.2g FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.19g MnCl2·4H2O.................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g H3BO3 ......................................................................................... 0.06g NaMoO4·2H2O............................................................................ 0.04g CuCl2·2H2O ................................................................................ 0.02g NiCl2·6H2O ................................................................................. 0.02g

Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 5.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ................................................................................ 2.0mg

Preparation of Vitamin B12 Solution: Add vitamin B12 to dis-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S·9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 7.3 with sterile H2SO4 or Na2CO3 . Aseptical© 2010 by Taylor and Francis Group, LLC

Marine Cytophaga Agar Agar ............................................................................................ 15.0g Nutrient broth................................................................................ 8.0g Yeast extract.................................................................................. 5.0g Salt solution ..................................................................................1.0L

Salt Solution: Composition per liter: NaCl.......................................................................................... 12.86g MgCl2.......................................................................................... 2.48g KCl.............................................................................................. 0.75g CaCl2 ........................................................................................... 0.56g Fe(SO4)2(NH4)2 ........................................................................ 0.048g

Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add solid components to 1.0L of salt solution. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Cytophaga species.

Marine Cytophaga Medium Composition per liter: NaCl............................................................................................ 24.7g Agar ............................................................................................ 15.0g MgSO4·7H2O ................................................................................ 6.3g MgCl2·6H2O ................................................................................. 4.6g Tryptic digest of casein................................................................. 1.0g Yeast extract.................................................................................. 1.0g KCl................................................................................................ 0.7g NaHCO3 solution .....................................................................10.0mL CaCl2·2H2O solution................................................................10.0mL pH 7.2 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 0.2g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

CaCl2·2H2O Solution: Composition per 10.0mL: CaCl2·2H2O .................................................................................. 1.2g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except NaHCO3 solution and CaCl2·2H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile NaHCO3 solution and 10.0mL of

Marine Flagellate Medium with B-Vitamins

1015

sterile CaCl2·2H2O solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Solution A: Composition per 980.0mL:

Use: For the cultivation of Cytophaga species, Flexibacter species,

NaCl............................................................................................ 25.0g DL-Sodium lactate......................................................................... 2.0g MgSO4·7H2O ................................................................................ 2.0g Na2SO4 .......................................................................................... 1.0g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g K2HPO4......................................................................................... 0.5g CaCl2·2H2O .................................................................................. 0.1g Resazurin ................................................................................... 1.0mg

Microscilla species, and Saprospira grandis.

Marine Cytophaga Medium A Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein ............................................................ 2.0g Beef extract ................................................................................... 0.5g Yeast extract.................................................................................. 0.5g Sodium acetate .............................................................................. 0.2g Seawater.................................................................................700.0mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation of Flexibacter maritimus.

Marine Cytophaga Medium B Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein ............................................................ 2.0g Beef extract ................................................................................... 0.5g Yeast extract.................................................................................. 0.5g Sodium acetate .............................................................................. 0.2g Seawater.................................................................................500.0mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation of Vibrio ordalii.

Marine Cytophaga Medium C Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein ............................................................ 2.0g Beef extract ................................................................................... 0.5g Yeast extract.................................................................................. 0.5g Sodium acetate .............................................................................. 0.2g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to seawater and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Cytophaga agarovorans, Cytophaga fermentans, and Cytophaga salmonicolor.

Marine Desulfovibrio Medium Composition per liter: Solution A ..............................................................................980.0mL Solution B ................................................................................10.0mL Solution C ................................................................................10.0mL pH 7.8 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3–4 min. Allow to cool to room temperature while gassing under 100% N2.

Solution B: Composition per 10.0mL: FeSO4·7H2O.................................................................................. 0.5g

Preparation of Solution B: Add FeSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Solution C: Composition per 10.0mL: Ascorbic acid ................................................................................ 0.1g Sodium thioglycolate .................................................................... 0.1g

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Preparation of Medium: To 980.0mL of cooled solution A, anaerobically add 10.0mL of solution B and 10.0mL of solution C. Mix thoroughly. Adjust pH to 7.8 with NaOH. Distribute into tubes or flasks. During distribution, swirl the medium to keep the precipitate in suspension. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Desulfovibrio desulfuricans, Desulfovibrio salexigens, and Desulfovibrio vulgaris.

Marine Flagellate Medium Composition per 15.0mL: Rice grains .................................................................................... 2.0g Seawater...................................................................................15.0mL

Preparation of Medium: Autoclave rice grains for 15 min at 15 psi pressure–121°C. Add 2.0g of sterile rice grains to 15.0mL of filter-sterilized seawater. Aseptically distribute into T-25 tissue culture flasks. Use: For the cultivation of Acanthoecopsis unguiculata, Amastigomonas species, Bicosoeca vacillans, Bodo designis, Bodo variabilis, Caecitellus parvulus, Choanoeca perplexa, Codosiga gracilis, Diaphanoeca grandis, Entosiphon species, Goniomonas species, Procryptobia species, Pseudobodo tremulans, Rhynchomonas nasuta, Salpingoeca urceolata, Stephanoeca diplocostata, and Stephanopogon apogon.

Marine Flagellate Medium with B-Vitamins Composition per liter: Seawater.................................................................................990.0mL Vitamin solution.......................................................................10.0mL

Vitamin Solution: Composition per 100.0mL: Thiamine·HCl ............................................................................. 0.15g Calcium D-(+)-pantothenate ....................................................... 0.05g Nicotinamide............................................................................... 0.05g

1016

Marine Glucose Trypticase™ Yeast Extract Agar

Pyridoxal·HCl ............................................................................. 0.05g Riboflavin ................................................................................... 0.05g Folic acid................................................................................... 0.025g Pyridoxamine·HCl .................................................................... 0.025g Biotin ....................................................................................... 12.5mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Allow natural seawater to age for 2 months. Filter sterilize. Aseptically add 100.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Oikomonas species.

Marine Glucose Trypticase™ Yeast Extract Agar (MGTY Agar) Composition per liter: Agar .............................................................................................. 8.0g Glucose ......................................................................................... 2.0g Pancreatic digest of casein ............................................................ 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl·H2O...................................................................... 0.5g Seawater.................................................................................750.0mL Tris-HCl buffer (5.0 mM, pH 7.5) ...........................................50.0mL Resazurin (0.1% solution)..........................................................1.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

NH4Cl ........................................................................................... 0.4g Sodium acetate·3H2O.................................................................... 0.4g KH2PO4......................................................................................... 0.2g CaCl2·2H2O .................................................................................. 0.1g NaHCO3 solution .....................................................................60.0mL 2-Propanol..................................................................................5.0mL Na2S·9H2O solution ...................................................................3.0mL Cyanocobalamin solution ..........................................................1.0mL Selenite-molybdate-tungstate solution.......................................1.0mL Thiamine solution ......................................................................1.0mL Trace elements solution .............................................................1.0mL Vitamin solution.........................................................................1.0mL

Trace Elements Solution: Composition per 100.0mL: FeSO4·7H2O......................................................................... 1400.0mg ZnSO4·7H2O .......................................................................... 145.0mg CoCl2·6H2O ........................................................................... 120.0mg MnCl2·4H2O .......................................................................... 100.0mg NiCl2·6H2O .............................................................................. 50.0mg H3BO3 ........................................................................................ 6.0mg CuSO4·5H2O .............................................................................. 3.0mg HCl (25%,w/v)...........................................................................8.0mL

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Selenite-Molybdate-Tungstate Solution: Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks under 97% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust.

NaOH............................................................................................ 0.2g Na2MoO4·2H2O ....................................................................... 40.0mg Na2WO4·2H2O ......................................................................... 33.0mg Na2SeO3·2H2O........................................................................... 5.0mg

Use: For the cultivation and maintenance of Spirochaeta isovalerica.

Preparation of Selenite-Molybdate-Tungstate Solution: Add

Marine Glucose Trypticase™ Yeast Extract Broth (MGTY Broth) Composition per liter: Glucose ......................................................................................... 2.0g Pancreatic digest of casein ............................................................ 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl·H2O...................................................................... 0.5g Seawater.................................................................................750.0mL Tris-HCl buffer (5.0 mM, pH 7.5) ...........................................50.0mL Resazurin (0.1% solution)..........................................................1.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks under 97% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust.

Use: For the cultivation and maintenance of Spirochaeta isovalerica.

Marine Methanogenium Alcohol Medium Composition per 1003.0mL: NaCl ............................................................................................ 21.0g MgCl2·6H2O.................................................................................. 3.0g NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.5g © 2010 by Taylor and Francis Group, LLC

components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

NaHCO3 Solution: Composition per liter: NaHCO3 ...................................................................................... 84.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................... 2.5g NaOH....................................................................................... 1 pellet

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N2. Dissolve 1 pellet of NaOH in the anaerobic water. Weigh out a little more than 2.5g of Na2S·9H2O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on paper towels or filter paper. Add 2.5g of washed Na2S·9H2O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Pressurize to 60kPa with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

Preparation of 2-Propanol: Filter sterilize 10.0mL of 2-propanol. Sparge with 100% N2.

Marine Methylotroph Broth

1017

Vitamin Solution: Composition per liter:

Trace Metals Solution: Composition per liter:

Sodium 2-mercaptoethanesulfonate............................................ 0.25g Pyridoxine·HCl ........................................................................... 0.15g Calcium pantothenate ................................................................... 0.1g Nicotinic acid ................................................................................ 0.1g p-Aminobenzoic acid............................................................... 40.0mg Biotin ....................................................................................... 10.0mg Potassium phosphate buffer (25mM solution, pH 7.0) ........................................................1.0L

ZnSO4·7H2O ................................................................................. 1.4g MnSO4·H2O ................................................................................ 0.84g FeSO4·7H2O................................................................................ 0.28g CuSO4·5H2O............................................................................... 0.25g Na2MoO4·2H2O .......................................................................... 0.24g CoCl2·6H2O ................................................................................ 0.24g CaCl2·2H2O ................................................................................ 0.15g

Preparation of Vitamin Solution: Combine components. Mix

tilled/deionized water and bring volume to 1.0L. Mix thoroughly.

thoroughly. Filter sterilize. Sparge with 100% N2.

Thiamine·HCl ............................................................................... 0.1g Sodium phosphate buffer (0.1M solution, pH 3.6) ..........................................................1.0L

Preparation of Medium: Add components, except vitamin B12 solution and methanol, to distilled/deionized water and bring volume to 980.0mL. Adjust pH to 7.0 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Filter sterilize methanol. Aseptically add sterile vitamin B12 solution and filter-sterilized methanol. Distribute into sterile tubes or flasks.

Preparation of Thiamine Solution: Combine components. Mix

Use: For the cultivation and maintenance of Methylophaga thalassica.

Thiamine Solution: Composition per liter:

Preparation of Trace Metals Solution: Add components to dis-

thoroughly. Filter sterilize. Sparge with 100% N2.

Cyanocobalamin Solution: Composition per liter: Cyanocobalamin ...................................................................... 50.0mg

Preparation of Cyanocobalamin Solution: Add cyanocobalamin to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N2.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except NaHCO3 solution, 2-propanol, Na2S·9H2O solution, cyanocobalamin solution, selenite-molybdate-tungstate solution, thiamine solution, trace elements solution, and vitamin solution, to distilled/deionized water and bring volume to 930.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 60.0mL of sterile NaHCO3 solution, 5.0mL of sterile 2-propanol, 3.0mL of sterile Na2S·9H2O solution, 1.0mL of sterile cyanocobalamin solution, 1.0mL of sterile selenite-molybdate-tungstate solution, 1.0mL of sterile thiamine solution, 1.0mL of sterile trace elements solution, and 1.0mL of sterile vitamin solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of marine Methanogenium species.

Marine Methanol Medium Composition per liter: NaCl ............................................................................................ 20.0g (NH4)2SO4 ..................................................................................... 2.0g K2HPO4 ......................................................................................... 2.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.3g Methanol ..................................................................................10.0mL Vitamin B12 solution ................................................................10.0mL Trace metals solution .................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ...............................................................................10.0μg

Preparation of Vitamin B12 Solution: Add the vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Adjust pH to 5. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC

Marine Methylotroph Agar Composition per 1003.0mL: Agarose ....................................................................................... 12.0g Bis (2-hydroxyethyl) aminotris (hydroxymethyl) methane ..................................................................... 2.0g KH2PO4....................................................................................... 0.14g Ferric ammonium citrate............................................................. 0.06g Methanol ....................................................................................2.0mL Vitamin B12 solution ..................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12................................................................................ 0.1mg

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Store at 5°C.

Preparation of Medium: Add components, except methanol and vitamin B12 solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 2.0mL of filter-sterilized methanol and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Alteromonas species, Methylophaga marina, Methylophaga thalassica, and Methylophilus species.

Marine Methylotroph Broth Composition per 1003.0mL: Bis (2-hydroxyethyl) aminotris (hydroxymethyl) methane ..................................................................... 2.0g KH2PO4....................................................................................... 0.14g Ferric ammonium citrate............................................................. 0.06g Methanol ....................................................................................2.0mL Vitamin B12 solution ..................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12................................................................................ 0.1mg

1018

Marine Oxidation Fermentation HiVeg Medium

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Store at 5°C.

Preparation of Medium: Add components, except methanol and vitamin B12 solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 2.0mL of filter-sterilized methanol and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Alteromonas species, Methylophaga marina, Methylophaga thalassica, and Methylophilus species.

Marine Oxidation Fermentation HiVeg Medium (MOF HiVeg Medium)

MgSO4·7H2O ................................................................................ 2.0g KCl................................................................................................ 1.0g CaCl2 ............................................................................................. 0.5g FeSO4 ......................................................................................... 1.0mg

Preparation of Synthetic Seawater: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to 1.0L of synthetic seawater. Mix thoroughly. Gently heat while stirring and bring to boiling. Adjust pH to 6.8 with KOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum.

Marine Peptone Yeast Medium with Magnesium Sulfate

Composition per liter: NaCl .............................................................................................. 9.7g MnCl2 ............................................................................................ 4.4g Agar .............................................................................................. 3.0g Na2SO4 .......................................................................................... 1.6g Plant hydrolysate........................................................................... 1.0g CaCl2 ............................................................................................. 0.9g (NH4)2SO4 ..................................................................................... 0.5g Tris hydroxymethyl aminomethane .............................................. 0.5g KCl............................................................................................ 0.275g Yeast extract.................................................................................. 0.1g NaHCO3 ...................................................................................... 0.08g KBr.............................................................................................. 0.04g SrCl2.......................................................................................... 0.017g H3BO3 ....................................................................................... 0.011g Phenol Red .................................................................................. 0.01g Na2HPO4 .................................................................................... 4.0mg Sodium silicate........................................................................... 2.0mg NaFl ........................................................................................... 1.2mg NH4NO3 ..................................................................................... 0.8mg pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the differentiation of marine bacteria on the basis of fermentative and oxidative metabolism of carbohydrates.

Marine Peptone Succinate Salts Medium (PSS Medium) Composition per liter: Peptone........................................................................................ 10.0g Succinic acid ................................................................................. 1.0g (NH4)2SO4 ..................................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g FeCl3·6H2O ................................................................................ 2.0mg MnSO4·H2O ............................................................................... 2.0mg Synthetic seawater ........................................................................1.0L pH 6.8 ± 0.2 at 25°C

Synthetic Seawater: Composition per liter: NaCl ............................................................................................ 27.5g MgCl2 ............................................................................................ 5.0g © 2010 by Taylor and Francis Group, LLC

Composition per liter: NaCl............................................................................................ 20.0g Peptone ....................................................................................... 10.0g MgSO4·7H2O ................................................................................ 2.0g (NH4)2SO4 .................................................................................... 2.0g Yeast extract.................................................................................. 1.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Oceanospirillum pusillum.

Marine Pseudomonas Medium Composition per liter: Agar ............................................................................................ 15.0g Nutrient broth................................................................................ 8.0g Yeast extract.................................................................................. 5.0g Salt solution ..................................................................................1.0L

Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to 1.0L of salt solution. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Alteromonas haloplanktis.

Marine Rhodococcus Medium Composition per liter: Yeast extract................................................................................ 10.0g Malt extract................................................................................... 4.0g Glucose ......................................................................................... 4.0g Seawater.................................................................................750.0mL

Marine Salts Medium for Sporosarcina halophila Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Rhodococcus marinonascens.

Marine Rhodopseudomonas Medium Composition per liter: NaCl ............................................................................................ 30.4g Yeast extract.................................................................................. 1.0g Disodium succinate....................................................................... 1.0g KH2PO4 ......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.4g NH4Cl ........................................................................................... 0.4g CaCl2·2H2O................................................................................. 0.05g Ferric citrate (0.1% solution) .....................................................5.0mL Trace elements solution SL-6 ....................................................1.0mL Ethanol .......................................................................................0.5mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O................................................................................. 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Use: For the cultivation and maintenance of Rhodopseudomonas marina.

Marine Rhodopseudomonas Medium Composition per liter: NaCl ............................................................................................ 30.0g Peptone.......................................................................................... 2.5g Yeast extract.................................................................................. 2.5g pH 7.0 ± 0.4 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into screwcapped tubes. Autoclave for 15 min at 15 psi pressure–121°C. Before inoculating, loosen the screw caps, heat the medium to drive out O2, and screw down the cap tightly.

Use: For the cultivation of Rhodopseudomonas marina.

1019

MgCl2............................................................................................ 7.0g Proteose peptone No.3 .................................................................. 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.36g NaHCO3 ...................................................................................... 0.06g NaBr.......................................................................................... 0.026g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of marine bacteria.

Marine Spirochete Medium (DSMZ Medium 1008) Composition per liter: Trypticase peptone........................................................................ 2.0g Yeast extract ................................................................................. 1.0g Na-thioglycolate .......................................................................... 1.0g Resazurin .................................................................................. 0.5mg Charcoal-filtered, natural seawater .......................................800.0mL Cellobiose solution ..................................................................20.0mL pH 7.5 ± 0.2 at 25°C

Cellobiose Solution: Composition per 100.0mL: Cellobiose ................................................................................... 10.0g

Preparation of Cellobiose Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Add components, except thioglycolate and cellobiose solution, to seawater and bring volume to 800.0mL. Mix thoroughly. Bring volume to 980.0mL with distilled/deionized water. (Note: Bottled water from Biomaris GmbH can be used instead of filtered seawater.) Gently heat and bring to boiling. Boil for 3 min. Cool to room temperature while sparging with 100% N2. Add the thioglycolate. Adjust pH to 7.5 with 10N NaOH. Dispense into tubes or bottles under 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically and anoxically add cellobiose solution. Use: For the cultivation of Spirochaeta bajacaliforniensis.

Marine Salts Medium for Sporosarcina halophila Composition per liter: Marine salts mix ....................................................................... 100.0g Agar ............................................................................................ 20.0g Yeast extract................................................................................ 10.0g Proteose peptone No. 3 ................................................................. 5.0g Glucose ......................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Marine Salts Medium Composition per liter: NaCl ............................................................................................ 81.0g Yeast extract................................................................................ 10.0g MgSO4 .......................................................................................... 9.6g © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Sporosarcina halophila.

1020

Marine Spirochete Medium

Marine Spirochete Medium Composition per liter: Cellobiose ..................................................................................... 2.0g Peptone.......................................................................................... 2.0g Yeast extract.................................................................................. 1.0g Sodium thioglycolate .................................................................... 1.0g Seawater, charcoal filtered.....................................................800.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except sodium thioglycolate, to glass-distilled water and bring volume to 1.0L. Mix thoroughly. Bubble 100% N2 into medium for 1.5 min. Add sodium thioglycolate. Adjust pH to 7.5 with 10N KOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Spirochaeta bajacaliforniensis.

Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl .......................................................................................... 19.45g MgCl2 ............................................................................................ 8.8g Sulfur ............................................................................................ 5.0g Peptone.......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg Na2S·9H2O solution ...................................................................0.5mL pH 6.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Thermococcus aegaeus DSM 12767.

Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl.......................................................................................... 19.45g MgCl2............................................................................................ 8.8g Sulfur ............................................................................................ 5.0g Peptone ......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg Na2S·9H2O solution ...................................................................0.5mL pH 6.5 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 1.0g

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl.

Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter through normal filter paper. An iron sediment will collect in the filter. Gently heat while stirring and bring to boiling. Boil for 5 min. Cool under an anaerobic gas mixture of N2. Adjust pH to 6.5. Distribute the medium into Hungate tubes or serum bottles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering.

Preparation of Medium: Add components, except sulfur and

Use: For the cultivation and maintenance of Thermococcus pacificus

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 1.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter through normal filter paper. An iron sediment will collect in the filter. Gently heat while stirring and bring to boiling. Boil for 5 min. Cool under an anaerobic gas mixture of N2. Adjust pH to 6.0. Distribute the medium into Hungate tubes or serum bottles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering. © 2010 by Taylor and Francis Group, LLC

DSM 10394 and Thermococcus gorgonarius DSM 10395.

Marine Thermococcus Medium

KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate .................................................................................. 0.1g KBr.............................................................................................. 0.08g SrCl2 ............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3 ...................................................................................... 4.0mg NaF............................................................................................. 2.4mg NH4NO3 ..................................................................................... 1.6mg Na2S·9H2O solution ...................................................................0.5mL pH 6.5 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 1.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl.

Preparation of Medium: Add components, except sulfur and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter through normal filter paper. An iron sediment will collect in the filter. Gently heat while stirring and bring to boiling. Boil for 5 min. Cool under an anaerobic gas mixture of 80% N2 + 20% CO2. Adjust pH to 6.5. Distribute the medium into Hungate tubes or serum bottles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering.

Use: For the cultivation and maintenance of Thermococcus stetteri DSM 5262.

1021

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 1.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter through normal filter paper. An iron sediment will collect in the filter. Gently heat while stirring and bring to boiling. Boil for 5 min. Cool under an anaerobic gas mixture of N2. Adjust pH to 5.8. Distribute the medium into Hungate tubes or serum bottles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering.

Use: For the cultivation and maintenance of Thermococcus celer DSM 2476.

Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl.......................................................................................... 19.45g MgCl2............................................................................................ 8.8g Sulfur ............................................................................................ 5.0g Peptone ......................................................................................... 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g Yeast extract.................................................................................. 1.0g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg Na2S·9H2O solution...................................................................0.5mL pH 7.2 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 1.0g

1022

Marine Thermococcus Medium

tles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering.

Use: For the cultivation and maintenance of Thermococcus profundus DSM 9503, Thermococcus peptonophilus DSM 10343, Thermococcus guaymasensis 11113, and Thermococcus aggregans DSM 12819.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 1.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl.

Preparation of Medium: Add components, except sulfur and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter through normal filter paper. An iron sediment will collect in the filter. Gently heat while stirring and bring to boiling. Boil for 5 min. Cool under an anaerobic gas mixture of N2. Adjust pH to 7.5. Distribute the medium into Hungate tubes or serum bottles containing finely divided sulfur (0.5% w/v). Seal the tubes or bottles under the same anaerobic gas used when cooling the medium. Sterilize the medium at 100°C for 3 hr on 3 consecutive days. Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05%. The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering.

Use: For the cultivation and maintenance of Thermococcus litoralis DSM 5473, Thermococcus litoralis 5474, Thermococcus fumicolans DSM 12820, and Thermococcus sibiricus DSM 12597. © 2010 by Taylor and Francis Group, LLC

Marinithermus hydrothermalis Medium (DSMZ Medium 973) Composition per liter: NaCl............................................................................................ 30.0g MgCl2·6H2O ............................................................................... 4.18g MgSO4·7H2O ................................................................................ 3.4g Yeast extract.................................................................................. 1.0g Tryptone........................................................................................ 1.0g KCl.............................................................................................. 0.33g NH4Cl ......................................................................................... 0.25g K2HPO4....................................................................................... 0.14g CaCl2·2H2O ................................................................................ 0.14g Fe(NH4)2(SO4)2·6H2O ............................................................. 10.0mg NiCl2·6H2O ................................................................................ 0.5mg Na2Se3·5H2O ............................................................................. 0.5mg Trace elements solution ...........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Use: For the cultivation of Marinithermus hydrothermalis.

Marinitoga Medium (DSMZ Medium 904) Composition per 1045.0mL: Sea salts ...................................................................................... 30.0g PIPES............................................................................................ 6.0g Yeast extract.................................................................................. 1.0g Tryptone........................................................................................ 1.0g Resazurin ................................................................................... 0.5mg Glucose solution ......................................................................25.0mL Na2S·9H2O solution .................................................................10.0mL L-Cysteine solution ..................................................................10.0mL pH 7.0 ± 0.2 at 25°C Glucose Solution: Composition per 25.0mL: Glucose ......................................................................................... 2.5g

Marinitoga piezophila Medium Preparation of Glucose Solution: Add sucrose to distilled/deionized water and bring volume to 25.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. L-Cysteine

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 100% N2. Add components, except glucose solution, L-cysteine-HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into anaerobe tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter, 50.0mL glucose solution, 10.0mL L-cysteine-HCl·H2O solution, and 10.0mL Na2S·9H2O. Mix thoroughly. The final pH should be 7.0.

Use: For the cultivation of Marinitoga camini and Caloranaerobacter azorensis.

Marinitoga piezophila Medium (DSMZ Medium 945) Composition per liter: NaCl ............................................................................................ 30.0g Yeast extract.................................................................................. 5.0g Trypticase™.................................................................................. 5.0g MES ............................................................................................ 1.95g NH4Cl ........................................................................................... 1.0g Na-acetate ................................................................................... 0.83g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.2g CaCl2·2H2O................................................................................... 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg Maltose solution.....................................................................100.0mL Na2S·9H2O solution .................................................................10.0mL Cysteine solution......................................................................10.0mL pH 6.0 ± 0.2 at 25°C

Maltose Solution: Composition per 100.0mL: Maltose........................................................................................ 4.96g

Preparation of Maltose Solution: Add maltose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

1023

ly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically. Preparation of Medium: Add components, except maltose solution, NaHCO3 solution, and Na2S·9H2O solution, to 880.0mL distilled/deionized water. Mix thoroughly. Sparge for 30 min with 100% N2. Adjust pH to 6.0 with concentrated NaOH. Distribute under 100% N2 into anaerobic tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 100.0mL sterile maltose solution, 10.0mL sterile Na2S·9H2O solution, and 10.0mL sterile cysteine solution per liter medium. Mix thoroughly.

Use: For the cultivation of Marinitoga piezophila.

Marinitoga piezophila Medium (DSMZ Medium 945) Composition per liter: NaCl............................................................................................ 30.0g Sulfur .......................................................................................... 10.0g Yeast extract.................................................................................. 5.0g Trypticase™.................................................................................. 5.0g MES ............................................................................................ 1.95g NH4Cl ........................................................................................... 1.0g Na-acetate ................................................................................... 0.83g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.2g CaCl2·2H2O .................................................................................. 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg Na2S·9H2O solution .................................................................10.0mL Cysteine solution .....................................................................10.0mL pH 6.0 ± 0.2 at 25°C

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Cysteine Solution: Composition per 10.0mL:

Preparation of Sulfur: Sterilize sulfur by steaming for 3 hr on each

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of Medium: Add components, except sulfur, cysteine solution, and Na2S·9H2O solution, to 980.0mL distilled/deionized water. Mix thoroughly. Sparge for 30 min with 100% N2. Adjust pH to 6.0

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thorough-

of 3 successive days.

1024

Marinobacter lutaoensis Medium

with concentrated NaOH. Distribute under 100% N2 into anaerobic tubes or bottles containing appropriate amounts of sterile sulfur (1g steam-sterilized sulfur per 100mL medium). Autoclave for 20 min at 110°C. Cool to room temperature. Aseptically and anaerobically add 10.0mL sterile Na2S·9H2O solution and 10.0mL sterile cysteine solution per liter medium. Mix thoroughly.

Use: For the cultivation of Marinitoga piezophila.

Marinobacter lutaoensis Medium (DSMZ Medium 1066) Composition per liter:

p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution, Concentrated: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Peptone ......................................................................................... 4.0g Yeast extract ................................................................................. 2.0g NaCl ........................................................................................... 25.0g MgCl2·6H2O.................................................................................. 2.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Marionobacter sp.

Preparation of Medium: Add components to distilled/deionized wa-

NaCl............................................................................................ 11.7g MgSO4 ........................................................................................ 7.85g TRIS.............................................................................................. 6.0g Yeast extract.................................................................................. 5.0g Peptone ......................................................................................... 5.0g NH4Cl ........................................................................................... 3.0g CaCl2 ........................................................................................... 1.47g KCl.............................................................................................. 0.74g pH 7.8 ± 0.2 at 25°C

ter and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Marinobacter lutaoensis.

Marinobacter Medium (DSMZ Medium 941) Composition per liter: NaCl .............................................................................................. 6.0g NH4Cl ........................................................................................... 1.0g Na-acetate ..................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g KCl................................................................................................ 0.1g KH2PO4 ......................................................................................... 0.1g Peptone.......................................................................................... 0.1g CaCl2·2H2O................................................................................. 0.04g Trace elements solution SL-7 ....................................................1.0mL Vitamin solution, concentrated ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution SL-7: Composition per liter: FeCl2·7H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ...................................................................................... 62.0mg CuCl2·2H2O ............................................................................. 17.0mg HCl (25% solution) ....................................................................6.5mL

Preparation of Trace Elements Solution SL-7: Add FeCl2·7H2O

Marinobacter Medium (DSMZ Medium 970) Composition per liter:

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Marinobacter sp.

Marinococcus albus Agar (LMG Medium 212) Composition per liter: NaCl............................................................................................ 81.0g Agar ............................................................................................ 15.0g Yeast............................................................................................ 10.0g MgSO4·7H2O ................................................................................ 9.6g MgCl2·6H2O ................................................................................. 7.0g Proteose peptone........................................................................... 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.36g NaB ........................................................................................ 226.0mg NaHCO3 ................................................................................... 60.0mg pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Marinococcus albus.

Marinococcus albus Medium

Vitamin Solution, Concentrated: Composition per 100.0mL:

Composition per liter:

NaCl............................................................................................ 81.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O ................................................................................ 9.6g MgCl2·6H2O ................................................................................. 7.0g Proteose peptone No. 3 ................................................................. 5.0g

Martin-Lewis Agar, Enriched

KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.36g NaHCO3 ...................................................................................... 0.06g NaBr.......................................................................................... 0.026g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Marinococcus albus. Marinomonas vaga Medium See: Nutrient Agar with 3% NaCl

Marinomonas vaga Medium (DSMZ Medium 617) Composition per liter: NaCl ............................................................................................ 30.0g Agar ............................................................................................ 15.0g Beef extract ................................................................................. 10.0g Peptone........................................................................................ 10.0g pH 7.1 ± 0.2 at 25°C

Preparation of Medium: Add components to tap water and bring

Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems.

Preparation of Supplement Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

VCAT Inhibitor: Composition per 10.0mL: Colistin....................................................................................... 7.5mg Trimethoprim lactate.................................................................. 5.0mg Vancomycin ............................................................................... 4.0mg Anisomycin................................................................................. 0.02g

Preparation of VCAT Inhibitor: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except supplement solution and VCAT inhibitor, to distilled/deionized water and bring volume to 980.0mL. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile supplement solution and sterile VCAT inhibitor. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the isolation and cultivation of pathogenic Neisseria from

volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

specimens containing mixed flora of bacteria and fungi.

Use: For the cultivation and maintenance of Marinomonas commu-

Composition per liter:

nis=Alteromonas communis and Marinomonas vaga=Alteromonas vaga.

Martin-Lewis Agar Composition per liter: Agar ............................................................................................ 12.0g Hemoglobin ................................................................................ 10.0g Pancreatic digest of casein ............................................................ 7.5g Selected meat peptone .................................................................. 7.5g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 4.0g Cornstarch ..................................................................................... 1.0g KH2PO4 ......................................................................................... 1.0g Supplement solution ................................................................10.0mL VCAT inhibitor ........................................................................10.0mL pH 7.2 ± 0.22 at 25°C

Source: Martin-Lewis agar is available as a prepared medium from BD Diagnostic Systems.

Supplement Solution: Composition per liter: Glucose ..................................................................................... 100.0g L-Cysteine·HCl............................................................................ 25.9g L-Glutamine................................................................................. 10.0g L-Cystine ....................................................................................... 1.1g Adenine ......................................................................................... 1.0g Nicotinamide adenine dinucleotide ............................................ 0.25g Vitamin B12 ................................................................................... 0.1g Thiamine pyrophosphate............................................................... 0.1g Guanine·HCl ............................................................................... 0.03g Fe(NO3)3·6H2O ........................................................................... 0.02g p-Aminobenzoic acid................................................................ 0.013g Thiamine·HCl ............................................................................ 3.0mg © 2010 by Taylor and Francis Group, LLC

1025

Martin-Lewis Agar, Enriched Agar ............................................................................................ 12.0g Pancreatic digest of casein............................................................ 7.5g Selected meat peptone .................................................................. 7.5g NaCl.............................................................................................. 5.0g K2HPO4......................................................................................... 4.0g Cornstarch..................................................................................... 1.0g KH2PO4......................................................................................... 1.0g Sarcina lutea suspension .........................................................20.0mL Horse serum, inactivated .........................................................20.0mL Supplement solution ................................................................10.0mL PCAT inhibitor.........................................................................10.0mL pH 7.2 ± 0.22 at 25°C

Source: The supplement solution (IsoVitaleX® enrichment) is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems. Sarcina lutea Suspension: Composition per 20.0mL: Sarcina lutea FDA 1001 ................................................. 106–107 cells

Preparation of Sarcina lutea Suspension: Aseptically wash the growth of 24-hr cultures of Sarcina lutea FDA 1001 cells from ThayerMartin plates with sterile soybean casein digest broth. Standardize the suspension by adding additional sterile tryptic soy broth to yield 40% light transmission at 530nm wavelength.

Soybean Casein Digest Broth: Composition per liter: Pancreatic digest of casein.......................................................... 17.0g NaCl.............................................................................................. 5.0g Papaic digest of soybean meal...................................................... 3.0g K2HPO4......................................................................................... 2.5g Glucose ......................................................................................... 2.5g pH 7.3 ± 0.2 at 25°C

1026

Mating Agar

Preparation of Soybean Casein Digest Broth: Add components

Use: For the cultivation and maintenance of Filobasidiella neofor-

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

mans.

Supplement Solution: Composition per liter:

Composition per liter:

Glucose ..................................................................................... 100.0g L-Cysteine·HCl............................................................................ 25.9g L-Glutamine................................................................................. 10.0g L-Cystine ....................................................................................... 1.1g Adenine ......................................................................................... 1.0g Nicotinamide adenine dinucleotide ............................................ 0.25g Vitamin B12 ................................................................................... 0.1g Thiamine pyrophosphate............................................................... 0.1g Guanine·HCl ............................................................................... 0.03g Fe(NO3)3·6H2O ........................................................................... 0.02g p-Aminobenzoic acid ................................................................ 0.013g Thiamine·HCl ............................................................................ 3.0mg

Maximum Recovery Diluent NaCl.............................................................................................. 8.5g Peptone ......................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: This diluent is a physiologically isotonic and protective medium for maximal recovery of microorganisms from a variety of sources.

M-Azide HiVeg Broth Base with Triphenyltetrazolium Chloride

Preparation of Supplement Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

PCAT Inhibitor: Composition per 10.0mL: Anisomycin ................................................................................. 0.02g Colistin....................................................................................... 7.5mg Trimethoprim lactate.................................................................. 5.0mg Penicillin G ............................................................................ 25,000U

Preparation of PCAT Inhibitor: Add components to distilled/de-

Composition per liter: Saccharose ................................................................................ 100.0g Plant hydrolysate No. 1............................................................... 40.0g Yeast extract................................................................................ 10.0g K2HPO4......................................................................................... 4.0g Glucose ......................................................................................... 2.0g NaN3 ............................................................................................. 0.4g Triphenyltetrazolium chloride solution .....................................5.0mL pH 7.2 ± 0.2 at 25°C

ionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Source: This medium is available as a premixed powder from Hi-

Preparation of Medium: Add components—except Sarcina lutea

Caution: Sodium azide is toxic. Azides also react with metals and

suspension, horse serum, supplement solution, and PCAT inhibitor—to distilled/deionized water and bring volume to 940.0mL. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 20.0mL of sterile Sarcina lutea suspension, 20.0mL of sterile horse serum, 10.0mL of supplement solution, and 10.0mL of sterile PCAT inhibitor. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the isolation and cultivation of pathogenic Neisseria, especially penicillinase-producing strains, from specimens containing mixed flora of bacteria and fungi.

Mating Agar Composition per liter: Agar ............................................................................................ 40.0g Sucrose........................................................................................ 10.0g Xylose ........................................................................................... 2.0g KH2PO4 ......................................................................................... 1.0g MgSO4 .......................................................................................... 0.5g Yeast extract.................................................................................. 0.5g CaCl2 ............................................................................................. 0.1g NaCl .............................................................................................. 0.1g Biotin ..........................................................................................5.0μg pH 5.7 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 5.7. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. © 2010 by Taylor and Francis Group, LLC

Media. disposal must be highly diluted.

Triphenyltetrazolium Choride Solution: Composition per 5.0mL: Triphenyltetrazolium chloride ...................................................... 0.1g

Preparation of Triphenyltetrazolium Choride Solution: Add triphenyltetrazolium chloride to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 1.0L.Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 5.0mL triphenyltetrazolium chloride solution. Mix thoroughly. Aseptically distribute into tubes.

Use: For the detection and enrichment of fecal streptococci in water and sewage by the membrane filtration method.

MB Medium

K2HPO4 ......................................................................................... 0.4g Resazurin ................................................................................... 0.5mg Sodium formate solution..........................................................50.0mL Na2S·9H2O solution .................................................................10.0mL Cysteine-HCl·H2O solution .....................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL Selenite-tungstate solution.........................................................1.0mL pH 7.2 ± 0.2 at 25°C

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.25g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Sodium Formate Solution: Composition per 50.0mL: Na-formate .................................................................................... 6.8g

Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Selenite-Tungstate Solution Composition per liter: NaOH ............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

1027

Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N2 + 20% CO2 gas mixture. Add components, except sodium formate solution, NaHCO3, cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N2 + 20% CO2. Add solid NaHCO3. Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. The final pH should be 7.2.

Use: For the cultivation of Methanocalculus taiwanensis, Methanococcus voltae (Methanococcus voltaei), and Methanofollis aquaemaris.

MB Medium (DSMZ Medium 924) Composition per liter: NaCl.............................................................................................. 5.0g NaHCO3 ........................................................................................ 4.0g Yeast extract.................................................................................. 2.0g Trypticase™.................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g MgCl2·6H2O ................................................................................. 1.0g KCl................................................................................................ 0.5g CaCl2·2H2O .................................................................................. 0.4g K2HPO4......................................................................................... 0.4g Resazurin ................................................................................... 0.5mg Sodium formate solution..........................................................50.0mL Na2S·9H2O solution .................................................................10.0mL Cysteine-HCl·H2O solution .....................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL Selenite-tungstate solution.........................................................1.0mL pH 6.5 ± 0.2 at 25°C

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.25g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate.................................................................................... 6.8g

1028

MB Medium

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N2 + 20% CO2 gas mixture. Add components, except sodium formate solution, NaHCO3, cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool © 2010 by Taylor and Francis Group, LLC

to 25°C while sparging with 80% N2 + 20% CO2. Add solid NaHCO3. Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. The final pH should be 6.5.

Use: For the cultivation of Methanofollis aquaemaris DSM 14661.

MB Medium (DSMZ Medium 924) Composition per liter: NaCl............................................................................................ 10.0g NaHCO3 ........................................................................................ 4.0g Yeast extract.................................................................................. 2.0g Trypticase™.................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g MgCl2·6H2O ................................................................................. 1.0g KCl................................................................................................ 0.5g CaCl2·2H2O .................................................................................. 0.4g K2HPO4......................................................................................... 0.4g Resazurin ................................................................................... 0.5mg Sodium acetate solution...........................................................50.0mL Sodium formate solution..........................................................50.0mL Na2S·9H2O solution .................................................................10.0mL Cysteine-HCl·H2O solution .....................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL Selenite-tungstate solution.........................................................1.0mL pH 7.2 ± 0.2 at 25°C

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.25g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate.................................................................................... 6.8g

Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Sodium Acetate Solution: Composition per 50.0mL: Na-acetate ..................................................................................... 1.6g

Preparation of Sodium Acetate Solution: Add sodium acetate to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Selenite-Tungstate Solution Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

MB Medium

Na2S·9H2O Solution: Composition per 10.0mL:

1029

MB Medium (DSMZ Medium 924)

Na2S·9H2O .................................................................................. 0.25g

Composition per liter:

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

NaCl.............................................................................................. 5.0g NaHCO3 ........................................................................................ 4.0g Yeast extract.................................................................................. 2.0g Trypticase™.................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g MgCl2·6H2O ................................................................................. 1.0g KCl................................................................................................ 0.5g CaCl2·2H2O .................................................................................. 0.4g K2HPO4......................................................................................... 0.4g Resazurin ................................................................................... 0.5mg Sodium acetate solution...........................................................50.0mL Sodium formate solution..........................................................50.0mL Na2S·9H2O solution .................................................................10.0mL Cysteine-HCl·H2O solution .....................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL Selenite-tungstate solution.........................................................1.0mL pH 7.2 ± 0.2 at 25°C

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ................................................................... 0.25g

Vitamin Solution: Composition per liter:

Sodium Formate Solution: Composition per 50.0mL:

Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N2 + 20% CO2 gas mixture. Add components, except sodium acetate solution, sodium formate solution, NaHCO3, cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 870.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N2 + 20% CO2. Add solid NaHCO3. Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium acetate solution, 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. The final pH should be 7.2.

Na-formate.................................................................................... 6.8g

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................. 0.25g

1030

M-BCG Yeast and Mold Agar

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Thiamine hydrochloride.............................................................. 0.05g Bromcresol Green..................................................................... 0.026g pH 4.6 ± 0.2 at 25°C

Use: For the detection of fungi in routine analysis of beverages using the membrane filter technique.

m-Bismuth Sulfite Broth See: Bismuth Sulfite Broth

M-Bismuth Sulfite Broth Composition per liter: Peptic digest of animal tissue ..................................................... 20.0g Bismuth sulfite indicator............................................................. 16.0g Glucose ....................................................................................... 10.0g Plant extract ................................................................................ 10.0g Na2HPO4 ....................................................................................... 8.0g FeSO4 ............................................................................................ 0.6g Brilliant Green ............................................................................ 0.05g pH 7.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly and heat with frequent agitation until boiling. Boil for 1 min. Do not autoclave. Cool to 45°– 50°C. Mix to disperse the precipitate and aseptically distribute into sterile tubes or flasks. Use 2.0–2.2mL of medium for each membrane filter.

Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method.

oxygen-free 80% N2 + 20% CO2 gas mixture. Add components, except sodium acetate solution, sodium formate solution, NaHCO3, cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 870.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N2 + 20% CO2. Add solid NaHCO3. Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium acetate solution, 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. The final pH should be 7.2.

Plant peptone .............................................................................. 20.0g Bismuth sulfite indicator............................................................. 16.0g Glucose ....................................................................................... 10.0g Plant extract ................................................................................ 10.0g Na2HPO4 ....................................................................................... 8.0g FeSO4 ............................................................................................ 0.6g Brilliant Green ............................................................................ 0.05g pH 7.7 ± 0.2 at 25°C

Use: For the cultivation of Methanocalculus taiwanensis DSM 14663.

Source: This medium is available as a premixed powder from Hi-

M-Bismuth Sulfite HiVeg Broth Composition per liter:

Media.

M-BCG Yeast and Mold Agar Composition per liter: Glucose ....................................................................................... 50.0g Agar ............................................................................................ 15.0g Biopeptone .................................................................................. 10.0g Yeast extract.................................................................................. 9.0g MgSO4·7H2O ................................................................................ 2.1g KH2PO4 ......................................................................................... 2.0g Diastase ....................................................................................... 0.05g © 2010 by Taylor and Francis Group, LLC

Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method.

M-Brilliant Green HiVeg Broth

MBM Acetate Medium (Mineral Base Medium with Acetate) Composition per liter: Agar ............................................................................................ 16.0g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 1.0g NH4H2PO4 .................................................................................... 1.0g Sodium acetate·3H2O.................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Bromthymol Blue ....................................................................... 0.01g pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Gently heat and bring to boiling. Distribute into screw-capped tubes in 3.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position.

1031

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Add components, except thiosfulfate solution and trace elements SL-4 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Sparge solution with 80% N2 and 20% CO2 gas mixture to make it anoxic. Distribute into culture vessels (e.g., 20mL in 120mL serum bottles) under a gas atmosphere of 80% N2 and 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Prior to inoculation, add the trace elements solution and thiosulfate solution to the medium. Adjust pH to 7.0. After inoculation, pressurize culture vessels to 0.5 bar overpressure with 100% H2 gas.

Use: For the cultivation of Sulfuricurvum kujiense. m-Brilliant Green Broth See: Brilliant Green Broth

Use: For determining the nutritional independence of bacteria. Bacteria that are nutritionally independent turn the medium blue.

MBM Medium See: Methylene Blue Milk Medium

MBM Medium (Modified) (DSMZ Medium 1020) Composition per liter: NaNO3........................................................................................... 0.2g KH2PO4 ........................................................................................ 0.2g NH4Cl .......................................................................................... 0.2g MgCl2·6H2O.................................................................................. 0.4g KCl ............................................................................................... 0.2g CaCl2·2H2O................................................................................... 0.1g Resazurin .................................................................................. 1.0mg Thiosulfate solution .................................................................10.0mL Trace elements solution SL-4 ...................................................2.0mL pH 7.0 ± 0.2 at 25°C

Thiosulfate Solution: Composition per 10.0mL: Na2S2O3 ........................................................................................ 2.5g

M-Brilliant Green Broth Composition per liter: Proteose peptone......................................................................... 20.0g Lactose........................................................................................ 20.0g Saccharose .................................................................................. 20.0g NaCl............................................................................................ 10.0g Yeast extract.................................................................................. 6.0g Phenol Red.................................................................................. 0.16g Brilliant Green .......................................................................... 0.025g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to1.0L. Mix thoroughly. Distribute into tubes containing inverted Durham tubes, in 10.0mL amounts for testing 1.0mL or less of sample. Gently heat and bring to boiling. Do not autoclave. Cool the broth rapidly. Medium is sensitive to light.

Use: For the detection of coliform microorganisms in foods, dairy

Preparation of Thiosulfate Solution: Add components to dis-

products, water, and wastewater, as well as in other materials of sanitary importance.

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

M-Brilliant Green HiVeg Broth

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Composition per liter: Plant peptone No. 3..................................................................... 20.0g Lactose........................................................................................ 20.0g Saccharose .................................................................................. 20.0g NaCl............................................................................................ 10.0g Yeast extract.................................................................................. 6.0g Phenol Red.................................................................................. 0.16g Brilliant Green .......................................................................... 0.025g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

1032

M-Broth, HiVeg

Use: For the detection of coliform microorganisms in foods, dairy products, water, and wastewater, as well as in other materials of sanitary importance.

M-Broth, HiVeg Composition per liter: Plant hydrolysate......................................................................... 12.5g K2HPO4 ......................................................................................... 5.0g NaCl .............................................................................................. 5.0g Sodium citrate ............................................................................... 5.0g Yeast extract.................................................................................. 5.0g D-Mannose .................................................................................... 2.0g MgSO4 .......................................................................................... 0.8g MnCl2.......................................................................................... 0.14g FeSO4 .......................................................................................... 0.04g Polysorbate 80..........................................................................0.75mL pH 7.0 ± 0.22 at 25°C

Preparation of Medium: Add components, except cycloheximide solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile cycloheximide solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation of Listeria monocytogenes from dairy products.

McBride Listeria Agar Composition per liter:

mixed powder from HiMedia.

Agar ............................................................................................ 15.0g Glycine........................................................................................ 10.0g Pancreatic digest of casein............................................................ 5.0g Peptic digest of animal tissue ....................................................... 5.0g NaCl.............................................................................................. 5.0g Beef extract................................................................................... 3.0g Phenylethyl alcohol ...................................................................... 2.5g LiCl ............................................................................................... 0.5g pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from BD Di-

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized

Source: This medium, without polysorbate 80, is available as a pre-

Use: For the detection of Salmonella in dried foods and feeds.

MCA Composition per liter: Carrots....................................................................................... 200.0g Agar ............................................................................................ 15.0g

Preparation of Medium: Peel and slice fresh carrots. Place carrots in a blender. Add 500.0mL of distilled/deionized water. Blend for 40 sec at high speed. Filter through four layers of cheesecloth. Squeeze out juice from the residue. Bring volume of filtrate to 1.0L with distilled/deionized water. Add agar. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Phytophthora megasperma.

McBride Agar, Modified Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g Tryptose ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Beef extract ................................................................................... 3.0g Phenylethanol................................................................................ 2.5g LiCl ............................................................................................... 0.5g Cycloheximide solution ...........................................................10.0mL pH 7.3 ± 0.2 at 25°C

Cycloheximide Solution: Composition per 10.0mL:

agnostic Systems. water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the selective isolation of Listeria monocytogenes from clinical and nonclinical specimens containing mixed flora.

McBride Listeria HiVeg Agar Base with Blood and Selective Supplement Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g Plant hydrolysate No. 1............................................................... 10.0g NaCl.............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Phenyl ethanol .............................................................................. 2.5g LiCl ............................................................................................... 0.5g Sheep blood, defibrinated ........................................................50.0mL Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without blood or selective supplement solution, is available as a premixed powder from HiMedia. Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately. Selective Supplement Solution: Composition per 10.0mL: Cycloheximide.............................................................................. 0.2g

Preparation of Selective Supplement Solution: Add cyclohex-

Cycloheximide .............................................................................. 0.2g

imide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Cycloheximide Solution: Add cycloheximide to

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

mation and inhalation.

Preparation of Medium: Add components, except blood and selective supplement solution, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling.

McClung-Toabe Agar

1033

Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood and 10.0mL selective supplement solution. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Cornstarch..................................................................................... 1.0g KH2PO4......................................................................................... 1.0g

Use: For the selective isolation of Listeria monocytogenes from clini-

Preparation of Medium: To 1.0L of GC agar base, add the cornstarch. Gently heat while stirring to dissolve. Add the naladixic acid and colistin. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

cal and nonclinical specimens containing mixed flora.

McBride Listeria HiVeg Agar Base, Modified, with Blood and Selective Supplement (Modified McBride Listeria HiVeg Agar Base) Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g NaCl .............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Phenyl ethanol............................................................................... 2.5g LiCl ............................................................................................... 0.5g Sheep blood, defibrinated ........................................................50.0mL Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without blood or selective supplement solution, is available as a premixed powder from HiMedia.

Caution: LiCl is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately. Selective Supplement Solution: Composition per 10.0mL: Cycloheximide .............................................................................. 0.2g

Preparation of Selective Supplement Solution: Add cycloheximide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation. Preparation of Medium: Add components, except blood and selective supplement solution, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood and 10.0mL selective supplement solution. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes. Use: For the selective isolation of Listeria monocytogenes from clinical and nonclinical specimens containing mixed flora.

McCarthy Agar Composition per liter: Cornstarch ................................................................................... 10.0g Naladixic acid ........................................................................... 0.015g Colistin........................................................................................ 0.01g GC agar base .................................................................................1.0L pH 7.2 ± 0.2 at 25°C

GC Agar Base: Composition per liter: Agar ............................................................................................ 10.0g Pancreatic digest of casein ............................................................ 7.5g Peptic digest of animal tissue........................................................ 7.5g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 4.0g © 2010 by Taylor and Francis Group, LLC

Preparation of GC Agar Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Use: For the isolation and differentiation of Gardnerella vaginalis (Haemophilus vaginalis, Corynebacterium vaginale) from genitourinary specimens. Bacteria that can utilize starch appear as colonies surrounded by a clear zone.

McClung Carbon-Free Broth Composition per liter: NaNO3 .......................................................................................... 2.0g K2HPO4 ........................................................................................ 0.8g MgSO4·7H2O ................................................................................ 0.5g FeCl3 ........................................................................................... 0.01g MnCl2·4H2O .............................................................................. 8.0mg ZnSO4 ........................................................................................ 2.0mg pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat without boiling until salts dissolve. Cool to 25°C. Adjust pH to 7.2. Filter sterilize.

Use: For use as a basal medium in determining the carbon assimilation capabilities of microorganisms.

McClung-Toabe Agar Composition per liter: Proteose peptone......................................................................... 40.0g Agar ............................................................................................ 25.0g Na2HPO4 ....................................................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl.............................................................................................. 2.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Egg yolk emulsion, 50%........................................................100.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Preparation of Medium: Add components, except egg yolk emulsion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti-

1034

McClung-Toabe Agar, Modified

cally add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 15.0mL volumes.

Use: For the isolation and cultivation of Clostridium perfringens in

Egg yolk emulsion, 50%........................................................100.0mL Hemin solution...........................................................................1.0mL pH 7.6 ± 0.2 at 25°C

Egg Yolk Emulsion, 50%: Composition per 100.0mL:

foods.

McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 ............................................................... 40.0g Agar ............................................................................................ 20.0g Na2HPO4 ....................................................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl .............................................................................................. 2.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Egg yolk emulsion, 50% ........................................................100.0mL Hemin solution...........................................................................1.0mL pH 7.6 ± 0.2 at 25°C

Hemin Solution: Composition per 100.0mL: Hemin............................................................................................ 0.5g NaOH (1N solution).................................................................20.0mL

Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water.

Preparation of Medium: Add components, except egg yolk emulsion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes.

Use: For the cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on lecithinase production and lipase production. Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen.

McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 ............................................................... 40.0g Agar ............................................................................................ 20.0g Na2HPO4 ....................................................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl .............................................................................................. 2.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Neomycin...................................................................................... 0.1g © 2010 by Taylor and Francis Group, LLC

Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Hemin Solution: Composition per 100.0mL: Hemin ........................................................................................... 0.5g NaOH (1N solution).................................................................20.0mL

Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water.

Use: For the cultivation of Clostridium species. For the differentiation of Clostridium species based on lecithinase production and lipase production. Bacteria that produce lecithinase appear as colonies surrounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen.

McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 ............................................................... 20.0g Agar ............................................................................................ 20.0g Yeast extract.................................................................................. 5.0g Pancreatic digest of casein............................................................ 5.0g NaCl.............................................................................................. 5.0g Sodium thioglycolate .................................................................... 1.0g Egg yolk emulsion, 50%..........................................................80.0mL pH 7.6 ± 0.2 at 25°C

Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Preparation of Medium: Add components, except egg yolk emulsion, 50%, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti-

M-CP Agar Base

1035

cally add 80.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes.

to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C.

Use: For the cultivation of Clostridium botulinum.

Preparation of Medium: Add components—except egg yolk

McClung-Toabe Egg Yolk Agar, CDC Modified (CDC Modified McClung-Toabe Egg Yolk Agar) Composition per liter: Pancreatic digest of casein .......................................................... 40.0g Agar ............................................................................................ 25.0g NaHPO4 ........................................................................................ 5.0g Yeast extract.................................................................................. 5.0g D-Glucose ...................................................................................... 2.0g NaCl .............................................................................................. 2.0g Egg yolk emulsion .................................................................100.0mL MgSO4 (5% solution) ................................................................0.2mL pH 7.4 ± 0.2 at 25°C

Egg Yolk Emulsion: Composition: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs. Separate yolks from whites for 11 eggs. Mix egg yolks with 1 chicken egg.

Preparation of Medium: Add components, except egg yolk emulsion, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C. Aseptically add 100.0mL of sterile egg yolk emulsion. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes. Use: For the isolation, cultivation, and differentiation of anaerobic bacteria from foods. Bacteria that produce lecithinase appear as colonies surrounded by an insoluble opaque precipitate. Bacteria that produce lipase activity appear as colonies with a sheen or “pearly” surface. Bacteria that possess proteolytic activity appear as colonies surrounded by a clear zone.

McClung-Toabe Egg Yolk Agar, CDC Modified (CDC Modified McClung-Toabe Egg Yolk Agar) Composition per liter: Pancreatic digest of casein .......................................................... 40.0g Agar ............................................................................................ 25.0g Na2HPO4 ....................................................................................... 5.0g Yeast extract.................................................................................. 5.0g Glucose ......................................................................................... 2.0g NaCl .............................................................................................. 2.0g KH2PO4 ......................................................................................... 1.0g Egg yolk emulsion, 50% ........................................................100.0mL MgSO4·7H2O (5% solution) ......................................................0.2mL pH 7.3 ± 0.2 at 25°C

emulsion, 50%—to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 15.0mL volumes.

McClung Toabe HiVeg Agar Base wtih Egg Yolk Composition per liter: Plant peptone No. 3..................................................................... 40.0g Agar ............................................................................................ 25.0g Na2HPO4 ....................................................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl.............................................................................................. 2.0g KH2PO4......................................................................................... 1.0g MgSO4 .......................................................................................... 0.1g Egg yolk emulsion, 50%........................................................100.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without egg yolk emulsion, is available as a premixed powder from HiMedia.

Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg ............................................................................ 1 NaCl (0.9% solution) ...............................................................50.0mL

Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Preparation of Medium: Add components, except egg yolk emulsion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 15.0mL volumes.

Use: For the isolation and cultivation of Clostridium perfringens in foods.

M-CP Agar Base Composition per liter: Tryptose ...................................................................................... 30.0g Yeast extract................................................................................ 20.0g Agar ............................................................................................ 15.0g Sucrose.......................................................................................... 5.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g FeCl3·6H2O................................................................................. 0.09g Indoxyl β-D-glucoside ................................................................ 0.06g

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M-CP HiVeg Agar Base with Phenolphthalein Diphosphate

Bromcresol Purple ...................................................................... 0.04g Selective supplement solution B..............................................20.0mL Selective supplement solution A..............................................10.0mL pH 7.6 ± 0.2 at 25°C

Use: For the cultivation and identification of Providencia stuartii.

Source: This medium is available from HiMedia.

Tryptose ...................................................................................... 30.0g Yeast extract................................................................................ 20.0g Agar ............................................................................................ 15.0g Sucrose.......................................................................................... 5.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g MgO4·7H2O .................................................................................. 0.1g Bromcresol Purple ...................................................................... 0.04g Phenolphthalein biphosphate tetrazolium salt solution .......................................................................10.0mL Selective supplement solution ...................................................4.0mL Indoxyl-β-D-glucoside solution .................................................4.0mL Ferric chloride solution..............................................................1.0mL pH 7.6 ± 0.2 at 25°C

Selective Supplement Solution A: Composition per 10.0mL: D-Cycloserine......................................................................... 400.0mg

Polymyxin B sulfate................................................................. 25.0mg

Preparation of Selective Supplement Solution A: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Selective Supplement Solution B: Composition per 20.0mL: Phenolphthalein diphosphate ........................................................ 0.1g

Preparation of Selective Supplement Solution B: Add phenolphthalein diphosphate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except selective supplement solutions A and B, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add selective supplement solutions A and B. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: Recommended by the Directive of the Council of the European Union 98/83/EC for the isolation and enumeration of Clostridium perfringens from water samples using the membrane filtration technique.

M-CP HiVeg Agar Base with Phenolphthalein Diphosphate Composition per liter:

m-CP Medium Composition per liter:

Selective Supplement Solution: Composition per 4.0mL: D-Cycloserine................................................................................ 0.4g Polymyxin B sulfate ................................................................ 25.0mg

Preparation of Selective Supplement Solution: Add components to 4.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Phenolphthalein Biphosphate Tetrazolium Salt Solution: Composition per 10.0mL: Phenolphthalein biphosphate tetrazolium salt ..................................................................................... 25.0mg

Preparation of Phenolphthalein Biphosphate Tetrazolium Salt Solution: Add phenolphthalein biphosphate tetrazolium salt to

Plant hydrolysate No. 1............................................................... 30.0g Agar ............................................................................................ 15.0g L-Cysteine·HCl.............................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.1g FeCl3·6H2O ................................................................................... 0.09 Indoxyl β-D-glucoside................................................................... 0.06 Bromcresol Purple ........................................................................ 0.04 Sucrose.......................................................................................... 5.0g Yeast extract................................................................................ 20.0g Phenolphthalein diphosphate solution .....................................10.0mL pH 7.2 ± 0.2 at 25°C

10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Source: This medium, without phenolphthalein diphosphate, is avail-

Preparation of Ferric Chloride Solution: Add FeCl3·6H2O to 4.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

able as a premixed powder from HiMedia.

Phenolphthalein Diphosphate Solution: Composition per 10.0mL: Phenolphthalein diphosphate ........................................................ 2.0g

Preparation of Phenolphthalein Diphosphate Solution: Add phenolphthalein diphosphate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except phenolphthalein diphosphate solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat while stirring until boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 4550°C. Aseptially add 10.0mL of sterile phenolphthalein diphosphate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. © 2010 by Taylor and Francis Group, LLC

Indoxyl-β-D-glucoside Solution: Composition per 10.0mL: Indoxyl-β-D-glucoside ................................................................ 0.45g

Preparation of Indoxyl-β-D-glucoside Solution: Add indoxylβ-D-glucoside to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Ferric Chloride Solution: Composition per 4.0mL: FeCl3·6H2O .............................................................................. 30.0mg

Preparation of Medium: Add components, except selective supplement solution, phenolphthalein biphosphate tetrazolium salt solution, indoxyl-β-D-glucoside solution, and ferric chloride solution, to distilled/deionized water and bring volume to 981.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 4.0mL of selective supplement solution. Mix thoroughly. Aseptically add 10.0mL phenolphthalein biphosphate tetrazolium salt solution, 4.0mL indoxyl-β-D-glucoside solution, and 1.0mL ferric chloride solution. Mix thoroughly. Pour into sterile Petri dishes or aseptically distribute into tubes. Use: A selective, chromogenic medium for the rapid identification and enumeration of Clostridium perfringens in water samples, including water used in food and beverage production.

MD1- Medium

MCP Medium (Modified MacConkey Medium) (MacConkey Phosphatase Medium) Composition per liter: Pancreatic digest of gelatin ......................................................... 17.0g Agar ............................................................................................ 13.5g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Bile salts........................................................................................ 1.5g Pancreatic digest of casein ............................................................ 1.5g Peptic digest of animal tissue........................................................ 1.5g Na2HPO4....................................................................................... 0.6g Glucose ......................................................................................... 0.2g Methyl Blue ................................................................................ 0.07g Neutral Red ................................................................................. 0.03g Crystal Violet ............................................................................. 1.0mg Phenolphthalein diphosphate solution .....................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium, without phenolphthalein diphosphate, is available as a premixed powder from HiMedia.

Phenolphthalein Diphosphate Solution: Composition per 10.0mL: Phenolphthalein diphosphate ........................................................ 2.0g

Preparation of Phenolphthalein Diphosphate Solution: Add phenolphthalein diphosphate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Use: For the cultivation and identification of Providencia stuartii.

MD Medium Composition per liter: Agar ............................................................................................ 20.0g L-Malic acid................................................................................. 20.0g Pancreatic digest of casein .......................................................... 10.0g D-Glucose ...................................................................................... 5.0g Casamino acids ............................................................................. 3.0g Pancreatic digest of soybean meal ................................................ 1.5g Tween™ 80 ................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Bromcresol Green solution ......................................................20.0mL pH 7.0 ± 0.2 at 25°C

Bromcresol Green Solution: Composition per 30.0mL: Bromcresol Green ......................................................................... 0.1g NaOH (0.01N solution)............................................................30.0mL

Preparation of Bromcresol Green Solution: Add Bromcresol Green to 30.0mL of NaOH solution. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Adjust pH to 7.0 with 10N KOH. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. © 2010 by Taylor and Francis Group, LLC

1037

Use: For the isolation and cultivation of Salmonella species from foods.

MD 1 Medium Composition per liter: Pancreatic digest of casein............................................................ 3.0g MgSO4·7H2O ................................................................................ 2.0g CaCl2 ............................................................................................. 0.5g Trace elements solution .............................................................1.0mL Vitamin B12 solution ..................................................................1.0mL

Trace Elements Solution: Composition per liter: EDTA ............................................................................................ 8.0g MnCl2·4H2O ................................................................................. 0.1g CoCl2 .......................................................................................... 0.02g KBr ............................................................................................. 0.02g ZnCl2 ........................................................................................... 0.02g CuSO4 ......................................................................................... 0.01g H3BO3 ......................................................................................... 0.01g NaMoO4·2H2O............................................................................ 0.01g BaCl2 .......................................................................................... 5.0mg LiCl ............................................................................................ 5.0mg SnCl2·2H2O................................................................................ 5.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Vitamin B12 Solution: Composition per 10.0mL: Vitamin B12 ................................................................................ 5.0mg

Preparation of Vitamin B12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of myxobacteria.

MD1- Medium (DSMZ Medium 1118) Composition per liter: Casitone ....................................................................................... 3.0g MgSO4·7H2O ................................................................................ 2.0g CaCl2·2H2O .................................................................................. 0.7g Vitamin solution ......................................................................10.0mL Trace elements solution SL-4 ...................................................1.0mL pH 7.1 ± 0.2 at 25°C

1038

MDPA with Calcium Carbonate

NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Vitamin Solution: Composition per 10.0ml: Vitamin B12 ................................................................................ 0.5mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Glucose ......................................................................................... 1.0g CaCl2 .......................................................................................... 0.54g NaBr.......................................................................................... 0.039g NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 0.9g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Preparation of Medium: Add components, except NaHCO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL NaHCO3 solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.1. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL sterile vitamin solution. Mix thorougly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Bacillus halophilus.

Use: For the cultivation of Myxococcus xanthus.

Composition per liter:

MDPA See: Malt Dextrose Peptone Agar

MDPA with Calcium Carbonate Composition per liter: Agar ............................................................................................ 25.0g Malt extract ................................................................................. 20.0g Glucose ....................................................................................... 20.0g CaCO3 ........................................................................................... 5.0g Peptone.......................................................................................... 1.0g

Use: For the cultivation of Dekkera species. MDPYA4 See: Malt 4% Dextrose Peptone Yeast Agar MDYA4 See: Malt 4% Dextrose Yeast Agar m-E Agar See: E Agar

Me15% MH Agar (DSMZ Medium 582) Composition per liter: NaCl .......................................................................................... 121.5g Agar ............................................................................................ 20.0g MgSO4 ........................................................................................ 14.4g MgCl2 .......................................................................................... 10.5g Yeast extract................................................................................ 10.0g Proteose peptone no. 3 .................................................................. 5.0g KCl................................................................................................ 3.0g © 2010 by Taylor and Francis Group, LLC

Me15% MH Medium (DSMZ Medium 582) NaCl.......................................................................................... 121.5g MgSO4 ........................................................................................ 14.4g MgCl2.......................................................................................... 10.5g Yeast extract................................................................................ 10.0g Proteose peptone no. 3.................................................................. 5.0g KCl................................................................................................ 3.0g Glucose ......................................................................................... 1.0g CaCl2 .......................................................................................... 0.54g NaBr.......................................................................................... 0.039g NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 0.9g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Preparation of Medium: Add components, except NaHCO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL NaHCO3 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Bacillus halophilus. MEA See: Malt Extract Agar

Meat Extract with Peptone (Pepted Meat Broth) Composition per liter: NaCl............................................................................................ 15.0g Peptic digest of animal tissue ..................................................... 10.0g Meat extract .................................................................................. 3.0g pH 7.2 ± 0.2 at 25°C

Medium A for Producing Lysates Source: This medium is available from HiMedia.

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MED IIa

Preparation of Medium: Add components to distilled/deionized

Composition per liter:

Tris buffer stock solution .........................................................10.0mL CaCl2 (5.0% solution)..............................................................10.0mL MgSO4·7H2O (3.33% solution) .................................................1.0mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Alcaligenes species.

Meat Extract with Peptone and 1.5% Salt Composition per liter: NaCl ............................................................................................ 15.0g Peptone........................................................................................ 10.0g Meat extract .................................................................................. 3.0g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Alcaligenes species.

Meat Infusion Agar, HiVeg (Standard Infusion Agar, HiVeg) Composition per liter: Agar ............................................................................................ 25.0g Plant infusion .............................................................................. 10.0g Plant peptone............................................................................... 10.0g NaCl .............................................................................................. 5.0g pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the mass cultivation of organisms for vaccine or toxin production.

M-EC Test Agar Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g NaCl .............................................................................................. 7.5g Proteose peptone ........................................................................... 5.0g Dipotassium phosphate ................................................................. 3.3g Yeast extract.................................................................................. 3.0g KH2PO4 ......................................................................................... 1.0g Sodium lauryl sulphate ................................................................. 0.2g Sodium deoxycholate.................................................................... 0.1g Bromcresol Purple ...................................................................... 0.08g Bromphenol Red ......................................................................... 0.08g pH 7.3 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Do not autoclave. Pour into sterile Petri dishes or leave in tubes.

Tris Buffer Stock Solution: Composition per 500.0mL: Tris(hydroxymethyl)aminomethane·HCl.................................. 35.01g Tris(hydroxymethyl)aminomethane ........................................... 3.35g

Preparation of Tris Buffer Stock Solution: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 7.2. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 20 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Vampirovibrio chlorellavorus.

Medium A Composition per liter: D-Glucose .................................................................................... 20.0g

Agar ............................................................................................ 20.0g Yeast extract................................................................................ 10.0g Biotin ......................................................................................... 1.0mg Calcium pantothenate ................................................................ 1.0mg pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components, except biotin and calcium pantothenate, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Add biotin and calcium pantothenate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Aseptically add the sterile biotin and calcium pantothenate solution to the cooled sterile basal medium. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Zymomonas mobilis.

Medium A for Producing Lysates Composition per liter: Nutrient broth................................................................................ 8.0g KCl................................................................................................ 1.0g MgSO4·7H2O .............................................................................. 0.25g MnCl2....................................................................................... 1.25mg FeSO4 (1.0mM solution)............................................................1.0mL Ca(NO3)2 (1.0M solution)..........................................................1.0mL pH 7.0–7.2 at 25°C

Preparation of Medium: Add components, except FeSO4 and

Ca(NO3)2, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Adjust pH to 7.0–7.2. Autoclave for 30 min at 15 psi pressure–115°C. Cool to 45°–50°C. Prepare 1.0mM FeSO4 solution and 1.0M Ca(NO3)2 solution separately. Filter sterilize both solutions. Aseptically add the sterile FeSO4 solution and sterile Ca(NO3)2 solution to the cooled sterile basal medium. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the cultivation of microorganisms to be lysed.

1040

Medium 2A

CaCl2·2H2O ................................................................................ 0.25g (NH4)2SO4 .................................................................................... 0.2g

Arginine ...................................................................................... 10.0g NaCl .............................................................................................. 5.0g Agar .............................................................................................. 4.0g Peptone.......................................................................................... 1.0g K2HPO4·3H2O............................................................................... 0.3g Phenol Red .................................................................................. 0.01g pH 7.2–7.4 at 25°C

Preparation of Solution A: Add components to distilled/deionized

Preparation of Medium: Add components to distilled/deionized

Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C.

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of Pseudomonas species based on their production of arginine dihydrolase activity.

water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 4.3. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C.

Solution B: Composition per 500.0mL: Agar ............................................................................................ 20.0g

Preparation of Medium: Aseptically combine 500.0mL of solution A and 500.0mL of solution B. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of aciduric, thermophilic Bacillus strains.

Medium AS4 Composition per liter: Sucrose........................................................................................ 80.0g PPLO broth without Crystal Violet........................................500.0mL Horse serum ...........................................................................200.0mL Phenol Red (0.5% solution) .......................................................5.0mL pH 7.2 ± 0.2 at 25°C

PPLO Broth without Crystal Violet: Composition per 500.0mL: Beef heart, solids from infusion................................................ 11.53g Peptone........................................................................................ 2.33g NaCl ............................................................................................ 1.15g

Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems.

Preparation of PPLO Broth without Crystal Violet: Add

Medium 2508-85-1 with Amino Acids Composition per liter: Agar ............................................................................................ 20.0g Nutrient broth................................................................................ 8.0g D-Glucose ...................................................................................... 5.0g Polypeptone™ .............................................................................. 5.0g Yeast extract.................................................................................. 5.0g L-Lysine......................................................................................... 0.1g L-Methionine ............................................................................... 0.05g Diaminopimelic acid................................................................... 0.05g

components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Beef heart for infusion may be substituted; 100.0g of beef heart for infusion is equivalent to 500.0g of fresh heart tissue.

Use: For the cultivation and maintenance of Escherichia coli.

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Adjust pH to 7.2. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 200.0mL of noninactivated, sterile horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Composition per liter:

Use: For the cultivation and maintenance of Spiroplasma melliferum. Medium for Acetivibrio cellulolyticus See: BC Medium

Medium for Aciduric, Thermophilic Bacillus Strains Composition per liter: Solution A ..............................................................................500.0mL Solution B ..............................................................................500.0mL pH 4.3 ± 0.2 at 25°C

Solution A: Composition per 500.0mL: KH2PO4 ......................................................................................... 3.0g Glucose ......................................................................................... 1.0g Starch ............................................................................................ 1.0g Tryptone ........................................................................................ 1.0g Yeast extract.................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.5g © 2010 by Taylor and Francis Group, LLC

Medium for Ammonia Oxidizers MgSO4·7H2O ................................................................................ 0.2g (NH4)2SO4 .................................................................................. 0.13g K2HPO4....................................................................................... 0.09g CaCl2·2H2O ................................................................................ 0.02g Chelated iron.............................................................................. 1.0mg MnCl2·4H2O .............................................................................. 0.2mg Na2MoO4·2H2O ......................................................................... 0.1mg ZnSO4·7H2O .............................................................................. 0.1mg CuSO4·5H2O ............................................................................ 0.02mg CoCl2·6H2O ................................................................................ 2.0μg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and enrichment of ammonia-oxidizing bacteria from soil.

Medium for Ammonia Oxidizers Composition per liter: (NH4)2SO4 .................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 0.02g K2HPO4....................................................................................... 0.02g Chelated iron.............................................................................. 1.0mg

Medium for Ammonia-Oxidizing Bacteria

MnCl2·4H2O............................................................................... 0.2mg Na2MoO4·2H2O ......................................................................... 0.1mg ZnSO4·7H2O .............................................................................. 0.1mg CuSO4·5H2O ............................................................................ 0.02mg CoCl2·6H2O ................................................................................2.0μg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and enrichment of ammonia-oxidizing bacteria from soil.

Medium for Ammonia Oxidizers Composition per liter: K2HPO4 ......................................................................................... 0.5g (NH4)2SO4 ..................................................................................... 0.5g Phenol Red .................................................................................... 0.5g MgSO4·7H2O .............................................................................. 0.05g CaCl2·2H2O................................................................................. 0.02g NaCl ............................................................................................ 0.02g Na2MoO4·2H2O ..........................................................................2.4μg Metals “44” ................................................................................1.0mL

Preparation of Metals “44”: Add a few drops of H2SO4 to dis-

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K2HPO4....................................................................................... 0.05g Seawater.................................................................................400.0mL

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and enrichment of ammonia-oxidizing bacteria from brackish specimens.

Medium for Ammonia Oxidizers, Marine Composition per liter: (NH4)2SO4 .................................................................................. 1.32g MgSO4·7H2O ................................................................................ 0.2g Chelated iron............................................................................... 0.13g K2HPO4....................................................................................... 0.11g CaCl2·2H2O ................................................................................ 0.02g ZnSO4·7H2O .............................................................................. 0.1mg CuSO4·5H2O............................................................................ 0.02mg CoCl2·6H2O ................................................................................ 2.0μg MnCl2·4H2O ............................................................................... 2.0μg Na2MoO4·2H2O .......................................................................... 1.0μg Seawater........................................................................................1.0L

Preparation of Medium: Combine components. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the isolation, cultivation, and enrichment of marine ammonia-oxidizing bacteria.

Medium for Ammonia-Oxidizing Bacteria Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

(NH4)2SO4 ............................................................................. 235.0mg KH2PO4.................................................................................. 200.0mg CaCl2·2H2O ............................................................................. 40.0mg MgSO4·7H2O ........................................................................... 40.0mg Iron-EDTA-Phenol Red solution ...............................................1.0mL Na2CO3 solution...................................................................... variable

Use: For the isolation, cultivation, and enrichment of ammonia-oxidizing bacteria from soil.

Na2CO3 Solution: Composition per 100.0mL:

tilled/deionized water to inhibit precipitate formation. Add components to acidified distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized

Medium for Ammonia Oxidizers Composition per liter: (NH4)2SO4 ..................................................................................... 0.5g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.04g MgSO4·7H2O .............................................................................. 0.04g Ferric citrate ............................................................................... 0.5mg Phenol Red ................................................................................. 0.5mg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and enrichment of ammonia-oxidizing bacteria from soil.

Medium for Ammonia Oxidizers, Brackish Composition per liter: CaCO3 ........................................................................................... 5.0g NH4Cl ........................................................................................... 0.5g © 2010 by Taylor and Francis Group, LLC

Na2CO3 ......................................................................................... 5.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Iron-EDTA-Phenol Red Solution: Composition per 100.0mL: FeSO4·7H2O............................................................................. 50.0mg Sodium EDTA.......................................................................... 50.0mg Phenol Red............................................................................... 50.0mg

Preparation of Iron-EDTA-Phenol Red Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Medium: Add components, except Na2CO3 solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Add enough sterile Na2CO3 solution to turn the medium pale pink. During incubation and growth of bacteria, add additional sterile Na2CO3 solution to restore the pale pink color. Growth is complete when no further color change is observed.

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Medium B for Sulfate Reducers

Use: For the cultivation of Nitrosolobus multiformis and Nitrosomonas europaea.

Medium B for Sulfate Reducers (Postgate’s Medium B for Sulfate Reducers) Composition per liter: Sodium lactate............................................................................... 3.5g MgSO4·7H2O ................................................................................ 2.0g NH4Cl ........................................................................................... 1.0g CaSO4 ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g FeSO4·7H2O.................................................................................. 0.5g Ascorbic acid ................................................................................ 0.1g Thioglycollic acid ......................................................................... 0.1g pH 7.0–7.5 at 25°C

Preparation of Medium: Add components, except ascorbic acid and thioglycollic acid, to tap water and bring volume to 1.0L. For marine bacteria, NaCl may be added or seawater used in place of tap water. Mix thoroughly. Adjust pH to 7.0–7.5. Thioglycolate and ascorbate should be added immediately prior to sterilization. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and maintenance of Desulfovibrio species and Desulfotomaculum species. This medium turns black as a result of H2S production due to bacterial growth.

Medium for Bacillus schlegelii Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4·2H2O............................................................................. 2.9g KH2PO4 ......................................................................................... 2.3g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaHCO3 ........................................................................................ 0.5g CaCl2·2H2O................................................................................. 0.01g MnSO4·H2O ............................................................................. 10.0mg Ferric ammonium citrate solution............................................20.0mL Trace elements solution SL-6 ....................................................5.0mL pH 6.8 ± 0.2 at 25°C

Ferric Ammonium Citrate Solution: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Add components, except ferric ammonium citrate solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile ferric ammonium citrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the chemolithotrophic growth of Bacillus schlegelii.

Medium for Bacillus stearothermophilus Composition per 1001.0mL: NH4Cl ........................................................................................... 1.0g K2HPO4......................................................................................... 0.5g Yeast extract.................................................................................. 0.2g Casamino acids ............................................................................. 0.1g MgSO4·7H2O .............................................................................. 0.02g Phenol solution ......................................................................100.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.4 ± 0.2 at 25°C

Phenol Solution: Composition per 100.0mL: Phenol ......................................................................................... 0.47g

Preparation of Phenol Solution: Add phenol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except phenol solution and trace elements solution SL-4, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 100.0mL of sterile phenol solution and 1.0mL of sterile trace elements solution SL-4. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Bacillus stearothermophilus. Medium BG11 for Cyanobacteria See: BG11 Agar and BG11 Medium Medium BG11 for Marine Cyanobacteria See: BG11 Marine Agar and BG11 Marine Broth

Medium for Carbon Monoxide Oxidizers

Medium with Biphenyl (DSMZ Medium 457d) Composition per liter:

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added or sea water used in place of distilled/deionized water. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Na2HPO4 ..................................................................................... 2.44g KH2PO4 ....................................................................................... 1.52g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O................................................................................. 0.05g Trace elements solution SL-4 ..................................................10.0mL Biphenyl solution .....................................................................25.0mL pH 6.9 ± 0.2 at 25°C

Use: For detection, culturing, and storage of Desulfovibrio species and many Desulfotomaculum species. This medium should be used when a clear culture medium is desired such as for chemostat culture. This medium may be cloudy after sterilization but usually clears on cooling. It turns black as a result of H2S production due to bacterial growth.

Trace Elements Solution SL-4: Composition per liter:

Composition per liter:

EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Biphenyl Solution: Composition per liter: Biphenyl...................................................................................... 10.0g

Preparation of Biphenyl Solution: Add biphenyl to 1.0L ethanol. Mix thoroughly. Filter sterilize using a cellulose filter membrane. Preparation of Medium: Add components, except biphenyl solution, to 1.0L distilled/deionized water. Adjust pH to 6.9. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Add an aliquot of the biphenyl solution to a sterile flask so that the final concentration will be 0.25g/L biphenyl, and let the ethanol evaporate. Aseptically add sterile medium to the crystal-layered flask.

Use: For the cultivation of biphenyl- utilizing bacteria.

Medium C for Sulfate Reducers (Postgate’s Medium C for Sulfate Reducers) Composition per liter: Sodium lactate............................................................................... 6.0g Na2SO4 .......................................................................................... 4.5g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g Sodium citrate·2H2O..................................................................... 0.3g CaCl2·6H2O................................................................................. 0.06g MgSO4·7H2O .............................................................................. 0.06g FeSO4·7H2O.............................................................................. 0.004g pH 7.5 ± 0.2 at 25°C

Medium for Campylobacter DSM 806 (DSMZ Medium 121) Na-aspartate ................................................................................ 10.0g MgSO4·7H2O ................................................................................ 1.0g Yeast extract.................................................................................. 0.2g CaCl2·2H2O ............................................................................. 28.0mg Resazurin ................................................................................... 1.0mg Cysteine phosphate solution ..................................................100.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Gas under 80% N2 + 20% CO2.

Cysteine Phosphate Solution: Composition per 100.0mL: K2HPO4....................................................................................... 0.75g NaH2PO4 ................................................................................... 0.25g Cysteine-HCl·H2O ...................................................................... 0.25g

Preparation of Cysteine Phosphate Solution: Add components to 100.0mL distilled/deionized water. Mix thoroughly. Gas under 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except cysteine phosphate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 100.0mL of sterile cysteine phosphate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Sulfurospirillum sp.

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Medium for Carbon Monoxide Oxidizers

CaCl2·2H2O................................................................................. 0.03g Ferric ammonium citrate........................................................... 0.018g Trace elements solution SL-6 ....................................................1.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes. After inoculation, incubate in an atmosphere of 80% CO +10% O2 + 10% N2.

Use: For the chemoautotrophic cultivation and maintenance of Alcaligenes species, Pseudomonas carboxydohydrogena, and other Pseudomonas species.

Medium for Carbon Monoxide Oxidizers Composition per liter: Agar ............................................................................................ 12.0g Na2HPO4·12H2O........................................................................... 4.5g Sodium acetate .............................................................................. 3.0g NH4Cl ........................................................................................... 1.5g KH2PO4 ....................................................................................... 0.75g MgSO4·7H2O ............................................................................... 0.2g CaCl2·2H2O................................................................................. 0.03g Ferric ammonium citrate........................................................... 0.018g Trace elements solution SL-6 ....................................................1.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes. After inoculation incubate in air.

Medium for Chlorate Respirers (DSMZ Medium 908) Composition per liter: Solution A.....................................................................................1.0L Solution B ................................................................................10.0mL Solution C ................................................................................10.0mL Vitamin solution.........................................................................5.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Solution A: Composition per liter: NaHCO3 ........................................................................................ 2.5g Na-acetate ................................................................................... 1.36g NaClO3 ......................................................................................... 1.0g NaH2PO4 ....................................................................................... 0.6g NH4Cl ......................................................................................... 0.25g KCl................................................................................................ 0.1g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Solution B: Composition per 10.0mL: MgSO4 ..................................................................................... 30.0mg CaCl2·2H2O ............................................................................. 10.0mg

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Solution C: Na2MoO4 ................................................................................. 25.0mg Na2WO4·2H2O ......................................................................... 25.0mg

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Vitamin Solution: Composition per liter: Vitamin B12.............................................................................. 50.0mg Pantothenic acid....................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg Alpha-lipoic acid ..................................................................... 50.0mg p-Aminobenzoic acid............................................................... 50.0mg Thiamine-HCl·2H2O................................................................ 50.0mg Nicotinic acid........................................................................... 25.0mg Nicotinamide............................................................................ 25.0mg Biotin ....................................................................................... 20.0mg Folic acid ................................................................................. 20.0mg Pyridoxamine-HCl................................................................... 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g H3BO3 .................................................................................... 300.0mg CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg

Medium for Chlorobium ferrooxidans

ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ....................................................................7.7mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/ deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Aseptically and anaerobically combine 1000.0mL solution A, 10.0mL solution B and 10.0mL solution C. Aseptically and anaerobically add 5.0mL vitamin solution and 1.0mL trace elements solution SL-10. Mix thoroughly. The pH should be 7.2. Use: For the cultivation of Dechloromonas agitata and Azospira oryzae.

Medium with Chloroacrylic Acid (DSMZ Medium 457c) Composition per liter: Na2HPO4 ..................................................................................... 2.44g KH2PO4 ....................................................................................... 1.52g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O................................................................................. 0.05g Chloroacrylic acid solution ......................................................20.0mL Trace elements solution SL-4 ..................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components

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Aseptically add 20.0mL sterile chloroacrylic acid solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Use: For the cultivation of chloroacrylic acid-utilizing Burkholderia sp. (Burkholderia cepacia), Rhodococcus erythropolis (Arthrobacter picolinophilus, and Nocardia spp.

Medium for Chlorobium ferrooxidans (DSMZ Medium 29a) Composition per 5.0L: Solution A.....................................................................................4.0L Solution B ..............................................................................860.0mL Solution E ..............................................................................100.0mL Solution F.................................................................................30.0mL Solution C ..................................................................................5.0mL Solution D..................................................................................5.0mL pH 6.8 at 25°C Solution A: Composition per 4.0L: MgSO4 .......................................................................................... 2.5g KH2PO4......................................................................................... 1.7g NH4Cl ........................................................................................... 1.7g KCl................................................................................................ 1.7g CaCl2·2H2O ................................................................................ 1.25g Na-acetate ................................................................................... 0.82g

Preparation of Solution A: Add components to 4.0L distilled water. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C in a 5-liter special bottle or flask with four openings at the top, together with a teflon-coated magnetic bar. In this 5-liter bottle, two openings are for tubes in the central, silicon rubber stopper; one is a short, gasinlet tube with a sterile cotton filter, and the other is an outlet tube for medium, which reaches the bottom of the vessel at one end and has, at the other end, a silicon rubber tube with a pinch cock and a bell for aseptic dispensing of the medium into bottles. The other two openings have gas-tight screw caps; one of these openings is for the addition of sterile solutions and the other serves as a gas outlet. After autoclaving, cool solution A to room temperature under a N2 atmosphere with a positive pressure of 0.05–0.1 atm (a manometer for low pressure will be required). Saturate the cold medium with CO2 by magnetic stirring for 30 min under a CO2 atmosphere of 0.05–0.1 atm. Solution B: Distilled water........................................................................860.0mL

Preparation of Solution B: Autoclave distilled water for 15 min at 15 psi pressure–121°C in a cotton-stoppered Erlenmeyer flask. Cool to room temperature under an atmosphere of N2 in an anaerobic jar.

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Solution C: Composition per 100.0mL:

Preparation of Trace Elements Solution SL-4: Add components

Vitamin B12 ................................................................................ 2.0mg

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Chloroacrylic Acid Solution: Composition per liter: 3-Chloroacrylic acid ..................................................................... 4.0g

Preparation of Chloroacrylic Acid Solution: Add 3-chloroacrylic acid to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Filter sterilize.

Preparation of Medium: Add components, except chloroacrylic acid solution, to 1.0L distilled/deionized water. Adjust pH to 6.9. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. © 2010 by Taylor and Francis Group, LLC

Preparation of Solution C: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge under 100% N2 gas for 3 min. Filter sterilize. Store under N2 gas. Solution D: Composition per liter: FeCl2·4H2O................................................................................... 1.5g H3BO3 .................................................................................... 300.0mg CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg

1046

Medium D for Sulfate Reducers

NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ....................................................................7.7mL

Use: For the cultivation of Desulfovibrio species and Desulfotomacu-

Preparation of Solution D: Add FeCl2·4H2O to 10.0mL of HCl so-

Medium D for Sulfate Reducers (Postgate’s Medium D for Sulfate Reducers)

lution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 4.2g

Preparation of Solution E: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO2 until saturated. Filter sterilize under 100% CO2 into a sterile, gas-tight 100.0mL screw-capped bottle.

Solution F: Composition per 100.0mL: FeSO4 .......................................................................................... 25.0g

Preparation of Solution F: Add FeSO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO2 until saturated. Filter sterilize under 100% CO2 into a sterile, gas-tight 100.0mL screw-capped bottle. Preparation of Medium: Add solutions B, C, D, and E to solution A through one of the screw-cap openings against a stream of either N2 gas or, better, a mixture of 95% N2 and 5% CO2 while the medium is magnetically stirred. Adjust the pH of the medium with sterile HCl or Na2CO3 solution (2M solutions) to pH 6.8. Distribute the medium aseptically through the medium outlet tube into sterile, 100mL bottles (with metal caps and autoclavable rubber seals) using the positive gas pressure (0.05– 0.1 atm) of the N2 /CO2 gas mixture: Leave a small air bubble in each bottle to meet possible pressure changes. The tightly sealed, screw-cap bottles can be stored for several weeks or months in the dark. Use: For the cultivation of Chlorobium ferrooxidans. Medium D See: Castenholz D Medium Medium D, Modified See: Castenholz D Medium, Modified

Medium D for Sulfate Reducers (Postgate’s Medium D for Sulfate Reducers) Composition per liter: Sodium pyruvate ........................................................................... 3.5g MgCl2·6H2O.................................................................................. 1.6g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.............................................................................. 0.004g pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Malate or fumarate may also be used as a carbon source. For marine bacteria, NaCl may be added or seawater used in place of distilled/deionized water. Mix thoroughly. Adjust pH to 7.5. Filter sterilize. Aseptically distribute into sterile tubes or flasks. © 2010 by Taylor and Francis Group, LLC

lum species that can grow in the absence of sulfate.

Composition per liter: MgCl2·6H2O ................................................................................. 1.6g Choline chloride............................................................................ 1.0g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g KH2PO4......................................................................................... 0.5g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.............................................................................. 0.004g pH 7.5 ± 0.2 at 25°C

Use: For the cultivation of Desulfovibrio species and Desulfotomaculum species that can grow in the absence of sulfate.

Medium D for Thermus Composition per liter: Pancreatic digest of casein............................................................ 1.0g Yeast extract.................................................................................. 1.0g NaNO3 .......................................................................................... 0.7g KNO3 ............................................................................................ 0.1g MgSO4·7H2O ................................................................................ 0.1g Na2HPO4 ....................................................................................... 0.1g Nitrilotriacetic acid ....................................................................... 0.1g CaSO4·2H2O ............................................................................... 0.06g NaCl........................................................................................... 8.0mg MnSO4·H2O ............................................................................... 2.2mg ZnSO4·7H2O .............................................................................. 0.5mg H3BO3 ........................................................................................ 0.5mg FeCl3 ........................................................................................ 0.28mg Na2MoO4·2H2O ....................................................................... 0.03mg CuSO4 ...................................................................................... 0.02mg pH 8.2 ± 0.2 at 25°C

Preparation of Medium: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Readjust pH to 8.2 with H2SO4 or KOH. Add distilled/deionized water to 1.0L. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Thermus species.

Medium D for Thermus, Modified Composition per liter: Agar ............................................................................................ 25.0g Pancreatic digest of casein............................................................ 1.0g Yeast extract.................................................................................. 1.0g Salt solution ...........................................................................100.0mL pH 8.2 ± 0.2 at 25°C

Salt Solution: Composition per liter: NaNO3 ........................................................................................ 6.89g Na2HPO4·12H2O........................................................................... 2.8g

Medium for DSM 14457 and DSM 14458

KNO3 .......................................................................................... 1.03g Nitrilotriacetic acid ....................................................................... 1.0g MgSO4·7H2O ................................................................................ 1.0g CaSO4·2H2O ................................................................................. 0.6g NaCl ............................................................................................ 0.08g FeCl3·6H2O solution ................................................................10.0mL Trace elements solution ...........................................................10.0mL

FeCl3·6H2O Solution: Composition per 100.0mL: FeCl3·6H2O .............................................................................. 47.0mg

Preparation of FeCl3·6H2O Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Trace Elements Solution: Composition per liter: MnSO4·4H2O ................................................................................ 1.7g ZnSO4·7H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.5g CoCl2·6H2O ............................................................................. 46.0mg CuSO4·5H2O ............................................................................ 25.0mg Na2MoO4·2H2O ....................................................................... 25.0mg H2SO4 .........................................................................................0.5mL

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

1047

Preparation of NaN3 Solution: Add NaN3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use freshly prepared solution. Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Preparation of Medium: Add components, except polymyxin sulfate solution and NaN3 solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile polymyxin sulfate solution and NaN3 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation and cultivation of Corynebacterium species.

Medium D4 Composition per liter: Agar ............................................................................................ 15.0g Sucrose........................................................................................ 10.0g NH4Cl ........................................................................................... 5.0g Na2HPO4, anhydrous .................................................................... 2.3g Pancreatic digest of casein............................................................ 1.0g Sodium dodecyl sulfate................................................................. 0.6g Glycerol ...................................................................................10.0mL

Preparation of Salt Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Readjust pH to 8.2 with H2SO4 or KOH. Add distilled/deionized water to 1.0L.

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium:Add components to distilled/deionized

Use: For the selective isolation and cultivation of Pseudomonas syrin-

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 8.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Thermus aquaticus.

Medium D2 Composition per liter: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g LiCl ............................................................................................... 5.0g Pancreatic digest of casein ............................................................ 4.0g Yeast extract.................................................................................. 2.0g Tris(hydroxymethyl)amino-methane·HCl buffer.......................... 1.2g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.3g Polymyxin sulfate solution ......................................................10.0mL NaN3 solution...........................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Polymyxin Sulfate Solution: Composition per 10.0mL: Polymyxin sulfate ....................................................................... 0.04g

Preparation of Polymyxin Sulfate Solution: Add polymyxin sulfate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Use freshly prepared solution. NaN3 Solution: Composition per 10.0mL: NaN3 .......................................................................................... 2.0mg © 2010 by Taylor and Francis Group, LLC

Medium DG See: Castenholz DG Medium Medium DGN See: Castenholz DGN Medium

Medium for DSM 14457 and DSM 14458 (DSMZ Medium 956) Composition per liter: KH2PO4......................................................................................... 2.0g (NH4)2SO4 .................................................................................... 2.0g NaCl.............................................................................................. 0.5g MgSO4·7H2O ............................................................................ 0.125g FeSO4·7H2O................................................................................ 0.02g Methanol solution ....................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Methanol Solution: Composition per 10.0mL: Methanol ....................................................................................5.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except methanol solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL sterile methanol solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

1048

Medium for DSM 14457 and DSM 14458

Use: For the cultivation of Methylobacterium lusitanum and Methylobacterium suomiense.

Medium for DSM 14457 and DSM 14458 (DSMZ Medium 956) Composition per liter: KH2PO4 ......................................................................................... 2.0g (NH4)2SO4 ..................................................................................... 2.0g NaCl .............................................................................................. 0.5g MgSO4·7H2O ............................................................................ 0.125g FeSO4·7H2O................................................................................ 0.02g Methylamine solution ..............................................................10.0mL pH 7.2 ± 0.2 at 25°C

Methylamine Solution: Composition per 10.0mL: Methylamine ................................................................................. 3.0g

Preparation of Methylamine Solution: Add methylamine to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except methylamine solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL sterile methylamine solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Methylobacterium lusitanum and Methy-

Medium E for Sulfate Reducers (Postgate’s Medium E for Sulfate Reducers) Composition per liter: Agar ............................................................................................ 15.0g Sodium lactate .............................................................................. 3.5g MgCl2·6H2O ................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g Na2SO4 .......................................................................................... 1.0g CaCl2·2H2O .................................................................................. 1.0g Yeast extract.................................................................................. 1.0g KH2PO4......................................................................................... 0.5g Ascorbic acid ................................................................................ 0.1g Thioglycollic acid ......................................................................... 0.1g FeSO4·7H2O.............................................................................. 0.004g pH 7.6 ± 0.2 at 25°C

Preparation of Medium: Add components, except ascorbic acid and thioglycollic acid, to tap water and bring volume to 1.0L. For marine bacteria, NaCl may be added or seawater used in place of tap water. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.6. Thioglycolate and ascorbate should be added immediately prior to sterilization. Distribute into screw-capped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and enumeration of Desulfovibrio species and Desulfotomaculum species as black colonies in deep agar cultures. Also used for the isolation of pure cultures of Desulfovibrio species and Desulfotomaculum species.

lobacterium suomiense.

Medium E-2 Medium E for Bacillus

Composition per liter:

NaCl ............................................................................................ 50.0g K2HPO4 ....................................................................................... 10.6g Sucrose........................................................................................ 10.0g KH2PO4 ......................................................................................... 5.3g (NH4)2SO4 ..................................................................................... 1.0g MgSO4 ........................................................................................ 0.25g Trace salts solution...................................................................10.0mL

K2HPO4·3H2O .............................................................................. 7.5g KH2PO4......................................................................................... 3.7g NaNH4HPO4·4H2O....................................................................... 3.5g Tap water ......................................................................................1.0L Thiamine solution ....................................................................10.0mL MgSO4·7H2O solution .............................................................10.0mL n-Octane.................................................................................. variable pH 7.0 ± 0.2 at 25°C

Trace Salts Solution: Composition per liter:

Thiamine Solution: Composition per 10.0mL:

MnSO4·H2O .................................................................................. 3.0g Disodium EDTA ........................................................................... 1.0g FeSO4·7H2O.................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g CoCl2·6H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Thiamine .................................................................................. 10.0mg

Composition per liter:

Preparation of Trace Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically distribute into sterile tubes or flasks.

Preparation of Thiamine Solution: Add thiamine to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. MgSO4·7H2O Solution: Composition per 10.0mL: MgSO4·7H2O ............................................................................ 0.246g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components, except octane, to tap water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Inoculate tubes and place in a desiccator to which n-octane has been added and evaporated.

Use: For the cultivation of a recombinant strain of Escherichia coli that utilizes hydrocarbons.

Medium with EDTA as Carbon Source

1049

Composition per 1001.0mL:

Use: For the cultivation and maintenance of Ectothiorhodospira halochloris.

Basal medium ........................................................................800.0mL Solution C ..............................................................................200.0mL Vitamin solution B .....................................................................1.0mL

Composition per liter:

Medium for Ectothiorhodospira

Basal Medium: Composition per 800.0mL: NaCl .......................................................................................... 180.0g Na2SO4 ........................................................................................ 20.0g Na2CO3 ......................................................................................... 6.0g Na2S·9H2O .................................................................................... 1.0g Sodium succinate .......................................................................... 1.0g NH4Cl ........................................................................................... 0.8g KH2PO4 ......................................................................................... 0.5g Yeast extract.................................................................................. 0.5g MgCl2·6H2O.................................................................................. 0.1g CaCl2·7H2O................................................................................. 0.05g Trace elements solution A..........................................................1.0mL pH 8.5 ± 0.2 at 25°C

Trace Elements Solution A: Composition per liter: FeCl2·4H2O ................................................................................... 1.8g H3BO3 .................................................................................... 500.0mg CoCl2·6H2O ........................................................................... 250.0mg ZnCl2 ...................................................................................... 100.0mg MnCl2·4H2O............................................................................. 70.0mg Na2MoO4·2H2O ....................................................................... 30.0mg CuCl2·2H2O ............................................................................. 10.0mg NiCl2·6H2O .............................................................................. 10.0mg Na2SeO3·5H2O......................................................................... 10.0mg

Preparation of Trace Elements Solution A: Add components to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 3 with 1N HCl. Bring volume to 1.0L with distilled/ deionized water.

Preparation of Basal Solution: Add components to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Adjust pH to 8.5. Distribute into screw-capped bottles. Autoclave for 15 min at 14 psi pressure–120°C.

Vitamin Solution B: Composition per 100.0mL: Nicotinamide............................................................................ 35.0mg Thiamine dichloride ................................................................. 30.0mg p-Aminobenzoic acid............................................................... 20.0mg Biotin ....................................................................................... 10.0mg Calcium DL-pantothenate......................................................... 10.0mg Pyridoxal·HCl .......................................................................... 10.0mg Vitamin B12 ................................................................................ 5.0mg

Preparation of Vitamin Solution B: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Solution C: Composition per 200.0mL: NaHCO3 ...................................................................................... 14.0g

Preparation of Solution C: Add NaHCO3 to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Filter sterilize.

Medium with EDTA as Carbon and Nitrogen Source Agar ............................................................................................ 15.0g MgSO4·7H2O ................................................................................ 0.3g Disodium ethylenediaminetetraacetate....................................... 0.25g CaCl2·2H2O .............................................................................. 0.244g Ferric ammonium citrate............................................................. 0.05g Phosphate solution ...................................................................50.0mL Trace elements solution SL-6 ....................................................5.0mL Schlegel’s vitamin solution........................................................5.0mL pH 7.6 ± 0.4 at 25°C

Phosphate Solution: Composition per 50.0mL: Na2HPO4·2H2O........................................................................... 3.57g KH2PO4....................................................................................... 0.67g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Adjust pH to 7.6. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Schlegel’s Vitamin Solution: Composition per 100.0mL: Nicotinic acid................................................................................ 2.0g Pyridoxamine............................................................................. 5.0mg Cyanocobalamin ........................................................................ 2.0mg p-Aminobenzoate....................................................................... 1.0mg Thiamine .................................................................................... 1.0mg Calcium DL-pantothenate........................................................... 0.5mg Biotin ......................................................................................... 0.2mg

Preparation of Schlegel’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Filter sterilize.

Preparation of Medium: Add components, except phosphate solution and Schlegel’s vitamin solution, to distilled/deionized water and bring volume to 945.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 50.0mL of sterile phosphate solution and 5.0mL of sterile Schlegel’s vitamin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of bacteria that can utilize EDTA as a carbon source.

Medium with EDTA as Carbon Source (DSMZ Medium 473)

Preparation of Medium: To 800.0mL of sterile basal solution,

Composition per liter:

aseptically add 200.0mL of sterile solution C and 1.0mL of sterile vitamin solution B. Mix thoroughly.

Agar ............................................................................................ 15.0g MgSO4·7H2O .............................................................................. 0.49g

1050

Medium for Erythrobacter longus

Na2-EDTA..................................................................................... 0.2g Ferric ammonium citrate............................................................. 0.08g Ca(NO3)2·4H2O........................................................................... 0.02g Phosphate solution ...................................................................10.0mL Trace elements solution SL-6 ....................................................5.0mL Vitamin solution.........................................................................5.0mL pH 7.5 ± 0.2 at 25°C

CaCl2 .......................................................................................... 1.12g KCl......................................................................................... 664.0mg NaHCO3 ................................................................................. 192.0mg H3BO3 ...................................................................................... 26.0mg SrCl2 ........................................................................................ 24.0mg KBr ............................................................................................ 6.0mg NaF ............................................................................................ 3.0mg

Phosphate Solution: Composition per 10.0mL:

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

KH2PO4 ..................................................................................... 0.272g

Preparation of Phosphate Solution: Add KH2PO4 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Vitamin Solution: Composition per 100.0mL: KH2PO4 ..................................................................................... 0.272g Biotin .......................................................................................... 0.08g Folic acid..................................................................................... 0.08g Thiamin-HCl ............................................................................... 0.08g

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components

Ferric Citrate Solution: Composition per 10.0mL: Ferric citrate.................................................................................. 0.5g

Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Preparation of Medium: Add components, except artificial sea water, to distilled/deionized water and bring volume to 300.0mL. Mix thoroughly. Adjust pH to 7.5. Aseptically add 700.0mL artificial sea water. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Erythrobacter longus.

Medium F Composition per liter: MgSO4·7H2O ................................................................................ 0.5g (NH4)2SO4 .................................................................................. 0.15g KCl.............................................................................................. 0.05g KH2PO4....................................................................................... 0.05g Ca(NO3)2..................................................................................... 0.01g FeSO4·7H2O solution...............................................................10.0mL pH 3.5 ± 0.2 at 25°C

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

FeSO4·7H2O Solution: Composition per 10.0mL:

Preparation of Medium: Add components, except vitamin solution

FeSO4·7H2O.................................................................................. 1.0g

and phosphate solution, to 985.0mL distilled/deionized water. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes (20.0mL per Petri dish). Cool to room temperature. Aseptically add 10.0mL sterile phosphate solution and 5.0mL sterile vitamin solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Preparation of FeSO4·7H2O Solution: Add the FeSO4·7H2O to

Use: For the cultivation of unclassified bacterium DSM6780.

Medium for Erythrobacter longus (DSMZ Medium 695) Composition per liter: Peptone.......................................................................................... 2.0g Soytone ......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g Proteose peptone No.3 .................................................................. 1.0g Ferric citrate solution .................................................................2.0mL Artificial seawater..................................................................700.0mL pH 7.5 ± 0.2 at 25°C

Artificial Seawater: Composition per liter: NaCl ........................................................................................ 23.477g MgCl2·6H2O.............................................................................. 4.981g Na2SO4 ...................................................................................... 3.917g © 2010 by Taylor and Francis Group, LLC

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except FeSO4·7H2O

solution, to tap water and bring volume to 990.0mL. Mix thoroughly. Gently heat until dissolved. Adjust pH to 3.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile FeSO4·7H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Thiobacillus species.

Medium F for Sulfate Reducers (Postgate’s Medium F for Sulfate Reducers) Composition per liter: Agar ............................................................................................ 12.0g Pancreatic digest of casein.......................................................... 10.0g Sodium lactate .............................................................................. 3.5g Ferrous citrate ............................................................................... 0.5g Na2SO3 .......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Ascorbic acid ................................................................................ 0.1g Sodium thioglycolate .................................................................... 0.1g pH 7.1 ± 0.2 at 25°C

Medium G for Sulfate Reducers Preparation of Medium: Add components, except ascorbic acid and thioglycollic acid, to tap water and bring volume to 1.0L. For marine bacteria, NaCl may be added or seawater used in place of tap water. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.1. Thioglycolate and ascorbate should be added immediately prior to sterilization. Distribute into screw-capped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For isolation and cultivation of Desulfotomaculum nigrificans, Desulfovibrio species, and other Desulfotomaculum species especially in food. These bacteria form black colonies in deep agar cultures.

Medium for Freshwater Flexibacteria Composition per 1002.0mL: Casamino acids ............................................................................. 1.0g MgSO4·7H2O ................................................................................ 1.0g Tris (hydroxymethyl) amino methane........................................... 1.0g CaCl2·2H2O................................................................................... 0.1g KNO3 ............................................................................................ 0.1g Sodium glycerophosphate............................................................. 0.1g Thiamine .................................................................................... 1.0mg Cobalamine .................................................................................1.0μg Glucose solution ........................................................................1.0mL Trace elements solution .............................................................1.0mL pH 7.5 ± 0.2 at 25°C

Glucose Solution: Composition per 100.0mL: Glucose ....................................................................................... 10.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution: Composition per liter: ZnCl2 ........................................................................................... 20.8g H3BO3 ......................................................................................... 2.85g MnCl2·4H2O.................................................................................. 1.8g Sodium tartrate............................................................................ 1.77g FeSO4 .......................................................................................... 1.36g CoCl2·6H2O ............................................................................. 40.4mg CuCl2·2H2O ............................................................................. 26.9mg Na2MoO4·2H2O ....................................................................... 25.2mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except glucose solution and trace elements solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 1.0mL of sterile glucose solution and 1.0mL of sterile trace elements solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Cytophaga psychrophila, Flectobacillus major, Flexibacter aurantiacus, Flexibacter aurantiacus, Flexibacter elegans, Flexibacter flexilis, Flexibacter roseolus, Flexibacter ruber, Flexibacter sancti, and Herpetosiphon geysericola.

Medium with Fluoranthene (DSMZ Medium 457b) Composition per liter: Na2HPO4 ..................................................................................... 2.44g © 2010 by Taylor and Francis Group, LLC

1051

KH2PO4....................................................................................... 1.52g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Twen 80 ........................................................................................ 0.2g Fluoranthene solution ..............................................................50.0mL CaCl2·2H2O ................................................................................ 0.05g Trace elements solution SL-4 ..................................................10.0mL Fluoranthene solution ..............................................................50.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Fluoranthene Solution: Add fluoranthene to 1.0L acetone. Mix thoroughly. Filter sterilize using a cellulose filter membrane. Preparation of Medium: Add components, except fluoranthene solution, to 1.0L distilled/deionized water. Adjust pH to 6.9. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Add an aliquot of the fluoranthene solution to a sterile flask so that the final concentration will be 0.1g/L fluoranthene, and let the acetone evaporate. Aseptically add sterile medium to the crystal-layered flask. Use: For the cultivation of fluoranthene-utilizing Pseudomonas frederiksbergensis Sphingomonas sp. (Pseudomonas paucimobilis), and other bacteria.

Medium G for Sulfate Reducers (Postgate’s Medium G for Sulfate Reducers) Composition per 1015.2mL: Solution 1...............................................................................970.0mL Solution 4.................................................................................30.0mL Solution 8A, 8B, 8C, 8D, or 8E...............................................10.0mL Solution 5...................................................................................3.0mL Solution 2...................................................................................1.0mL Solution 3...................................................................................1.0mL Solution 6...................................................................................0.1mL Solution 7...................................................................................0.1mL pH 7.2 ± 0.2 at 25°C

1052

Medium for Halophilic Archaea

Solution 1: Composition per 970.0mL:

Solution 7: Composition per 100.0mL:

Na2SO4 .......................................................................................... 3.0g NaCl .............................................................................................. 1.2g MgCl2·6H2O.................................................................................. 0.4g KCl................................................................................................ 0.3g NH4Cl ........................................................................................... 0.3g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g

Succinic acid................................................................................. 0.6g Isobutyric acid .............................................................................. 0.5g Valeric acid ................................................................................... 0.5g 2-Methylbutyric acid .................................................................... 0.5g 3-Methylbutyric acid .................................................................... 0.5g Caproic acid .................................................................................. 0.2g

Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Adjust pH to 7.2 with 2N HCl. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution 2: Composition per 10.0mL: NaOH ......................................................................................... 5.0mg Na2SeO3 ................................................................................... 0.03mg

Preparation of Solution 2: Add NaOH and Na2SeO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution 3: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.12g MnCl2·4H2O.................................................................................. 0.1g ZnCl2 ........................................................................................... 0.07g H3BO3 ......................................................................................... 0.06g NiCl2·6H2O ............................................................................... 0.025g NaMoO4·2H2O.......................................................................... 0.025g CuCl2·2H2O .............................................................................. 0.015g

Preparation of Solution 3: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution 4: Composition per 30.0mL: NaHCO3 ...................................................................................... 2.55g

Preparation of Solution 4: Add NaHCO3 to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Gas with 100% CO2 for 10–15 min. Filter sterilize. Solution 5: Composition per 3.0mL: Na2S·9H2O .................................................................................. 0.36g

Preparation of Solution 5: Add Na2S·9H2O to distilled/deionized water and bring volume to 3.0mL. Mix thoroughly. Gas with 100% N2 for 5–10 min. Cap tube with a rubber stopper. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution 6: Composition per 100.0mL: Thiamine·HCl ............................................................................. 0.01g Cyanocobalamin ........................................................................ 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Biotin ......................................................................................... 1.0mg

Preparation of Solution 7: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 9.0 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution 8A: Composition per 100.0mL: Sodium acetate·3H2O.................................................................. 20.0g

Solution 8B: Composition per 100.0mL: Propionic acid ............................................................................... 7.0g

Solution 8C: Composition per 100.0mL: n-Butyric acid ............................................................................... 8.0g

Solution 8D: Composition per 100.0mL: Benzoic acid.................................................................................. 5.0g

Solution 8E: Composition per 100.0mL: n-Palmitic acid .............................................................................. 5.0g

Preparation of Solutions 8A–E: Add the appropriate amount of component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 9.0 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: To 970.0mL of cooled, sterile solution 1, aseptically add 1.0mL of sterile solution 2, 1.0mL of sterile solution 3, 30.0mL of sterile solution 4, 3.0mL of sterile solution 5, 0.1mL of sterile solution 6, 0.1mL of sterile solution 7, and 10.0mL of sterile solution 8A, 8B, 8C, 8D, or 8E. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the isolation and cultivation of Desulfovibrio baarsii, Desulfovibrio sapovorans, Desulfobacter species, Desulfonema species, Desulfobulbus species, and Desulfotomaculum acetoxidans.

Medium for Halophilic Archaea (DSMZ Medium 1184) Composition per liter: NaCl ......................................................................................... 195.0g MgSO4·7H2O .............................................................................. 50.8g MgCl2·6H2O ............................................................................... 32.5g Yeast extract ................................................................................. 5.0g KCl ............................................................................................... 5.0g CaCl2·2H2O .................................................................................. 0.8g NaBr ............................................................................................. 0.6g NaHCO3 ...................................................................................... 0.16g pH 6.7 ± 0.3 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5–7.0. Distribute into tubes or flasks. Gently heat while stirring and bring to

Medium 4 m 1

boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of halophilic archaea. For the cultivation of Pycnoporus cinnabarinus and Natrinema ejinorense.

Medium for Halophilic Bacilli Composition per liter: NaCl .......................................................................................... 100.0g Casamino acids ........................................................................... 10.0g Yeast extract................................................................................ 10.0g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except naphthalene, to distilled/deionized water and bring volume to 998.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 2.0mL of sterile naphthalene to 20.0mL of sterile basal salts. Ultrasonically homogenize the solution. Add the naphthalene–basal salts homogenate back to the remainder of the sterile basal salts medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and enrichment of hydrocarbon-degrading bacteria.

Medium K See: Kievskaya Broth

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Medium K (DSMZ Medium 1122)

Use: For the cultivation of halophilic Bacillus species.

Medium for Hydrocarbon-Degrading Bacteria Composition per 1020.0mL: NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g NaCl .............................................................................................. 0.4g Hydrocarbon ............................................................................20.0mL KH2PO4 solution........................................................................0.5mL Na2HPO4·H2O solution..............................................................0.5mL

KH2PO4 Solution: Composition per 100.0mL: KH2PO4 ....................................................................................... 10.0g

Preparation of KH2PO4 Solution: Add KH2PO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Na2HPO4·H2O Solution: Composition per 100.0mL: Na2HPO4·H2O............................................................................. 10.0g

Preparation of Na2HPO4·H2O Solution: Add Na2HPO4·H2O to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Add components—except hydrocarbon, KH2PO4 solution, and Na2HPO4·H2O solution—to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 0.5mL of sterile KH2PO4 solution and 0.5mL of the sterile Na2HPO4·H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes in 10.0mL volumes. Add 0.2mL of sterile hydrocarbon to each tube.

Use: For the cultivation and enumeration of hydrocarbon-degrading bacteria in fresh water.

Medium for Hydrocarbon-Degrading Bacteria (Naphthalene Mineral Salts Medium) Composition per liter: K2HPO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.3g CaCl2 ............................................................................................. 0.1g FeSO4·7H2O................................................................................ 0.02g Naphthalene ...............................................................................2.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

1053

Composition per liter: Agar ............................................................................................ 20.0g (NH4)2SO4 .................................................................................... 2.0g KH2PO4 ........................................................................................ 2.0g NaCl.............................................................................................. 0.5g MgSO4·7H2O ............................................................................ 0.125g FeSO4·7H2O.............................................................................. 0.002g Methanol, sterilized by filtration .............................................10.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.2 0. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0 sterile methanol. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the cultivation of Methylobacillus pratensis and Methylovorus mays.

Medium for Lactobacilli (ATCC Medium 980) Composition per liter: Agar ............................................................................................ 20.0g Peptone ....................................................................................... 12.5g Glucose ....................................................................................... 11.0g Sodium acetate............................................................................ 10.0g Yeast extract.................................................................................. 5.5g KH2PO4....................................................................................... 0.25g K2HPO4....................................................................................... 0.25g MgSO4 .......................................................................................... 0.1g MnSO4·4H2O .............................................................................. 0.05g FeSO4·7H2O................................................................................ 0.05g pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Autoclave for 10 min at 15 psi pressure–120°C. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Pediococcus acidilactici and Bacillus species.

Medium 4 m 1 Composition per liter: Agar ............................................................................................ 15.0g Peptone ......................................................................................... 3.0g

1054

Medium M71

Pancreatic digest of casein ............................................................ 3.0g Yeast extract.................................................................................. 3.0g Maltose.......................................................................................... 2.0g Lactose .......................................................................................... 1.0g Sodium dichromate solution ..................................................100.0mL pH 7.0 ± 0.2 at 25°C

Sodium Dichromate Solution: Composition per 100.0mL: Sodium dichromate ..................................................................... 0.05g

Preparation of Sodium Dichromate Solution: Add sodium dichromate to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Preparation of Medium: Add components, except sodium dichromate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. Aseptically add sterile sodium dichromate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and cultivation of Corynebacterium sepedonicum.

Medium M71 Composition per liter: Agar ............................................................................................ 20.0g Peptone........................................................................................ 10.0g Glucose ......................................................................................... 5.0g H3BO3 ........................................................................................... 1.0g Pancreatic digest of casein ............................................................ 1.0g Cycloheximide ............................................................................ 0.05g 2,3,5-Triphenyltetrazolium·HCl solution.................................10.0mL

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation. 2,3,5-Triphenyltetrazolium·HCl Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium·HCl.................................................. 0.05g

Preparation of 2,3,5-Triphenyltetrazolium·HCl Solution: Add 2,3,5-triphenyltetrazolium·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium·HCl solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile 2,3,5-triphenyltetrazolium·HCl solution. Mix thoroughly. Pour into sterile Petri dishes. Use: For the selective isolation and cultivation of Pseudomonas syringae.

Medium 523M Composition per liter: Agar ............................................................................................ 15.0g Sucrose........................................................................................ 10.0g Casamino acids ............................................................................. 2.0g K2HPO4 ......................................................................................... 2.0g Yeast extract.................................................................................. 2.0g MgSO4·7H2O ................................................................................ 0.3g © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation of Clavibacter toxicus.

Medium for Marine Flexibacteria Composition per 1001.0mL: Pancreatic digest of casein............................................................ 5.0g Yeast extract.................................................................................. 5.0g Tris (hydroxymethyl) amino methane .......................................... 1.0g KNO3 ............................................................................................ 0.5g Sodium glycerophosphate............................................................. 0.1g Trace elements solution .............................................................1.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: ZnCl2 ........................................................................................... 20.8g H3BO3 ......................................................................................... 2.85g MnCl2·4H2O ................................................................................. 1.8g Sodium tartrate............................................................................ 1.77g FeSO4 .......................................................................................... 1.36g CoCl2·6H2O ............................................................................. 40.4mg CuCl2·2H2O ............................................................................. 26.9mg Na2MoO4·2H2O ....................................................................... 25.2mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except trace elements solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 1.0mL of sterile trace elements solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Cytophaga aprica, Cytophaga diffluens, Cytophaga johnsonae, Cytophaga lytica, Cytophaga species, Flexibacter aggregans, Flexibacter aurantiacus, Flexibacter litoralis, Flexibacter tractuosus, Flexithrix dorotheae, Herpetosiphon cohaerens, Herpetosiphon nigricans, Herpetosiphon persicus, Microscilla arenaria, Microscilla furvescens, Microscilla marina, Microscilla sericea, and Saprospira grandis.

Medium for Marine Methylotrophs (DSMZ Medium 750) Composition per liter: NaCl............................................................................................ 25.0g Agar ............................................................................................ 20.0g Peptone ....................................................................................... 10.0g Beef extract................................................................................... 7.0g K2HPO4......................................................................................... 1.0g (NH4)2SO4 .................................................................................... 1.0g Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL filter sterilized methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Medium for Nitrite Oxidizers Use: For the cultivation and maintenance of Methylophaga marina and Methylophaga thalassica.

1055

Deoxythymidine-5´-Monophosphate Solution: Composition per 10.0mL: Deoxythymidine-5´-monophosphate ....................................... 15.0mg

Medium for Methylobacterium podarium (DSMZ Medium 1032) Composition per liter:

Preparation of Deoxythymidine-5´-Monophosphate Solution: Add deoxythymidine-5´-monophosphate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Agar ............................................................................................ 15.0g Na2HPO4·2H2O............................................................................. 7.9g KH2PO4 ......................................................................................... 1.5g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Methylamine solution ..............................................................30.0mL Trace metal solution (Kelly solution T)...................................10.0mL pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components, except deoxythymidine-5´-monophosphate solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C. Aseptically add 10.0mL of sterile deoxythymidine-5´-monophosphate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Methylamine Solution: Composition per 10.0ml:

siae.

Methylamine ................................................................................. 0.5g

Preparation of Methylamine Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Trace Metal Solution (Kelly Solution T): Composition per liter: EDTA .......................................................................................... 50.0g NaOH ............................................................................................ 9.0g CaCl2·2H2O................................................................................. 7.34g FeSO4·7H2O.................................................................................. 5.0g MnCl2·4H2O.................................................................................. 2.5g ZnSO4·7H2O ................................................................................. 1.0g CoCl2·6H2O .................................................................................. 0.5g (NH4)2MoO4 ................................................................................. 0.5g CuSO4·5H2O ................................................................................. 0.2g

Preparation of Trace Metal Solution: Add EDTA to 400.0mL distilled/deionized water. Add NaOH with constant mixing. This is best done in a 1–2L beaker on a magnetic stirrer. Add the other salts individually to about 30-40mL water to dissolve before adding to the EDTA-NaOH solution. Allow each component to mix thoroughly before adding the next component. Adjust pH to 6.0 using 1M NaOH (approximately 24.0mL). Bring volume to 1.0L with distilled/deionized water. Filter sterilize. Do not autoclave! Store in a dark bottle.

Preparation of Medium: Add components, except trace metal solution and methylamine solution, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Adjust pH to 7.4. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 10 min at 105 psi pressure–115°C. Cool to 50°C. Aseptically add methylamine solution and trace metal solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the cultivation of Methylobacterium podarium.

Medium N Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g Yeast nitrogen base without amino acids...................................... 6.7g Casamino acids, vitamin free........................................................ 2.0g Isoleucine ...................................................................................... 0.1g Valine ............................................................................................ 0.1g Deoxythymidine-5´-monophosphate solution .........................10.0mL © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Saccharomyces cerevi-

Medium N for Sulfate Reducers (Postgate’s Medium N for Sulfate Reducers) Composition per liter: (NH4)2SO4 .................................................................................... 7.0g Sodium lactate .............................................................................. 6.0g NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g KH2PO4......................................................................................... 0.5g Sodium citrate·2H2O..................................................................... 0.3g FeSO4·7H2O.................................................................................. 0.1g CaCl2·6H2O ................................................................................ 0.06g MgSO4·7H2O .............................................................................. 0.06g pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. For marine bacteria, NaCl may be added or seawater used in place of distilled/deionized water. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the detection, culturing, and storage of Desulfovibrio species and many Desulfotomaculum species. This medium should be used when a clear culture medium is desired such as for chemostat culture. This medium may be cloudy after sterilization but usually clears on cooling. It turns black as a result of H2S production due to bacterial growth.

Medium ND See: Castenholz ND Medium

Medium for Nitrite Oxidizers Composition per liter: KHCO3.......................................................................................... 1.5g KH2PO4......................................................................................... 0.5g K2HPO4......................................................................................... 0.5g KNO2 ............................................................................................ 0.3g MgSO4·7H2O ................................................................................ 0.2g NaCl.............................................................................................. 0.2g CaCl2·2H2O ................................................................................ 0.01g FeSO4·7H2O................................................................................ 0.01g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

1056

Medium for Nitrite Oxidizers, Marine

Use: For the isolation, cultivation, and enrichment of nitrate-oxidizing bacteria.

Medium for Nitrite Oxidizers, Marine Composition per liter:

ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

MgSO4·7H2O ................................................................................ 0.1g NaNO2......................................................................................... 0.07g CaCl2·2H2O................................................................................ 6.0mg K2HPO4 .................................................................................... 1.74mg Chelated iron.............................................................................. 1.0mg MnCl2·4H2O..............................................................................66.0μg Na2MoO4·2H2O ........................................................................30.0μg ZnSO4·7H2O .............................................................................30.0μg CuSO4·5H2O ...............................................................................6.0μg CoCl2·6H2O ................................................................................0.6μg Seawater.................................................................................700.0mL

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Medium: Add components to distilled/deionized

lution, to 1.0L distilled/deionized water. Adjust pH to 6.9. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Add an aliquot of the phenanthrene solution to a sterile flask so that the final concentration will be 0.1g/L phenanthrene, and let the acetone evaporate. Aseptically add sterile medium to the crystal-layered flask.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and enrichment of marine nitrateoxidizing bacteria.

Medium for Osmophilic Fungi (M 40 Y) Composition per liter: Sucrose...................................................................................... 400.0g Agar ............................................................................................ 20.0g Malt extract ................................................................................. 20.0g Yeast extract.................................................................................. 5.0g

Use: For the cultivation of osmophilic fungi.

Medium with Phenanthrene (DSMZ Medium 457b) Composition per liter: Na2HPO4 ..................................................................................... 2.44g KH2PO4 ....................................................................................... 1.52g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Tween 80....................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.05g Phenanthrene solution..............................................................50.0mL Trace elements solution SL-4 ..................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Phenanthrene Solution: Composition per liter: Phenanthrene................................................................................. 2.0g

Preparation of Phenanthrene Solution: Add phenanthrene to 1.0L acetone. Mix thoroughly. Filter sterilize using a cellulose filter membrane.

Preparation of Medium: Add components, except phenanthrene so-

Use: For the cultivation of phenanthrene-utilizing Sphingomonas sp. (Pseudomonas paucimobilis), Pseudomonas frederiksbergensis, and other bacteria.

Medium with Polyhydroxybutyric Acid as Carbon Source (DSMZ Medium 474) Composition per liter: Agar ............................................................................................ 16.0g Na2HPO4 ..................................................................................... 2.44g KH2PO4....................................................................................... 1.52g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 0.05g PHB solution............................................................................66.0mL Trace elements solution SL-4 ..................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Medium for Prosthecomicrobium and Ancalomicrobium

PHB Solution: Composition per 100.0mL:

1057

Preparation of Vitamin Solution: Add components to distilled/

Poly-ß-hydroxybutyric acid (PHB)............................................... 3.0g

deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of PHB Solution: Add poly-ß-hydroxybutyric acid

Preparation of Medium: Add components, except vitamin solu-

(PHB) to 100.0mL distilled/deionized water. Stir overnight. Sonicate until a white homogenous suspension is obtained. Autoclave for 5 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Add components, except PHB solution, to 1.0L distilled/deionized water. Adjust pH to 6.9. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Use 500.0mL to prepare bottom layer of a double agar plate by aseptically pouring 10.0mL amounts into sterile Petri dishes. Allow to solidify. Warm the PHB solution to 50°C. Aseptically add 33 mL of sterile PHB solution to the remaining 500.0mL of the medium. Mix thoroughly. Pour the PHB containing agar as a top layer over the solidified base agar. Use: For the cultivation of Comamonas testosteroni.

Medium for Prosthecomicrobium and Ancalomicrobium Composition per liter: Agar ............................................................................................ 15.0g Peptone.......................................................................................... 0.1g Hutner’s mineral base solution ................................................20.0mL Vitamin solution.......................................................................10.0mL

Hutner’s Mineral Base Solution: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O................................................................................. 3.34g FeSO4·7H2O.................................................................................. 0.1g (NH4)2MoO4 ............................................................................ 9.25mg Metals “44” ..............................................................................50.0mL

Preparation of Hutner’s Mineral Base Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Metals “44”: Composition per 100.0mL: ZnSO4·7H2O ................................................................................. 1.1g FeSO4·7H2O.................................................................................. 0.5g EDTA .......................................................................................... 0.25g MnSO4·7H2O ............................................................................ 0.154g CuSO4·5H2O ............................................................................... 0.04g Co(NO3)2·6H2O ........................................................................ 0.025g Na2B4O7·10H2O........................................................................ 0.018g

Preparation of Metals “44”: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................... 0.01g Calcium pantothenate ................................................................ 5.0mg Nicotinamide.............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg © 2010 by Taylor and Francis Group, LLC

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile vitamin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation of Prosthecomicrobium species and Ancalomicrobium species.

Medium for Prosthecomicrobium and Ancalomicrobium Composition per liter: (NH4)2SO4 .................................................................................. 0.25g Glucose ....................................................................................... 0.25g Na2HPO4 ................................................................................... 0.071g Modified Hutner’s basal salts ..................................................20.0mL Vitamin solution.......................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O ................................................................................ 3.34g FeSO4·7H2O.................................................................................. 0.1g (NH4)2MoO4 ............................................................................ 9.25mg Metals “44”..............................................................................50.0mL

Metals “44”: Composition per 100.0mL: ZnSO4·7H2O ................................................................................. 1.1g FeSO4·7H2O.................................................................................. 0.5g EDTA .......................................................................................... 0.25g MnSO4·7H2O ............................................................................ 0.154g CuSO4·5H2O............................................................................... 0.04g Co(NO3)2·6H2O ........................................................................ 0.025g Na2B4O7·10H2O........................................................................ 0.018g

Preparation of Metals “44”: Add a few drops of H2SO4 to distilled/deionized water to inhibit precipitate formation. Add components to acidified distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Vitamin Solution: Composition per liter: Thiamine·HCl ............................................................................ 5.0mg D-Calcium pantothenate ............................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

1058

Medium for Prosthecomicrobium and Ancalomicrobium, Modified

Preparation of Medium: Add components, except vitamin solution, to distilled deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Prosthecomicrobium enhydrum, Prosthecomicrobium pneumaticum, and Ancalomicrobium species.

Medium for Prosthecomicrobium and Ancalomicrobium, Modified Composition per liter: Agar ............................................................................................ 15.0g Glucose ......................................................................................... 1.0g (NH4)2SO4 ................................................................................... 0.25g Peptone........................................................................................ 0.15g Yeast extract................................................................................ 0.15g Modified Hutner’s basal salts ..................................................20.0mL Vitamin solution.......................................................................10.0mL

Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O................................................................................. 3.34g FeSO4·7H2O.................................................................................. 0.1g (NH4)2MoO4 ............................................................................ 9.25mg Metals “44” ..............................................................................50.0mL

Preparation of Metals “44”: Add a few drops of H2SO4 to dis-

tilled/deionized water to inhibit precipitate formation. Add components to acidified distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Vitamin Solution: Composition per liter: Thiamine·HCl ............................................................................ 5.0mg D-Calcium pantothenate ............................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vitamin solution, to distilled deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to © 2010 by Taylor and Francis Group, LLC

room temperature. Aseptically add 10.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Ancalomicrobium adetum, Prosthecomicrobium hirschii, and Prosthecomicrobium species.

Medium for Prosthecomicrobium and Ancalomicrobium with Nicotinamide Composition per liter: (NH4)2SO4 .................................................................................. 0.25g Glucose ....................................................................................... 0.25g Na2HPO4 ................................................................................... 0.071g Modified Hutner’s basal salts ..................................................20.0mL Vitamin solution.......................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O ................................................................................ 3.34g FeSO4·7H2O.................................................................................. 0.1g (NH4)2MoO4 ............................................................................ 9.25mg Metals “44”..............................................................................50.0mL

Preparation of Modified Hutner’s Basal Salts: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Readjust pH to 7.2 with H2SO4 or KOH. Add distilled/deionized water to 1.0L. Store at 5°C. Metals “44”: Composition per 100.0mL: ZnSO4·7H2O ................................................................................. 1.1g FeSO4·7H2O.................................................................................. 0.5g EDTA .......................................................................................... 0.25g MnSO4·7H2O ............................................................................ 0.154g CuSO4·5H2O ............................................................................... 0.04g Co(NO3)2·6H2O ........................................................................ 0.025g Na2B4O7·10H2O........................................................................ 0.018g

Preparation of Metals “44”: Add a few drops of H2SO4 to distilled/deionized water to inhibit precipitate formation. Add components to acidified distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Vitamin Solution: Composition per liter: Thiamine·HCl ............................................................................ 5.0mg D-Calcium pantothenate ............................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinamide.............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Medium for Roseospira Use: For the cultivation and maintenance of Ancalomicrobium adetum and Prosthecomicrobium species.

1059

Acetate Solution: Composition per 10.0mL: Sodium acetate............................................................................ 0.41g

Medium R Composition per liter: Na2S2O3·5H2O .............................................................................. 5.0g KNO3 ............................................................................................ 2.0g MgCl2·6H2O.................................................................................. 0.5g NH4Cl ........................................................................................... 0.5g KH2PO4 solution......................................................................10.0mL NaHCO3 solution .....................................................................10.0mL FeSO4·7H2O solution...............................................................10.0mL pH 7.0 ± 0.2 at 25°C

KH2PO4 Solution: Composition per 10.0mL: KH2PO4 ......................................................................................... 2.0g

Preparation of KH2PO4 Solution: Add KH2PO4 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.0g

Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. FeSO4·7H2O Solution: Composition per 10.0mL: FeSO4·7H2O............................................................................. 10.0mg

Preparation of FeSO4·7H2O Solution: Add the FeSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components—except KH2PO4 solu-

tion, NaHCO3 solution, and FeSO4·7H2O solution—to tap water and bring volume to 970.0mL. Mix thoroughly. Gently heat until dissolved. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile KH2PO4 solution, 10.0mL of NaHCO3 solution, and 10.0mL of FeSO4·7H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Thiobacillus denitrificans.

Medium for Roseospira (DSMZ Medium 998) Composition per liter: NaCl ............................................................................................ 20.0g MgCl2·6H2O.................................................................................. 1.0g MgSO4·7H2O .............................................................................. 0.25g NH4Cl ........................................................................................... 0.5g Yeast extract.................................................................................. 0.5g KH2PO4 ......................................................................................... 0.3g CaCl2·2H2O................................................................................. 0.05g NaHCO3 soltuion .....................................................................10.0mL Acetate solution .......................................................................10.0mL Succinate solution ....................................................................10.0mL Trace elements solution SL-12 ..................................................1.0mL Vitamin V7 solution...................................................................1.0mL pH 6.9 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Acetate Solution: Add sodium acetate to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% N2. Filter sterilize.

Succinate Solution: Composition per 10.0mL: Sodium succinate ........................................................................ 0.85g

Preparation of Succinate Solution: Add sodium succinate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% N2. Filter sterilize. NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% N2. Filter sterilize. Vitamin Solution V7: Composition per liter: Pyridoxine-HCl........................................................................ 50.0mg Nicotinic acid........................................................................... 20.0mg Vitamin B12 .............................................................................. 20.0mg Thiamine-HCl·2H2O................................................................ 10.0mg p-Aminobenzoic acid............................................................... 10.0mg D-Ca-pantothenate ..................................................................... 5.0mg Biotin ......................................................................................... 2.0mg

Preparation of Vitamin Solution V7: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-12: Composition per liter: FeSO4·7H2O.................................................................................. 1.1g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O ................................................................................ 0.19g MnCl2·2H2O ............................................................................... 0.05g ZnCl2 ........................................................................................ 42.0mg NiCl2·6H2O.............................................................................. 24.0mg Na2MoO4·4H2O ....................................................................... 18.0mg CuCl2·2H2O ............................................................................... 2.0mg

Preparation of Trace Elements Solution Sl-12: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except bicarbonate, vitamin, acetate, and succinate solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Boil for 1 min. Cool to room temperature while sparging with 90% N2 + 10% CO2 gas. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add bicarbonate and vitamin solutions. Mix thoroughly. Adjust pH to 6.9. Distribute into sterile 50mL screwcapped bottles. Add the organic acetate and succinate substrates.

Use: For the cultivation of Roseospira spp.

Medium for Roseospira Composition per liter: NaCl............................................................................................ 20.0g MgCl2·6H2O ................................................................................. 1.0g

1060

Medium S

MgSO4·7H2O .............................................................................. 0.25g NH4Cl ........................................................................................... 0.5g Yeast extract.................................................................................. 0.5g KH2PO4 ......................................................................................... 0.3g CaCl2·2H2O................................................................................. 0.05g NaHCO3 solution .....................................................................10.0mL Acetate solution .......................................................................10.0mL Succinate solution ....................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-12 ..................................................1.0mL Vitamin V7 solution...................................................................1.0mL pH 6.9 ± 0.2 at 25°C

Acetate Solution: Composition per 10.0mL: Sodium acetate ............................................................................ 0.41g

Preparation of Acetate Solution: Add sodium acetate to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% N2. Filter sterilize. Succinate Solution: Composition per 10.0mL: Sodium succinate ........................................................................ 0.85g

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.2g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Add components, except sulfide, bicarbonate, vitamin, acetate, and succinate solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Boil for 1 min. Cool to room temperature while sparging with 90% N2 + 10% CO2 gas. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add bicarbonate and vitamin solutions. Mix thoroughly. Adjust pH to 6.9. Distribute into sterile 50mL screw-capped bottles. Add sulfide and organic acetate and succinate substrates.

Use: For the cultivation of Roseospira navarrensis.

Medium S Composition per liter: Na2S2O3·5H2O .............................................................................. 5.0g (NH4)2SO4 .................................................................................... 4.0g KH2PO4......................................................................................... 4.0g MgSO4 .......................................................................................... 0.5g CaCl2 ........................................................................................... 0.25g FeSO4 .......................................................................................... 0.01g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% N2. Filter sterilize.

Use: For the cultivation of Thiobacillus species.

Vitamin Solution V7: Composition per liter:

Composition per liter:

Pyridoxine-HCl ........................................................................ 50.0mg Nicotinic acid ........................................................................... 20.0mg Vitamin B12 .............................................................................. 20.0mg Thiamine-HCl·2H2O ................................................................ 10.0mg p-Aminobenzoic acid ............................................................... 10.0mg D-Ca-pantothenate...................................................................... 5.0mg Biotin ......................................................................................... 2.0mg

Preparation of Vitamin Solution V7: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-12: Composition per liter: FeSO4·7H2O.................................................................................. 1.1g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O ................................................................................ 0.19g MnCl2·2H2O................................................................................ 0.05g ZnCl2 ........................................................................................ 42.0mg NiCl2·6H2O .............................................................................. 24.0mg Na2MoO4·4H2O ....................................................................... 18.0mg CuCl2·2H2O ............................................................................... 2.0mg

Medium S Glucose ....................................................................................... 10.0g K2HPO4........................................................................................ .4.0g Peptone ......................................................................................... 4.0g Yeast extract.................................................................................. 4.0g KH2PO4......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g pH 7.7 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the general cultivation of a wide variety of bacteria.

Medium SP 4 Composition per liter: Pancreatic digest of casein.......................................................... 11.0g Peptone ......................................................................................... 5.3g Glucose ......................................................................................... 5.0g NaCl.......................................................................................... 0.875g Beef extract............................................................................... 0.525g Yeast extract.............................................................................. 0.525g Beef heart, solids from infusion.................................................. 0.35g Fetal bovine serum, heat inactivated .....................................170.0mL Yeast extract solution.............................................................100.0mL CMRL 1066, 10X solution ......................................................50.0mL Fresh yeast extract solution .....................................................35.0mL

Medium for Sulfate Reducers

Phenol Red solution .................................................................20.0mL Penicillin solution ....................................................................10.0mL pH 7.6 ± 0.2 at 25°C

Yeast Extract Solution: Composition per 100.0mL: Yeast extract.................................................................................. 2.0g

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. CMRL 1066, 10X Solution: Composition per liter: NaCl .............................................................................................. 6.8g NaHCO3 ........................................................................................ 2.2g D-Glucose ...................................................................................... 1.0g KCl................................................................................................ 0.4g L-Cysteine·HCl·H2O.................................................................... 0.26g CaCl2, anhydrous .......................................................................... 0.2g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4·H2O............................................................................. 0.14g L-Glutamine................................................................................... 0.1g Sodium acetate·3H2O................................................................ 0.083g L-Glutamic acid ......................................................................... 0.075g L-Arginine·HCl............................................................................ 0.07g L-Lysine·HCl ............................................................................... 0.07g L-Leucine..................................................................................... 0.06g Glycine........................................................................................ 0.05g Ascorbic acid .............................................................................. 0.05g L-Proline ...................................................................................... 0.04g L-Tyrosine.................................................................................... 0.04g L-Aspartic acid ............................................................................ 0.03g L-Threonine ................................................................................. 0.03g L-Alanine................................................................................... 0.025g L-Phenylalanine......................................................................... 0.025g L-Serine ..................................................................................... 0.025g L-Valine ..................................................................................... 0.025g L-Cystine ..................................................................................... 0.02g L-Histidine·HCl·H2O ................................................................... 0.02g L-Isoleucine ................................................................................. 0.02g Phenol Red .................................................................................. 0.02g L-Methionine ............................................................................. 0.015g Deoxyadenosine.......................................................................... 0.01g Deoxycytidine ............................................................................. 0.01g Deoxyguanosine.......................................................................... 0.01g Glutathione, reduced ................................................................... 0.01g Thymidine ................................................................................... 0.01g Hydroxy-L-proline....................................................................... 0.01g L-Tryptophan ............................................................................... 0.01g Nicotinamide adenine dinucleotide ........................................... 7.0mg Tween™ 80 ................................................................................ 5.0mg Sodium glucoronate·H2O ........................................................... 4.2mg Coenzyme A .............................................................................. 2.5mg Cocarboxylase............................................................................ 1.0mg Flavin adenine dinucleotide ....................................................... 1.0mg Nicotinamide adenine dinucleotide phosphate ........................................................ 1.0mg Uridine triphosphate .................................................................. 1.0mg Choline chloride......................................................................... 0.5mg Cholesterol ................................................................................. 0.2mg 5-Methyldeoxycytidine .............................................................. 0.1mg Inositol ..................................................................................... 0.05mg © 2010 by Taylor and Francis Group, LLC

1061

p-Aminobenzoic acid............................................................... 0.05mg Niacin..................................................................................... 0.025mg Niacinamide........................................................................... 0.025mg Pyridoxine.............................................................................. 0.025mg Pyridoxal·HCl ........................................................................ 0.025mg Biotin ....................................................................................... 0.01mg D-Calcium pantothenate ........................................................... 0.01mg Folic acid ................................................................................. 0.01mg Riboflavin ................................................................................ 0.01mg Thiamine·HCl .......................................................................... 0.01mg

Source: CMRL 1066, 10X medium is available as a premixed powder from BD Diagnostics.

Preparation of CMRL 1066, 10X Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Filter sterilize. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free...................................... 25.0g

Phenol Red Solution: Composition per 100.0mL: Phenol Red.................................................................................. 0.01g

Preparation of Phenol Red Solution: Add Phenol Red to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Penicillin Solution: Composition per 10.0mL: Penicillin ........................................................................... 1,000,000U

Preparation of Penicillin Solution: Add penicillin to distilled/deionized water and bring volume to 10.0mL. Filter sterilize.

Preparation of Medium: Add components—except fetal bovine serum, yeast extract solution, CMRL 1066, 10X solution, fresh yeast extract solution, Phenol Red solution, and penicillin solution—to distilled/deionized water and bring volume to 615.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 170.0mL of sterile fetal bovine serum, 100.0mL of sterile yeast extract solution, 50.0mL of sterile CMRL 1066, 10X solution, 35.0mL of sterile fresh yeast extract solution, 20.0mL of sterile Phenol Red solution, and 10.0mL of sterile penicillin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the isolation and cultivation of Spiroplasma species from ticks.

Medium for Sulfate Reducers (ATCC Medium 1282) Composition per 1050.0mL: Modified Baar’s medium for sulfate reducers ........................................................1020.0mL Organic acid solution ...............................................................10.0mL Wolfe’s vitamin solution..........................................................10.0mL Wolfe’s mineral solution..........................................................10.0mL pH 7.5 ± 0.2 at 25°C

1062

Medium for Sulfate Reducers

Modified Baar’s Medium for Sulfate Reducers: Composition per 1020.0mL: Component I ..........................................................................400.0mL Component III........................................................................400.0mL Component II .........................................................................200.0mL Fe(NH4)2(SO4)2 (5% solution).................................................20.0mL

Component I: Composition per 400.0mL: Sodium citrate ............................................................................... 5.0g MgSO4 .......................................................................................... 2.0g CaSO4 ........................................................................................... 1.0g NH4Cl ........................................................................................... 1.0g

Preparation of Component I: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C.

Component II: Composition per 200.0mL: K2HPO4 ......................................................................................... 0.5g

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic

Preparation of Component II: Add K2HPO4 to distilled/deion-

acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Filter sterilize.

Component III: Composition per 400.0mL:

Preparation of Medium: To each test tube containing 10.0mL of modified Baar’s medium for sulfate reducers, aseptically add 0.1mL of sterile organic acid solution, 0.1mL of sterile Wolfe’s vitamin solution, and 0.1mL of sterile Wolfe’s mineral solution immediately prior to inoculation.

ized water and bring volume to 200.0mL. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C.

Sodium lactate............................................................................... 3.5g Yeast extract.................................................................................. 1.0g

Preparation of Component III: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Desulfotomaculum ther-

Preparation of Modified Baar’s Medium for Sulfate Reducers: Aseptically combine the three sterile solutions, except the

Medium for Sulfate Reducers (Postgate’s Medium for Sulfate Reducers) (ATCC Medium 1283)

Fe(NH4)2(SO4)2 solution. Mix thoroughly. Distribute 5.0mL volumes into tubes under 97% N2 + 3% H2. Add medium to tubes while still warm to exclude as much O2 as possible. Prepare a 5% solution of ferrous ammonium sulfate, Fe(NH4)2(SO4)2. Sterilize by filtration. Add 0.2mL of sterile Fe(NH4)2(SO4)2 solution to 10.0mL of medium immediately prior to inoculation.

Organic Acid Solution: Composition per 100.0mL: Butyric acid..............................................................................5.18mL Caproic acid ...............................................................................2.4mL Octanoic acid ...........................................................................1.25mL

Preparation of Organic Acid Solution: Add components to distilled/deionized water and bring volume to 75.0mL. Adjust pH to 7.0 with 5N NaOH. Bring volume to 100.0mL with distilled/deionized water. Filter sterilize.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Cyanocobalamin .....................................................................100.0μg © 2010 by Taylor and Francis Group, LLC

mobenzoicum and Desulfovibrio sapovorans.

Composition per liter: Part A .....................................................................................869.0mL Part C .....................................................................................100.0mL Part D .......................................................................................10.0mL Part E .......................................................................................10.0mL Part F........................................................................................10.0mL Part B .........................................................................................1.0mL pH 7.7 ± 0.2 at 25°C

Part A: Composition per 869.0mL: Na2SO4 .......................................................................................... 3.0g NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ........................................................................................... 0.3g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g

Preparation of Part A: Add components to distilled/deionized water and bring volume to 869.0mL. Mix thoroughly. Prepare and autoclave part A under 90% N2 + 10% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Part B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ................................................................................ 0.19g MnCl2·4H2O ................................................................................. 0.1g

Medium VTY

ZnCl2 ........................................................................................... 0.07g H3BO3 ......................................................................................... 0.06g Na2MoO4·2H2O .......................................................................... 0.04g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.02g HCl, 25% .................................................................................10.0mL

Preparation of Part B: Add the FeCl2·4H2O to the HCl. Add dis-

Peptone ......................................................................................... 5.0g CaCl2 ............................................................................................. 0.5g pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

tilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Use: For the cultivation of thermophilic actinomycetes.

Part C: Composition per 100.0mL:

Composition per liter:

NaHCO3 ........................................................................................ 5.0g

Preparation of Part C: Add the NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Gas with 90% N2 + 10% CO2 to remove residual O2. Part D: Composition per 10.0mL: Sodium butyrate ............................................................................ 0.7g Sodium caproate ........................................................................... 0.3g Sodium octanoate........................................................................ 0.15g

Preparation of Part D: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Part E: Composition per 10.0mL: Yeast extract.................................................................................. 1.0g Thiamine·HCl .........................................................................100.0μg p-Aminobenzoic acid................................................................40.0μg D(+)-Biotin ...............................................................................10.0μg

Preparation of Part E: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Part F: Composition per 10.0mL:

1063

Medium for Treponema pectinovorum Polypeptone™ .............................................................................. 5.0g Heart infusion broth...................................................................... 5.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 5.0g K2HPO4......................................................................................... 2.0g (NH4)2SO4 .................................................................................... 2.0g Agar .............................................................................................. 1.0g Pectin ............................................................................................ 0.8g L-Cysteine·HCl·H2O ................................................................... 0.68g Rumen fluid ...........................................................................500.0mL Resazurin (25.0 mg/100.0mL water) .........................................4.0mL pH 7.0–7.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Prepare and distribute anaerobically under 90% N2 + 10% CO2. Mix thoroughly. Adjust pH to 7.0–7.2. Distribute into screw-capped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Treponema pectinovorum.

Medium for Ureaplasma See: B Broth

Medium VTY Composition per 100.0mL:

Preparation of Medium: To 869.0mL of sterile cooled part A, aseptically add the remaining sterile solutions in the following order: part B, part C, part D, part E, and part F. Mix thoroughly. Adjust pH to 7.7. Anaerobically distribute under 80% N2 + 20% CO2 into sterile tubes or flasks.

Peptone ......................................................................................... 1.0g Noble agar..................................................................................... 0.7g Yeast extract.................................................................................. 0.5g L-Cysteine·HCl·H2O ..................................................................... 0.1g Salts A......................................................................................20.0mL Salts B......................................................................................20.0mL Glucose solution ........................................................................5.0mL NaHCO3 (5% solution) ..............................................................1.0mL Hemin solution...........................................................................1.0mL Volatile fatty acid solution .......................................................0.31mL Resazurin (0.1% solution) .........................................................0.1mL pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Desulfovibrio baarsii and Desulfovibrio sapovorans.

NaHCO3 Solution: Composition per 10.0mL:

Na2S·9H2O .................................................................................... 0.4g

Preparation of Part F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Medium for Thermophilic Actinomycetes Composition per liter: Agar ............................................................................................ 20.0g Soluble starch.............................................................................. 10.0g Maize extract................................................................................. 5.0g NaCl .............................................................................................. 5.0g © 2010 by Taylor and Francis Group, LLC

NaHCO3 ........................................................................................ 0.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Glucose Solution: Composition per 10.0mL: Glucose ......................................................................................... 1.0g

1064

Megasphaera Medium

Preparation of Glucose Solution: Add glucose to distilled/deion-

Preparation of Medium: Add components to distilled/deionized

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Salts A: Composition per liter:

Use: For the cultivation and maintenance of Megasphaera elsdenii.

CaCl2·2H2O................................................................................... 0.6g MgSO4 ........................................................................................ 0.45g

Mehlman's Maintenance HiVeg Medium

Preparation of Salts A: Add components to distilled/deionized wa-

Composition per liter:

ter and bring volume to 1.0L. Mix thoroughly.

Plant peptone No. 3..................................................................... 15.0g Yeast extract.................................................................................. 7.5g K2HPO4......................................................................................... 5.0g Plant hydrolysate .......................................................................... 5.0g Agar .............................................................................................. 3.0g (NH4)2SO4 .................................................................................... 1.5g Starch, soluble............................................................................... 1.0g Neutral Red................................................................................. 0.02g pH 7.3 ± 0.22 at 25°C

Salts B: Composition per liter: NaCl .............................................................................................. 4.5g (NH4)2SO4 ..................................................................................... 4.5g Potassium phosphate buffer (0.05M, pH 7.4) ...........................................................1.0L

Preparation of Salts B: Add NaCl and (NH4)2SO4 to 1.0L of 0.05M potassium phosphate buffer, pH 7.4. Mix thoroughly.

Hemin Solution: Composition per liter: Hemin............................................................................................ 0.5g NaOH (0.01N solution)..............................................................1.0mL

Preparation of Hemin Solution: Add hemin to 1.0mL of 0.01N NaOH solution. Mix thoroughly.

Volatile Fatty Acid Solution: Composition per 31.0mL: Acetic acid ...............................................................................17.0mL Propionic acid ............................................................................6.0mL n-Butyric acid ............................................................................4.0mL n-Valeric acid .............................................................................1.0mL Isovaleric acid ............................................................................1.0mL Isobutyric acid............................................................................1.0mL DL-α-Methylbutyric acid............................................................1.0mL

Preparation of Volatile Fatty Acid Solution: Combine components. Mix thoroughly.

Preparation of Medium: Add components, except glucose and NaHCO3 solutions, to distilled/deionized water and bring volume to 94.0mL. Mix thoroughly. Adjust pH to 7.2. Gently heat and gas with 95% N2 + 5% CO2 until reduced. Anaerobically distribute into tubes or flasks. Cap with rubber stoppers. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 50°C. Filter sterilize glucose solution and NaHCO3 solution separately. Aseptically and anaerobically add sterile glucose solution and sterile NaHCO3 solution to cooled, sterile basal medium.

Use: For the cultivation and maintenance of Roseburia cecicola.

Megasphaera Medium Composition per liter: Yeast extract.................................................................................. 4.0g K2HPO4 ......................................................................................... 3.2g KH2PO4 ......................................................................................... 1.6g Agar .............................................................................................. 1.0g NH4Cl ........................................................................................... 0.5g Sodium thioglycolate .................................................................. 0.45g CaCl2 ............................................................................................. 0.2g MgCl2 ............................................................................................ 0.2g Sodium lactate (60% solution).................................................16.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Campylobacter spp.

Melin–Norkrans Medium (MN) Composition per 1001.2mL: Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Malt extract................................................................................... 2.8g KH2PO4......................................................................................... 0.5g (NH4)2HPO4................................................................................ 0.25g MgSO4·7H2O.............................................................................. 0.15g CaCl2 ........................................................................................... 0.05g NaCl.......................................................................................... 0.025g Thiamine .................................................................................... 0.1mg Biotine.................................................................................... 0.005mg Oligo solution ..........................................................................1.66mL FeCl3 solution.............................................................................1.2mL

FeCl3 Solution: Composition per 10.0mL: FeCl3 ............................................................................................. 1.0g

Preparation of FeCl3 Solution: Add FeCl3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Oligo Solution: Composition per 1.66mL: Lilly and Barnett solution ..........................................................1.0mL Hoagland 1% solution..............................................................0.66mL

Hoagland Solution: Composition per 100.0mL: Fe(NO3)3·9H2OH3BO3 ............................................................... 2.86g MnCl2.......................................................................................... 1.81g ZnSO4·7H2O.............................................................................. 0.22g CuSO4·5H2O .............................................................................. 0.08g H2MoO4·H20.............................................................................. 0.01g

Preparation of Hoagland Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly.

Membrane Clostridium perfringens Medium

Melissococcus plutonius Agar (LMG Medium 110)

Lilly and Barnett Solution: Composition per 100.0mL: Fe(NO3)3·9H2O...................................................................... 723.5mg ZnSO4·7H2O .......................................................................... 439.8mg

Preparation of Lilly and Barnett Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Oligo Solution: Combine 1.0mL of Lilly and Barnett solution and 0.66mL of 1% Hoagland solution.

Use: For the cultivation of Agaricus xanthoderma, Agaricus macrosporus, Antrodia serialis, Armillaria mellea, Auricularia fuscosuccinea, Boletinellus merulioides, Boletus leucophaeus, Cephaliophora irregularis, Circinella umbellata, Kuehneromyces mutabilis, Laccaria laccata, Lentinus tigrinus, Lenzites betulina, Leucogyrophana mollusca, Lycoperdon foetidum, Macrolepiota rhacodes, Macrolepiota procera, Pholiota lenta, Phoma exigua, and many other fungi.

Melissococcus pluton Medium Composition per liter: Glucose ....................................................................................... 10.0g Neopeptone ................................................................................... 5.0g Peptone.......................................................................................... 2.5g Yeast extract.................................................................................. 2.5g Soluble starch................................................................................ 2.0g Pancreatic digest of casein ............................................................ 2.0g L-Cysteine·HCl·H2O.................................................................... 0.25g Phosphate buffer (1M, pH 6.7) ................................................50.0mL pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks that have been flushed with 90% N2 + 10% CO2. Cap with butyl rubber stoppers. Place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Melissococcus pluton.

Melissococcus pluton Medium Composition per liter: Agar ............................................................................................ 20.0g KH2PO4 ....................................................................................... 13.5g Glucose ....................................................................................... 10.0g Peptone........................................................................................ 10.0g Soluble starch.............................................................................. 10.0g pH 6.6 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.6 with KOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

1065

Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 10.0g Neopeptone ................................................................................... 5.0g Peptone ......................................................................................... 2.5g Yeast extract.................................................................................. 2.5g Starch, soluble............................................................................... 2.0g Trypticase™.................................................................................. 2.0g L-Cysteine·HCl ........................................................................... 0.25g Phosphate buffer solution ........................................................50.0mL

Phosphate Buffer Solution: Composition per liter: KH2PO4......................................................................................... 4.5g Na2HPO4·2H2O............................................................................. 5.8g

Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.7.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Anaerobically distribute into tubes sparged with a gas mixture of 100% N2 + 10% CO2. Immediately plug with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Melissococcus plutonius.

Membrane Clostridium perfringens Medium (m-CP Medium) Composition per 1040.0mL: Tryptose ...................................................................................... 30.0g Yeast extract................................................................................ 20.0g Agar ............................................................................................ 15.0g Sucrose.......................................................................................... 5.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.1g Bromcresol Purple ...................................................................... 0.04g Phenolphthalein solution .........................................................20.0mL Indoxyl-β-D-glucoside solution .................................................8.0mL Selective supplement solution ...................................................8.0mL Ferric chloride solution..............................................................4.0mL pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Selective Supplement Solution: Composition per 8.0mL: D-Cycloserine................................................................................ 0.8g Polymyxin B sulfate ................................................................ 50.0mg

Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 8.0mL. Mix thoroughly. Filter sterilize.

Indoxyl-β-D-glucoside Solution: Composition per 10.0mL: Indoxyl-β-D-glucoside ............................................................. 75.0mg

Preparation of Indoxyl-β-D-glucoside Solution: Add indoxylβ-D-glucoside to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

1066

Membrane Lactose Glucuronide Agar

Ferric Chloride Solution: Composition per 10.0mL:

pear yellow on this medium and as E. coli colonies are both lactose and β-glucuronidase positive, they will appear green.

FeCl3·6H2O ................................................................................. 0.45g

Preparation of Ferric Chloride Solution: Add ferric chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Membrane Lauryl Sulfate Broth Composition per liter:

Phenolphthalein biphosphate tetrasodium salt..................................................................... 0.15g

Peptone ....................................................................................... 39.0g Lactose........................................................................................ 30.0g Yeast extract.................................................................................. 6.0g Sodium lauryl sulfate.................................................................... 1.0g Phenol Red.................................................................................... 0.2g pH 7.4 ± 0.2 at 25°C

Preparation of Phenolphthalein Solution: Add phenolphthalein

Preparation of Medium: Add components to distilled/deionized

Phenolphthalein Solution: Composition per 20.0mL:

biphosphate tetrasodium salt to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except selective supplement solution, phenolphthalein solution, ferric chloride solution, and indoxyl-β-D-glucoside solution to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 8.0mL selective supplement solution, 20.0mL phenolphthalein solution, 4.0mL ferric chloride solution, and 8.0mL indoxyl-β-Dglucoside solution. Mix thoroughly. Pour into sterile Petri dishes or aseptically distribute into sterile tubes. Use: For the rapid isolation and presumptive identification of Clostridium perfringens from food and water samples. A selective and chromogenic medium for the presumptive identification of Clostridium perfringens, especially from water samples. Recommended in European Council Directive 98/83/EC for testing the quality of water intended for human consumption. C. perfringens colonies have a characteristic opaque yellow appearance. Most other Clostridium spp. will appear as either purple colonies, due to the lack of sucrose fermentation, or blue/green colonies where the organism is still cleaving indoxyl-β-D-glucoside and also fermenting sucrose.

Membrane Lactose Glucuronide Agar (MLGA) Composition per liter: Peptone........................................................................................ 40.0g Lactose ........................................................................................ 30.0g Agar ............................................................................................ 10.0g Yeast extract.................................................................................. 6.0g Sodium lauryl sulfate .................................................................... 1.0g Sodium pyruvate ........................................................................... 0.5g 5-Bromo-4-chloro-3-indoxylβ-D-glucuronic acid ................................................................ 0.2g Phenol Red .................................................................................... 0.2g pH 7.4 ± 0.2 at 25°C

Source: This medium is available from Oxoid Unipath. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Pour into sterile Petri dishes.

Use: For the direct enumeration of E. coli and coliforms in foods by the membrane filtration method. The chromogenic substrate 5-bromo4-chloro-3-indoxyl-β-D-glucuronic acid (BCIG) is cleaved by the enzyme β-glucuronidase and produces a blue chromophore that builds up within the bacterial cells. In addition, the incorporation of Phenol Red detects lactose fermentation and results in yellow colonies when acid is produced. Since coliform colonies are lactose positive, they will ap© 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the enumeration of coliform organisms and Escherichia coli in water. m-Endo Agar, LES See: Endo Agar, LES m-Endo Broth See: Endo Broth

M-Endo HiVeg Agar LES Composition per liter: Agar ............................................................................................ 15.0g Lactose.......................................................................................... 9.4g Plant hydrolysate No. 1................................................................. 7.5g NaCl.............................................................................................. 3.7g Plant hydrolysate .......................................................................... 3.7g Plant peptone ................................................................................ 3.7g K2HPO4......................................................................................... 3.3g Na2SO3 ......................................................................................... 1.6g Yeast extract.................................................................................. 1.2g KH2PO4......................................................................................... 1.0g Basic Fuchsin................................................................................ 0.8g Synthetic detergent No. III............................................................ 0.1g Sodium lauryl sulfate.................................................................. 0.05g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin.

Preparation of Medium: Add ethanol to approximately 900.0mL of distilled/deionized water. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile 60mm Petri dishes in 4.0mL volumes. Protect from the light. Use: For the cultivation and enumeration of coliform bacteria by the membrane filter method.

M-Endo HiVeg Broth Composition per liter: Lactose........................................................................................ 25.0g Plant peptone .............................................................................. 20.0g K2HPO4......................................................................................... 7.0g Yeast extract.................................................................................. 6.0g

Meniscus glaucopis Broth

Na2SO3 ......................................................................................... 2.5g Basic Fuchsin................................................................................ 1.0g pH 7.2 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin. Preparation of Medium: Add ethanol to approximately 900.0mL of distilled/deionized water. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Rapidly cool broth below 45°C. Do not autoclave. Use 1.8–2.0mL for each filter pad. Protect from the light. Prepare broth freshly.

Use: For the cultivation and enumeration of coliform bacteria from water by the membrane filter method.

M-Endo HiVeg Broth MF (MF Endo HiVeg Medium) (M-Coliform HiVeg Broth) Composition per liter: Lactose ........................................................................................ 12.5g Plant hydrolysate No. 1............................................................... 10.0g Plant hydrolysate........................................................................... 5.0g Plant special peptone .................................................................... 5.0g NaCl .............................................................................................. 5.0g K2HPO4 ..................................................................................... 4.375g Na2SO3 ......................................................................................... 2.1g Yeast extract.................................................................................. 1.5g KH2PO4 ..................................................................................... 1.375g Basic Fuchsin .............................................................................. 1.05g Synthetic detergent No. III............................................................ 0.1g Sodium lauryl sulfate .................................................................. 0.05g pH 7.2 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from Hi-

1067

MgSO4·7H2O ................................................................................ 0.2g CaCl2·H2O .................................................................................. 0.01g FeSO4·7H2O............................................................................... 1.0mg Resazurin (0.025% solution) .....................................................4.0mL Trace elements solution SL-6 ....................................................1.0mL Vitamin solution.......................................................................10.0mL pH 7.3 ± 0.2 at 25°C

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Vitamin Solution: Composition per liter: Vitamin B12 ................................................................................ 2.8mg Thiamine·HCl .......................................................................... 0.28mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.3 with 10% Na2CO3. Gently heat and bring to boiling. Continue boiling until resazurin changes color. Cool to 50°C. Distribute into tubes in 7.0mL volumes under O2-free 97% N2 + 3% H2. Cap with rubber stoppers under O2-free 97% N2 + 3% H2. Place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C using fast exhaust. Cool to 50°C. Aseptically add 0.25mL of sterile vitamin solution to each tube.

Media.

Use: For the cultivation and maintenance of Meniscus glaucopis.

Caution: Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin.

Composition per liter:

Preparation of Medium: Add ethanol to approximately 900.0mL of distilled/deionized water. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Rapidly cool broth below 45°C. Do not autoclave. Use 1.8–2.0mL for each filter pad. Protect from the light. Prepare broth freshly.

Use: For the cultivation and enumeration of coliform bacteria from water by the membrane filter method.

Meniscus glaucopis Agar Composition per liter: Agar ............................................................................................ 15.0g CaCO3 ......................................................................................... 10.0g Maltose.......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g KH2PO4 ......................................................................................... 0.5g NaCl .............................................................................................. 0.4g NH4Cl ........................................................................................... 0.4g Sodium thioglycolate .................................................................... 0.3g © 2010 by Taylor and Francis Group, LLC

Meniscus glaucopis Broth Maltose ......................................................................................... 5.0g Agar .............................................................................................. 3.0g Yeast extract.................................................................................. 1.0g KH2PO4......................................................................................... 0.5g NaCl.............................................................................................. 0.4g NH4Cl ........................................................................................... 0.4g Sodium thioglycolate .................................................................... 0.3g MgSO4·7H2O ................................................................................ 0.2g CaCl2·H2O .................................................................................. 0.01g FeSO4·7H2O............................................................................... 1.0mg Resazurin (0.025% solution) .....................................................4.0mL Trace elements solution SL-6 ....................................................1.0mL Vitamin solution.......................................................................10.0mL pH 7.3 ± 0.2 at 25°C

1068

M-Enrichment Broth

Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O................................................................................. 0.01g

Caution: Sodium azide has a tendency to form explosive metal azides

Preparation of Trace Elements Solution SL-6: Add components

Sodium Carbonate Solution: Composition per 10.0mL:

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Vitamin Solution: Composition per liter: Vitamin B12 ................................................................................ 2.8mg Thiamine·HCl .......................................................................... 0.28mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 7.3 with 10% Na2CO3. Gently heat and bring to boiling. Continue boiling until resazurin changes color. Cool to 50°C. Distribute into tubes in 7.0mL volumes under O2-free 97% N2 + 3% H2. Cap with rubber stoppers under O2-free 97% N2 + 3% H2. Place tubes in a press. Autoclave for 15 min at 15 psi pressure–121°C using fast exhaust. Cool to 50°C. Aseptically add 0.25mL of sterile vitamin solution to each tube. Use: For the cultivation and maintenance of Meniscus glaucopis.

M-Enrichment Broth Composition per liter: Proteose peptone ......................................................................... 40.0g Yeast extract.................................................................................. 6.0g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 3.0g pH 7.0 ± 0.2 at 25°C

with plumbing materials. It is advisable to use enough water to flush off the disposables.

Na2CO3 ......................................................................................... 1.0g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except polysorbate 80 and sodium carbonate solution, to distilled/deionized water and bring volume to 997.5mL. Mix thoroughly. Gently heat to dissolve components. Do not autoclave. Cool to 50°C. Add polysorbate 80 and sodium carbonate solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the selective isolation and enumeration of enterococci from water, sewage, food, or other materials.

M-Enterococcus Agar Base, Modified Composition per liter: Yeast extract................................................................................ 30.0g Pancreatic digest of gelatin......................................................... 10.0g Agar ............................................................................................ 15.0g NaCl............................................................................................ 15.0g Esculin .......................................................................................... 1.0g Nalidixic acid.............................................................................. 0.25g NaN3 ........................................................................................... 0.15g Cycloheximide............................................................................ 0.05g Selective supplement solution .................................................15.0mL pH 7.1 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

Caution: Sodium azide has a tendency to form explosive metal azides

Preparation of Medium: Add components to distilled/deionized

with plumbing materials. It is advisable to use enough water to flush off the disposables.

Use: For the preliminary enrichment of organisms on membrane filter prior to using selective media.

m-Enterococcus Agar See: Enterococcus Agar

M-Enterococcus Agar Base with Polysorbate 80 and Sodium Carbonate Composition per liter: Agar ............................................................................................ 10.0g Casein enzymic hydrolysate ....................................................... 15.0g Papaic digest of soybean meal ...................................................... 5.0g Yeast extract.................................................................................. 5.0g K2HPO4 ......................................................................................... 4.0g Glucose ......................................................................................... 2.0g NaN3.............................................................................................. 0.4g Triphenyl tetrazolium chloride...................................................... 0.1g Sodium carbonate solution.........................................................2.0mL Polysorbate 80............................................................................0.5mL pH 7.2 ± 0.2 at 25°C

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol formation and inhalation.

Selective Supplement Solution: Composition per 20.0mL: 2,3,5-Triphenyl tetrazolium chloride ............................................ 0.2g

Preparation of Selective Supplement Solution: Add 2,3,5-triphenyl tetrazolium chloride to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the recovery of enterococci in water samples using the membrane filter technique.

M-Enterococcus HiVeg Agar Base Composition per liter: Plant hydrolysate ........................................................................ 15.0g Agar ............................................................................................ 10.0g Papaic digest of soybean meal...................................................... 5.0g Yeast extract.................................................................................. 5.0g

Metal Acetate Agar

KH2PO4 ......................................................................................... 4.0g Glucose ......................................................................................... 2.0g NaN3 ............................................................................................. 0.4g Triphenyl tetrazolium chloride...................................................... 0.1g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Cool to 45°–50°C. Do not autoclave. Pour into sterile Petri dishes.

Use: For the isolation, cultivation, and enumeration of entercocci in water, sewage, and feces by the membrane filter method. For the direct plating of specimens for the detection and enumeration of fecal streptococci.

MeReSa Agar Base with Methicillin (Methicillin-Resistant Staphylococcus aureus Agar) Composition per liter: Agar ............................................................................................ 20.0g Casein enzymic hydrolysate ....................................................... 10.0g Glycine........................................................................................ 10.0g Mannitol...................................................................................... 10.0g NaCl ............................................................................................ 10.0g Sodium pyruvate ......................................................................... 10.0g LiCl ............................................................................................... 5.0g Beef extract ................................................................................... 5.0g Indicator mix............................................................................... 0.13g MRSA selective supplement....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without MRSA selective supplement, is available as a premixed powder from HiMedia. Caution: Lithium chloride is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

MRSA Selective Supplement: Composition per 10.0mL: Methicillin.................................................................................. 4.0mg

Preparation of MRSA Selective Supplement: Add methicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except MRSA selective supplement, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL MRSA selective supplement. Pour into sterile Petri dishes or leave in tubes.

Use: For the isolation and cultivation of methicillin-resistant Staphylococcus aureus (MRSA).

MeReSa Agar Base with Oxacillin (Methicillin-Resistant Staphylococcus aureus Agar)

1069

Glycine........................................................................................ 10.0g Mannitol...................................................................................... 10.0g NaCl............................................................................................ 10.0g Sodium pyruvate......................................................................... 10.0g LiCl ............................................................................................... 5.0g Beef extract................................................................................... 5.0g Indicator mix............................................................................... 0.13g MRSA selective supplement....................................................10.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without MRSA supplement solution, is available as a premixed powder from HiMedia. Caution: Lithium chloride is harmful. Avoid bodily contact and inhalation of vapors. On contact with skin wash with plenty of water immediately.

MRSA Selective Supplement: Composition per 10.0mL: Oxacillin .................................................................................... 2.0mg

Preparation of MRSA Selective Supplement: Add oxacillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Metal Acetate Agar Composition per liter: Agar ............................................................................................ 15.0g Sodium acetate.............................................................................. 2.0g Beijerinck's solution ................................................................50.0mL Phosphate buffer solution ........................................................50.0mL Trace elements solution .............................................................1.0mL pH 6.8 ± 0.2 at 25°C

Beijerinck's Solution: Composition per liter: NH4Cl ......................................................................................... 10.0g MgSO4·7H2O ................................................................................ 0.4g CaCl2·2H2O .................................................................................. 0.2g

Preparation of Beijerinck’s Solution: Add CaCl2·2H2O to dis-

tilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Add the remaining components to distilled/deionized water and bring volume to 500.0mL in a separate flask. Combine the two solutions.

Phosphate Buffer Solution: Composition per liter: K2HPO4....................................................................................... 28.8g KH2PO4....................................................................................... 14.4g

Composition per liter:

Preparation of Phosphate Buffer Solution: Add components to

Agar ............................................................................................ 20.0g Casein enzymic hydrolysate ....................................................... 10.0g

distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C.

1070

Metal Acetate Broth

Trace Elements Solution: Composition per liter: EDTA .......................................................................................... 50.0g H3BO3 solution ......................................................................200.0mL ZnSO4·7H2O solution ............................................................100.0mL CoCl2·6H2O solution................................................................50.0mL CuSO4·5H2O solution ..............................................................50.0mL FeSO4·7H2O solution...............................................................50.0mL MnCl2·4H2O solution...............................................................50.0mL (NH4)6Mo7O24·4H2O solution .................................................50.0mL

H3BO3 Solution: Composition per 200.0mL:

lution. Add distilled/deionized water and bring volume to 1.0L. Allow solution to stand in a 2.0L cotton-stoppered flask at room temperature until the solution turns purple (approximately 2 weeks). Filter using two layers of Whatman #1 filter paper. Filter until clear.

Preparation of Medium: Add components, except phosphate buffer solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 50.0mL of sterile phosphate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Chlamydomonas reinhardtii.

H3BO3 ......................................................................................... 11.4g

Preparation of H3BO3 Solution: Add H3BO3 to distilled/deion-

Metal Acetate Broth

ized water and bring volume to 200.0mL. Mix thoroughly.

Composition per liter:

ZnSO4·7H2O Solution: Composition per 100.0mL:

Sodium acetate.............................................................................. 2.0g Beijerinck's solution ................................................................50.0mL Phosphate buffer solution ........................................................50.0mL Trace elements solution .............................................................1.0mL pH 6.8 ± 0.2 at 25°C

ZnSO4·7H2O ............................................................................... 22.0g

Preparation of ZnSO4·7H2O Solution: Add ZnSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. MnCl2·4H2O Solution: Composition per 50.0mL: MnCl2·4H2O................................................................................ 5.06g

Preparation of MnCl2·4H2O Solution: Add MnCl2·4H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. FeSO4·7H2O Solution: Composition per 50.0mL: FeSO4·7H2O................................................................................ 4.99g

Preparation of Beijerinck’s Solution: Add CaCl2·2H2O to dis-

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

Phosphate Buffer Solution: Composition per liter:

CoCl2·6H2O Solution: Composition per 50.0mL:

K2HPO4....................................................................................... 28.8g KH2PO4....................................................................................... 14.4g

CoCl2·6H2O ................................................................................ 1.61g

Preparation of CoCl2·6H2O Solution: Add CoCl2·6H2O to dis-

tilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

CuSO4·5H2O Solution: Composition per 50.0mL: CuSO4·5H2O ............................................................................... 1.57g

Preparation of CuSO4·5H2O Solution: Add CuSO4·5H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

(NH4)6Mo7O24·4H2O Solution: Composition per 50.0mL: (NH4)6Mo7O24·4H2O .................................................................... 1.1g

Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per liter: EDTA .......................................................................................... 50.0g H3BO3 solution ......................................................................200.0mL ZnSO4·7H2O solution ............................................................100.0mL CoCl2·6H2O solution ...............................................................50.0mL CuSO4·5H2O solution ..............................................................50.0mL FeSO4·7H2O solution...............................................................50.0mL MnCl2·4H2O solution ..............................................................50.0mL (NH4)6Mo7O24·4H2O solution .................................................50.0mL

Preparation of (NH4)6Mo7O24·4H2O Solution: Add 1.1 g of (NH4)6Mo7O24·4H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

H3BO3 Solution: Composition per 200.0mL:

Preparation of Trace Elements Solution: Add EDTA to dis-

Preparation of H3BO3 Solution: Add H3BO3 to distilled/deion-

tilled/deionized water and bring volume to 250.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until dissolved. Add 200.0mL of H3BO3 solution, 100.0mL of ZnSO4·7H2O solution, 50.0mL of MnCl2·4H2O solution, 50.0mL of FeSO4·7H2O solution, 50.0mL of CoCl2·6H2O solution, 50.0mL of CuSO4·5H2O solution, and 50.0mL of (NH4)6Mo7O24·4H2O solution. Gently heat and bring to boiling. Cool to 70°C. Adjust pH to 6.8 with hot (70°C) 20% KOH so© 2010 by Taylor and Francis Group, LLC

H3BO3 ......................................................................................... 11.4g ized water and bring volume to 200.0mL. Mix thoroughly.

ZnSO4·7H2O Solution: Composition per 100.0mL: ZnSO4·7H2O ............................................................................... 22.0g

Preparation of ZnSO4·7H2O Solution: Add ZnSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Metal Acetate Yeast Broth with Arginine

1071

MnCl2·4H2O Solution: Composition per 50.0mL:

Beijerinck’s Solution: Composition per liter:

MnCl2·4H2O................................................................................ 5.06g

NH4Cl ......................................................................................... 10.0g MgSO4·7H2O ................................................................................ 0.4g CaCl2·2H2O .................................................................................. 0.2g

Preparation of MnCl2·4H2O Solution: Add MnCl2·4H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

FeSO4·7H2O Solution: Composition per 50.0mL: FeSO4·7H2O................................................................................ 4.99g

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

Preparation of Beijerinck’s Solution: Add CaCl2·2H2O to dis-

Phosphate Buffer Solution: Composition per liter:

CoCl2·6H2O Solution: Composition per 50.0mL:

K2HPO4....................................................................................... 28.8g KH2PO4....................................................................................... 14.4g

CoCl2·6H2O ................................................................................ 1.61g

Preparation of Phosphate Buffer Solution: Add components to

Preparation of CoCl2·6H2O Solution: Add CoCl2·6H2O to dis-

tilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C.

CuSO4·5H2O Solution: Composition per 50.0mL:

Trace Elements Solution: Composition per liter:

CuSO4·5H2O ............................................................................... 1.57g

Preparation of (NH4)6Mo7O24·4H2O Solution: Add 1.1 g of

EDTA .......................................................................................... 50.0g H3BO3 solution ......................................................................200.0mL ZnSO4·7H2O solution ............................................................100.0mL CoCl2·6H2O solution ...............................................................50.0mL CuSO4·5H2O solution ..............................................................50.0mL FeSO4·7H2O solution...............................................................50.0mL MnCl2·4H2O solution ..............................................................50.0mL (NH4)6Mo7O24·4H2O solution .................................................50.0mL

(NH4)6Mo7O24·4H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

H3BO3 Solution: Composition per 200.0mL:

Preparation of Trace Elements Solution: Add EDTA to distilled/

H3BO3 ......................................................................................... 11.4g

deionized water and bring volume to 250.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until dissolved. Add 200.0mL of H3BO3 solution, 100.0mL of ZnSO4·7H2O solution, 50.0mL of MnCl2·4H2O solution, 50.0mL of FeSO4·7H2O solution, 50.0mL of CoCl2·6H2O solution, 50.0mL of CuSO4·5H2O solution, and 50.0mL of (NH4)6Mo7O24·4H2O solution. Gently heat and bring to boiling. Cool to 70°C. Adjust pH to 6.8 with hot (70°C) 20% KOH solution (approximately 80.0–90.0mL). Add distilled/deionized water and bring volume to 1.0L. Allow solution to stand in a 2.0L cotton-stoppered flask at room temperature until the solution turns purple (approximately 2 weeks). Filter using two layers of Whatman #1 filter paper. Filter until clear. Store at 4°C or at −20°C.

Preparation of H3BO3 Solution: Add H3BO3 to distilled/deion-

Preparation of CuSO4·5H2O Solution: Add CuSO4·5H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. (NH4)6Mo7O24·4H2O Solution: Composition per 50.0mL: (NH4)6Mo7O24·4H2O .................................................................... 1.1g

Preparation of Medium: Add components, except phosphate buffer solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 50.0mL of sterile phosphate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Chlamydomonas reinhardtii.

Metal Acetate Yeast Broth with Arginine Composition per liter: Yeast extract.................................................................................. 4.0g Sodium acetate .............................................................................. 2.0g Arginine ........................................................................................ 0.1g Beijerinck's solution.................................................................50.0mL Phosphate buffer solution ........................................................50.0mL Trace elements solution .............................................................1.0mL pH 6.8 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

ized water and bring volume to 200.0mL. Mix thoroughly.

ZnSO4·7H2O Solution: Composition per 100.0mL: ZnSO4·7H2O ............................................................................... 22.0g

Preparation of ZnSO4·7H2O Solution: Add ZnSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. MnCl2·4H2O Solution: Composition per 50.0mL: MnCl2·4H2O ............................................................................... 5.06g

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. CoCl2·6H2O Solution: Composition per 50.0mL: CoCl2·6H2O ................................................................................ 1.61g

Preparation of CoCl2·6H2O Solution: Add CoCl2·6H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

CuSO4·5H2O Solution: Composition per 50.0mL: CuSO4·5H2O............................................................................... 1.57g

1072

Metallogenium Cultivation Broth

Preparation of (NH4)6Mo7O24·4H2O Solution: Add 1.1 g of

(NH4)6Mo7O24·4H2O to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly.

Preparation of Trace Elements Solution: Add EDTA to distilled/ deionized water and bring volume to 250.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until dissolved. Add 200.0mL of H3BO3 solution, 100.0mL of ZnSO4·7H2O solution, 50.0mL of MnCl2·4H2O solution, 50.0mL of FeSO4·7H2O solution, 50.0mL of CoCl2·6H2O solution, 50.0mL of CuSO4·5H2O solution, and 50.0mL of (NH4)6Mo7O24·4H2O solution. Gently heat and bring to boiling. Cool to 70°C. Adjust pH to 6.8 with hot (70°C) 20% KOH solution (approximately 80.0–90.0mL). Add distilled/deionized water and bring volume to 1.0L. Allow solution to stand in a 2.0L cotton-stoppered flask at room temperature until the solution turns purple (approximately 2 weeks). Filter using two layers of Whatman #1 filter paper. Filter until clear. Store at 4°C or − 20°C.

Use: For the cultivation of Chlamydomonas reinhardtii.

Metallogenium Cultivation Broth Composition per liter: Gum arabic.................................................................................. 20.0g MnCO3 .......................................................................................... 0.5g

MnCO3: Composition per 100.0mL: MnCl2 .......................................................................................... 20.0g NaHCO3 (25% solution) ..........................................................25.0mL

Preparation of MnCO3: Add MnCl2 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Add NaHCO3 solution. Filter through Whatman #1 filter paper. Save the MnCO3 precipitate. Wash and store under distilled/deionized water.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Metallogenium species.

Metallogenium Cultivation Broth Composition per liter: Starch, hydrolyzed ...................................................................... 20.0g MnCO3 .......................................................................................... 0.5g

Preparation of Medium: Hydrolyze starch with HCl. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Metallogenium species.

Metallogenium Isolation Agar Composition per liter: Agar ............................................................................................ 15.0g Manganese acetate ........................................................................ 0.1g

Use: For the isolation and cultivation of Metallogenium species.

Metallogenium Medium Composition per liter: MnCO3 .......................................................................................... 2.0g Starch, hydrolyzed ........................................................................ 1.0g DNA............................................................................................ 0.01g Catalase...................................................................................... 5.0mg Mycoplasma broth base .........................................................100.0mL Yeast extract, ultrafiltrate.......................................................100.0mL Horse serum .............................................................................10.0mL

Mycoplasma Broth Base: Composition per liter: Pancreatic digest of casein............................................................ 7.0g NaCl.............................................................................................. 5.0g Beef extract................................................................................... 3.0g Yeast extract.................................................................................. 3.0g Beef heart, solids from infusion.................................................... 2.0g

Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

MnCO3: Composition per 100.0mL: MnCl2.......................................................................................... 20.0g NaHCO3 (25% solution) ..........................................................25.0mL

Preparation of MnCO3: Add MnCl2 to distilled/deionized water

and bring volume to 100.0mL. Mix thoroughly. Add NaHCO3 solution. Filter through Whatman #1 filter paper. Save the MnCO3 precipitate. Wash and store under distilled/deionized water.

Preparation of Medium: Add MnCO3, hydrolyzed starch, and DNA to 25.0mL of distilled/deionized water. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile Mycoplasma broth base, 100.0mL of ultrafiltrate of yeast extract, 10.0mL of horse serum, and 5.0mg of catalase. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Metallogenium species.

Metallosphaera Medium Composition per liter: (NH4)2SO4 .................................................................................... 1.3g Yeast extract.................................................................................. 1.0g KH2PO4....................................................................................... 0.28g MgSO4·7H2O .............................................................................. 0.25g

Methanobacteria Medium

CaCl2·2H2O................................................................................. 0.07g FeCl3·6H2O ................................................................................. 0.02g Na2B4·10H2O ............................................................................. 4.5mg MnCl2·4H2O............................................................................... 1.8mg ZnSO4·7H2O ............................................................................ 0.22mg CuCl2·2H2O ............................................................................. 0.05mg Na2MoO4·2H2O ....................................................................... 0.03mg VOSO4·2H2O ........................................................................... 0.03mg CoSO4 ...................................................................................... 0.01mg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 2.0 using 10N H2SO4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Metallosphaera sedula.

Methanobacillus Medium Composition per liter: KH2PO4 ......................................................................................... 9.0g K2HPO4 ......................................................................................... 6.0g NH4Cl ........................................................................................... 5.0g MgCl2 ............................................................................................ 1.0g CaCl2 ........................................................................................... 0.01g FeSO4·7H2O................................................................................ 0.01g Ethanol .....................................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Filter sterilize ethanol. Add components, except ethanol, to tap water and bring volume to 990.0mL. Mix thoroughly. Gently heat until dissolved. Autoclave for 20 min at 10psi pressure–115°C. Cool to 45°–50°C. Aseptically add sterile ethanol. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the selective isolation and cultivation of Methanobacillus species from mixed cultures.

Methanobacteria Medium Composition per liter: Mineral solution 2....................................................................50.0mL Sodium carbonate solution.......................................................50.0mL Mineral solution 1....................................................................25.0mL L-Cysteine-sulfide reducing agent ...........................................20.0mL Wolfe’s mineral solution ..........................................................10.0mL Wolfe’s vitamin solution ..........................................................10.0mL Resazurin (0.025% solution)......................................................4.0mL pH 7.2 ± 0.2 at 25°C

1073

Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 8.0g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. L-Cysteine-Sulfide

Reducing Agent: Composition per 20.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g Na2S·9H2O.................................................................................... 0.3g

Preparation of L-Cysteine-Sulfide Reducing Agent: Add

L-

cysteine·HCl·H2O to 10.0mL of distilled/deionized water. Mix thoroughly. In a separate tube, add Na2S·9H2O to 10.0mL of distilled/deionized water. Mix thoroughly. Gas both solutions with 100% N2 and cap tubes. Autoclave both solutions for 15 min at 15 psi pressure– 121°C using fast exhaust. Cool to 50°C. Aseptically combine the two solutions under 100% N2.

Wolfe’s Mineral Solution: Composition per liter MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Wolfe’s Vitamin Solution: Composition per liter:

Preparation of Medium: Add K2HPO4 to distilled/deionized water

Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Cyanocobalamin ..................................................................... 100.0μg

Mineral Solution 2: Composition per liter:

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Mineral Solution 1: Composition per liter: K2HPO4 ......................................................................................... 6.0g and bring volume to 1.0L. Mix thoroughly.

NaCl ............................................................................................ 12.0g KH2PO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.4g CaCl2·2H2O................................................................................... 1.6g

Preparation of Medium: Add components, except vitamin solution and L-cysteine-sulfide reducing agent, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Aseptically add the sterile vitamin solution and then the sterile L-cysteine-sulfide reducing agent. Adjust the pH to 7.2. Distribute aseptically and anaerobically into sterile tubes.

1074

Methanobacteria Medium with Glucose and Yeast Extract

Use: For the cultivation and maintenance of Acetogenium kivui, Methanobacterium formicicum, Methanobacterium thermoautotrophicum, and Methanobrevibacter arboriphilicus.

Methanobacteria Medium with Glucose and Yeast Extract

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic

Composition per liter: Glucose ......................................................................................... 5.0g Yeast extract.................................................................................. 2.0g Mineral solution 2....................................................................50.0mL Sodium carbonate solution.......................................................50.0mL Mineral solution 1....................................................................25.0mL L-Cysteine-sulfide reducing agent ...........................................20.0mL Wolfe’s mineral solution ..........................................................10.0mL Wolfe’s vitamin solution ..........................................................10.0mL Resazurin (0.025% solution)......................................................4.0mL pH 7.2 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4 ......................................................................................... 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Mineral Solution 2: Composition per liter: NaCl ............................................................................................ 12.0g KH2PO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.4g CaCl2·2H2O................................................................................... 1.6g

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 8.0g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

L-Cysteine-Sulfide

Reducing Agent: Composition per 20.0mL:

L-Cysteine·HCl·H2O...................................................................... 0.3g Na2S·9H2O .................................................................................... 0.3g

Preparation of L-Cysteine-Sulfide Reducing Agent: Add

L-

ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Cyanocobalamin ..................................................................... 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Use: For the cultivation and maintenance of Clostridium saccharolyticum, Clostridium thermoaceticum, and Clostridium thermohydrosulfuricum.

Methanobacteria Medium with Xylose, Yeast Extract, and Tryptone Composition per liter: Pancreatic digest of casein.......................................................... 10.0g Xylose ........................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Mineral solution 2....................................................................50.0mL Sodium carbonate solution ......................................................50.0mL Mineral solution 1....................................................................25.0mL L-Cysteine-sulfide reducing agent ...........................................20.0mL Wolfe’s mineral solution..........................................................10.0mL Wolfe’s vitamin solution..........................................................10.0mL Resazurin (0.025% solution) .....................................................4.0mL pH 7.2 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4......................................................................................... 6.0g

Preparation of Medium: Add K2HPO4 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Mineral Solution 2: Composition per liter: NaCl............................................................................................ 12.0g KH2PO4......................................................................................... 6.0g

Methanobacteria Medium with Yeast Extract, Sodium Acetate, and Methanol

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Aseptically add the sterile vitamin solution and then the sterile L-cysteine-sulfide reducing agent. Adjust the pH to 7.2. Distribute aseptically and anaerobically into sterile tubes.

Use: For the cultivation and maintenance of Thermobacteroides acetoethylicus.

Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 8.0g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

L-Cysteine-Sulfide

Reducing Agent: Composition per 20.0mL:

L-Cysteine·HCl·H2O...................................................................... 0.3g Na2S·9H2O .................................................................................... 0.3g

Preparation of L-Cysteine-Sulfide Reducing Agent: Add

1075

L-

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Methanobacteria Medium with Yeast Extract, Sodium Acetate, and Methanol Composition per liter: Glucose ......................................................................................... 5.0g Sodium acetate.............................................................................. 4.1g Yeast extract.................................................................................. 2.0g Mineral solution 2....................................................................50.0mL Sodium carbonate solution ......................................................50.0mL Mineral solution 1....................................................................25.0mL L-cysteine-sulfide reducing agent ............................................20.0mL Wolfe’s mineral solution..........................................................10.0mL Wolfe’s vitamin solution..........................................................10.0mL Methanol ....................................................................................4.0mL Resazurin (0.025% solution) .....................................................4.0mL pH 7.2 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4......................................................................................... 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Mineral Solution 2: Composition per liter: NaCl............................................................................................ 12.0g KH2PO4......................................................................................... 6.0g (NH4)2SO4 .................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.4g CaCl2·2H2O .................................................................................. 1.6g

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 8.0g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. L-Cysteine-Sulfide

Reducing Agent: Composition per 20.0mL:

L-Cysteine·HCl·H2O .............................................................. 300.0mg Na2S·9H2O............................................................................. 300.0mg

Preparation of L-Cysteine-Sulfide Reducing Agent: Add

L-

Preparation of Medium: Add components, except vitamin solu-

Wolfe’s Mineral Solution: Composition per liter:

tion and L-cysteine-sulfide reducing agent, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min

MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g

1076

Methanobacterium alcaliphilum Medium

NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

ZnCl2 ........................................................................................... 0.02g AlCl3·6H2O ................................................................................ 8.0mg CuCl2·2H2O ............................................................................... 4.0mg NiSO4·6H2O............................................................................... 4.0mg Na2SeO3 ..................................................................................... 2.7mg FeSO4·7H2O............................................................................... 2.0mg H3BO3 ........................................................................................ 2.0mg NaMoO4·2H2O........................................................................... 2.0mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic

Preparation of Medium: Add components, except NaHCO3, yeast

acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Cyanocobalamin .....................................................................100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except vitamin solution, L-cysteine-sulfide reducing agent, and methanol, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Filter sterilize methanol. Aseptically add 4.0mL of sterile methanol to cooled, sterile basal medium. Aseptically add the sterile vitamin solution and then the sterile L-cysteine-sulfide reducing agent. Adjust the pH to 7.2. Distribute aseptically and anaerobically into sterile tubes.

Use: For the cultivation and maintenance of Butyribacterium methylotrophicum.

Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. extract, peptone, and L-cysteine·HCl·H2O, to distilled/deionized water and bring volume to 990.0mL. Gently heat and bring to boiling under O2-free 100% N2. Continue boiling until resazurin becomes pale, indicating partial reduction. Add the yeast extract, peptone, and Lcysteine·HCl·H2O and continue boiling under O2-free 100% N2 until resazurin becomes colorless, indicating complete reduction. Cool to room temperature under O2-free 100% N2. Add NaHCO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Gas with O2-free 100% N2 in a sealed tube. Add reduced NaHCO3 solution to cooled reduced medium. Distribute anaerobically into tubes. Cap with butyl rubber stoppers and secure with closures. Autoclave for 15 min at 15 psi pressure– 121°C with fast exhaust.

Use: For the cultivation and maintenance of Methanobacterium alcaliphilum.

Methanobacterium Enrichment Medium Composition per liter: CaCO3 ....................................................................................... 100.0g K2HPO4......................................................................................... 5.0g (NH4)2SO4 .................................................................................... 0.3g MgSO47H2O ................................................................................. 0.1g FeSO4·7H2O................................................................................ 0.02g Na2CO3 solution ......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Ethanol.....................................................................................10.0mL Yeast autolysate .........................................................................5.0mL pH 7.2 ± 0.2 at 25°C

Na2CO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 0.5g

Methanobacterium alcaliphilum Medium Composition per liter: NaHCO3 ...................................................................................... 10.0g Yeast extract.................................................................................. 2.0g Peptone.......................................................................................... 2.0g NH4Cl ........................................................................................... 1.0g L-Cysteine·HCl·H2O...................................................................... 0.5g K2HPO4 ......................................................................................... 0.4g MgCl2·6H2O.................................................................................. 0.1g CaCl2 ........................................................................................... 0.02g Resazurin ................................................................................... 1.0mg Salt solution ...............................................................................5.0mL pH 8.4 ± 0.2 at 25°C

Salt Solution: Composition per 100.0mL: Sodium EDTA·2H2O .................................................................... 0.1g CoCl2·6H2O ................................................................................ 0.03g MnCl2·4H2O................................................................................ 0.02g © 2010 by Taylor and Francis Group, LLC

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deion-

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.1g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Filter sterilize ethanol. Add components—except ethanol, Na2CO3 solution, and Na2S·9H2O solution—to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile ethanol, Na2CO3 solution, and Na2S·9H2O solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and enrichment of Methanobacterium species.

Methanobacterium Mass-Culturing Medium

Methanobacterium espanolae Medium

1077

Methanobacterium formicicum Medium

Composition per 1020.0mL:

Composition per liter:

Sodium acetate·3H2O.................................................................... 2.5g Na2CO3 ......................................................................................... 0.5g (NH4)2SO4 ................................................................................... 0.45g K2HPO4 ....................................................................................... 0.29g KH2PO4 ....................................................................................... 0.18g MgSO4·7H2O .............................................................................. 0.12g CaCl2·2H2O................................................................................. 0.06g NaCl ............................................................................................ 0.05g L-Cysteine·HCl............................................................................ 0.25g Na2S·9H2O .................................................................................. 0.25g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 5.5 ± 0.2 at 25°C

NaCH3CO2 .................................................................................... 2.0g (NH4)2SO4 .................................................................................. 1.48g NaCl............................................................................................ 0.45g L-Cysteine·HCl·H2O ................................................................... 0.27g CaCl2·2H2O ................................................................................ 0.06g Fe(NH4)(SO4)2 ............................................................................ 0.06g MgSO4 ..................................................................................... 45.0mg Na2MoO4 ................................................................................. 24.0mg K2HPO4.................................................................................... 21.0mg KH2PO4.................................................................................... 21.0mg Resazurin ................................................................................... 1.0mg Na2SeO3 ..................................................................................... 0.2mg Na2S·9H2O solution...................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................. 25.0g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N2. Add Na2S·9H2O to the 100.0mL of anaerobic water. Mix thoroughly. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Autoclave for 20 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8 with concentrated HCl. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool medium while sparging with 100% N2. Prior to inoculation, aseptically and anaerobically add 1.0mL of sterile Na2S·9H2O solution per liter of medium. Repeat the addition of 1.0mL of sterile Na2S·9H2O solution per liter of medium every 48 hr during growth.

acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Use: For the cultivation of Methanobacterium formicicum.

Vitamin Solution: Composition per liter:

Composition per liter:

Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Preparation of Medium: Prepare and dispense the medium anaerobically with 80% N2 and 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.5–6.0. Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Check the pH of the medium after autoclaving.

Methanobacterium Mass-Culturing Medium NH4Cl ........................................................................................... 2.0g NaCl.............................................................................................. 0.6g KH2PO4....................................................................................... 0.42g K2HPO4....................................................................................... 0.23g Na2CO3 ....................................................................................... 0.16g MgCl2·2H2O ............................................................................... 0.04g CaCl2·2H2O ................................................................................ 0.03g Resazurin .................................................................................. 0.001g Na2S·9H2O solution .................................................................10.0mL Trace elements solution ...........................................................10.0mL

Na2S·9H2O Solution: Composition per 100.0mL: NaOH....................................................................................... 1 pellet Na2S·9H2O.................................................................................... 2.5g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

1078

Methanobacterium Medium

pers. Pressurize to 60kPa with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

Trace Elements Solution: Composition per liter: Sodium nitrilotriacetate............................................................... 1.67g CoCl2·6H2O ................................................................................ 0.18g FeCl2·4H2O ................................................................................. 0.14g MnCl2·4H2O................................................................................ 0.09g NiCl2·6H2O ................................................................................. 0.09g ZnCl2 ........................................................................................... 0.09g CaCl2 ........................................................................................... 0.06g Na2MoO4·2H2O ........................................................................ 0.046g CuSO4 ....................................................................................... 0.045g

Preparation of Trace Elements Solution: Add sodium nitrilotriacetate to 500.0mL of distilled/deionized water. Adjust pH to 6.5. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except Na2CO3 and Na2S·9H2O solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add Na2CO3. Mix thoroughly. Anaerobically distribute 9.9mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically and anaerobically add 0.1mL of sterile Na2S·9H2O solution to each tube. Mix thoroughly.

Use: For the cultivation of Methanobacterium species.

Methanobacterium Medium Composition per 1010.0mL: NaHCO3 ........................................................................................ 4.0g Sodium formate............................................................................. 2.0g Sodium acetate .............................................................................. 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl.............................................................................. 0.5g KH2PO4 ......................................................................................... 0.5g Na2S·9H2O .................................................................................... 0.5g MgSO4·7H2O ............................................................................... 0.4g NaCl .............................................................................................. 0.4g NH4Cl ........................................................................................... 0.4g CaCl2·2H2O................................................................................. 0.05g FeSO4·7H2O............................................................................... 2.0mg Resazurin ................................................................................... 1.0mg Sludge fluid..............................................................................50.0mL Fatty acid mixture ....................................................................20.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.7 ± 0.2 at 25°C

Sludge Fluid: Composition per 100.0mL: Yeast extract.................................................................................. 0.4g Sludge ....................................................................................100.0mL

Preparation of Sludge Fluid: To 100.0mL of sludge from an anaerobic digester, add 0.4g of yeast extract. Sparge with 100% N2 for a few minutes. Incubate at 37°C for 24 hr. Centrifuge the sludge at 13,000 × g for 15 min. Decant the clear supernatant solution. Sparge with 100% N2 for a few minutes. Store in screw-capped bottles at room temperature in the dark. © 2010 by Taylor and Francis Group, LLC

Fatty Acid Mixture: Composition per 20.0mL:

α-Methylbutyric acid.................................................................... 0.5g Isobutyric acid .............................................................................. 0.5g Isovaleric acid............................................................................... 0.5g Valeric acid ................................................................................... 0.5g

Preparation of Fatty Acid Mixture: Add components to distilled/ deionized water and bring volume to 20.0mL. Mix thoroughly. Adjust pH to 7.5 with concentrated NaOH. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium anaerobically under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1010.0mL. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation and maintenance of Methanobacterium species.

Methanobacterium ruminantium Medium Composition per liter: NaHCO3 ........................................................................................ 6.0g NaCl.............................................................................................. 2.0g L-Cysteine·HCl·H2O ..................................................................... 1.0g K2HPO4·3H2O .............................................................................. 1.0g KH2PO4......................................................................................... 1.0g NH4Cl ........................................................................................... 1.0g CaCl2·2H2O .................................................................................. 0.1g MgSO4·7H2O ................................................................................ 0.1g Resazurin ................................................................................... 1.0mg Rumen fluid ...........................................................................300.0mL Na2S·9H2O solution .................................................................10.0mL 6.8 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and distribute medium anaerobically under 80% H2 + 20% CO2. Add components, except rumen fluid and Na2S·9H2O solution, to distilled/deionized water and bring volume to 690.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until resazurin turns colorless, indicating reduction. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL of sterile Na2S·9H2O solution and 300.0mL of sterile ru-

Methanobacterium thermoautotrophicum Marburg Medium

1079

men fluid. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Use: For the cultivation of Methanobacterium ruminantium.

Methanobacterium subterraneum Medium (DSMZ Medium 814) Composition per liter: Na-formate .................................................................................... 3.2g Na-acetate ..................................................................................... 1.0g Tryptone ........................................................................................ 1.0g K2HPO4 ......................................................................................... 0.5g NaCl ............................................................................................ 0.45g NH4Cl ........................................................................................... 0.4g MgCl2·6H2O................................................................................ 0.03g FeSO4·7H2O.............................................................................. 0.003g Resazurin ................................................................................... 0.5mg Trace elements solution ...........................................................10.0mL Cysteine solution......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL NaHCO3 solution .....................................................................10.0mL Seven vitamin solution...............................................................1.0mL pH 8.3 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Cysteine Solution: Composition per 10.0mL: L-Cysteine-HCl·H2O ................................................................... 0.25g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 100% N2. Add components, except Na2S·9H2O solution, seven vitamin solution, cysteine solution, and NaHCO3 solution, to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Flush medium with N2 for 5 min. Adjust medium pH to 8.3. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 1.0mL seven vitamin mix, 10.0mL cysteine solution, 10.0mL Na2S·9H2O, and 10.0mL NaHCO3 solution. Mix thoroughly. Adjust pH to 8.3. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Methanobacterium subterraneum.

Methanobacterium thermoautotrophicum Marburg Medium Composition per liter: L-Cysteine·HCl·H2O ................................................................... 12.5g

Na2S·9H2O.................................................................................. 12.5g KH2PO4......................................................................................... 6.8g NH4Cl ........................................................................................... 2.1g Na2CO3 (1M solution) .............................................................32.0mL Trace elements solution ...........................................................10.0mL Resazurin (0.2% solution) .........................................................0.3mL

Trace Elements Solution: Composition per liter: MgCl2·6H2O ................................................................................. 4.0g Nitrilotriacetic acid ....................................................................... 3.0g FeCl2·4H2O................................................................................... 1.0g NiCl2·6H2O................................................................................. 0.12g CoCl2·6H2O ................................................................................ 0.02g NaMoO4·2H2O............................................................................ 0.02g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with NaOH. Add remaining components. Mix thoroughly. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except L-cysteine·HCl·H2O and Na2S·9H2O, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add L-cysteine·HCl·H2O and Na2S·9H2O. Mix thoroughly. Anaerobically distribute into serum bottles fitted with butyl rubber stop-

1080

Methanobacterium thermoautotrophicum Medium

pers and aluminum seals. Do not grease stoppers. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum Medium (DSMZ Medium 131) Composition per liter: Na2CO3 ......................................................................................... 4.0g (NH4)2SO4 ..................................................................................... 1.5g NaCl .............................................................................................. 0.6g KH2PO4 ......................................................................................... 0.3g K2HPO4 ....................................................................................... 0.15g MgSO4·7H2O .............................................................................. 0.12g CaCl2·2H2O................................................................................. 0.08g FeSO4·7H2O............................................................................... 4.0mg Resazurin ................................................................................... 1.0mg Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL L-Cysteine solution ..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Ca-pantothenate ......................................................................... 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg p-Aminobenzoic acid ................................................................. 1.0mg Vitamin B12 .............................................................................. 0.01mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 6.2g NaCl .............................................................................................. 1.0g Na2-EDTA................................................................................... 0.64g MnSO4·4H2O .............................................................................. 0.55g ZnSO4·7H2O ............................................................................... 0.18g CoCl2·6H2O ................................................................................ 0.17g CaCl2·2H2O................................................................................. 0.13g FeSO4·7H2O.................................................................................. 0.1g CuSO4......................................................................................... 0.05g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O..................................................................... 0.018g Na2MoO4·4H2O ........................................................................ 0.011g H3BO3 ......................................................................................... 0.01g

Preparation of Trace Elements Solution: Add Na2-EDTA to

500.0mL distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 1.5g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% H2 + 20% CO2 gas mixture. Add components, except vitamin solution, L-cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C while sparging with 80% H2 + 20% CO2. Aseptically and anaerobically add 10.0mL vitamin solution, 10.0mL of sterile L-cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks. Alternately the medium can be distributed to tubes under anaerobic conditions and autoclaved in tubes prior to addition of substrate solution, vitamin solution, and Na2S·9H2O solution. Appropriate amounts of these solutions can then be added to each tube by syringes to yield the desired concentrations.

Use: For the cultivation of Ectothiorhodospira shaposhnikovii and Methanothermobacter thermautotrophicus.

Methanobacterium thermoautotrophicum Medium Composition per liter: Na2CO3 ......................................................................................... 4.0g (NH4)2SO4 .................................................................................... 1.5g NaCl.............................................................................................. 0.6g KH2PO4......................................................................................... 0.3g K2HPO4....................................................................................... 0.15g MgSO4·7H2O ............................................................................. 0.12g CaCl2·2H2O ................................................................................ 0.08g FeSO4·7H2O.............................................................................. 4.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: NaCl.............................................................................................. 1.0g Disodium EDTA ......................................................................... 0.64g MnSO4·4H2O ............................................................................. 0.55g MgSO4·7H2O ............................................................................... 0.2g ZnSO4·7H2O .............................................................................. 0.18g CoCl2·6H2O ................................................................................ 0.17g CuSO4 ......................................................................................... 0.15g CaCl2·2H2O ................................................................................ 0.13g FeSO4·7H2O................................................................................. 0.1g NiCl2·H2O................................................................................. 0.025g KAl(SO4)2·12H2O..................................................................... 0.018g Na2MoO4·2H2O ....................................................................... 0.011g H3BO3 ......................................................................................... 0.01g

Methanobacterium thermoautotrophicum MS Medium Preparation of Trace Elements Solution: Add disodium EDTA to 500.0mL of distilled/deionized water. Mix thoroughly to dissolve. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg p-Aminobenzoic acid................................................................. 1.0mg Cyanocobalamin ...................................................................... 0.01mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thoroughly. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 1.5g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure– 121°C. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 1.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except L-cysteine·HCl solution and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile Lcysteine·HCl solution and sterile Na2S·9H2O solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanobacterium thermoautotrophicus and Methanoculleus oldenburgensis.

Methanobacterium thermoautotrophicum Medium, Taylor and Pirt Composition per liter: Na2CO3 ......................................................................................... 4.0g (NH4)2SO4 ..................................................................................... 3.0g NaCl .............................................................................................. 1.2g KH2PO4 ......................................................................................... 0.6g L-Cysteine·HCl·H2O ..................................................................... 0.5g K2HPO4 ......................................................................................... 0.3g Nitrilotriacetic acid ..................................................................... 0.03g CoCl2........................................................................................... 0.02g CaCl2 ........................................................................................... 0.01g FeSO4 .......................................................................................... 0.01g © 2010 by Taylor and Francis Group, LLC

1081

MgSO4 ........................................................................................ 0.01g MnSO4 ........................................................................................ 0.01g ZnSO4 ........................................................................................ 2.0mg Resazurin ................................................................................... 1.0mg AlK(SO4)2 .................................................................................. 0.2mg CuSO4 ........................................................................................ 0.2mg H3BO3 ........................................................................................ 0.2mg Na2MoO4 ................................................................................... 0.2mg Na2S·9H2O solution .................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and distribute medium anaerobically under 80% H2 + 20% CO2. Add components, except Na2S·9H2O solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until resazurin turns colorless, indicating reduction. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum MS Medium Composition per liter: NaHCO3 ........................................................................................ 8.4g Pancreatic digest of casein............................................................ 2.0g Yeast extract.................................................................................. 2.0g MgCl2·6H2O ................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g K2HPO4....................................................................................... 0.84g CaCl2·2H2O .................................................................................. 0.4g Reazurin.................................................................................... 0.001g Trace minerals solution..............................................................1.0mL pH 7.1 ± 0.2 at 25°C

Trace Minerals Solution: Composition per liter: Disodium EDTA·2H2O ................................................................. 0.5g ZnSO4·2H2O ............................................................................... 0.21g CoCl2·6H2O ................................................................................ 0.15g AlK(SO4)2 ................................................................................... 0.14g FeSO4·7H2O.................................................................................. 0.1g MnSO4·H2O .................................................................................. 0.1g Na2WO4·2H2O ............................................................................ 0.03g CuCl2·2H2O ................................................................................ 0.02g NiCl2·6H2O................................................................................. 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO2·2H2O .......................................................................... 0.01g Na2SeO4 ...................................................................................... 0.01g

Preparation of Trace Minerals Solution: Add components, except MnSO4·H2O, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1. Add MnSO4·H2O. Mix thoroughly.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Con-

1082

Methanobacterium Transduction Agar

tinue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Adjust pH to 7.1 with 6N HCl. Anaerobically distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

cysteine·HCl and Na2S·9H2O. Mix thoroughly. Cool while sparging with 100% N2. Bring volume to 50.0mL with distilled/deionized water. Sparge with 100% N2. Adjust pH to 10.0 with 3N NaOH.

Use: For the cultivation of Methanobacterium thermoautotrophicum.

Sodium citrate·9H2O..................................................................... 2.9g Titanium (III) chloride ................................................................ 0.75g

Methanobacterium Transduction Agar Composition per liter: Agar solution..........................................................................500.0mL Basal salts solution.................................................................100.0mL Na2CO3 solution.......................................................................24.0mL Titanium (III) citrate solution ..................................................16.0mL Trace elements solution ...........................................................10.0mL L-Cysteine/Na2S solution.........................................................10.0mL

Agar Solution: Composition per 500.0mL: Agar ............................................................................................ 15.0g

Preparation of Agar Solution: Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C while sparging with 80% N2 + 20% CO2.

Basal Salts Solution: Composition per liter: KH2PO4 ....................................................................................... 68.0g NH4Cl ......................................................................................... 21.2g Resazurin ................................................................................. 10.0mg

Preparation of Basal Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ..................................................................... 9.55g MgCl2·7H2O................................................................................ 4.06g FeCl2·7H2O ................................................................................. 0.98g NaWO4·6H2O............................................................................ 0.264g NiCl2·6H2O ............................................................................... 0.118g Na2Se2O3 ....................................................................................... 0.1g NaMoO4·2H2O.......................................................................... 0.024g CoCl2·6H2O .............................................................................. 0.023g

Titanium (III) Citrate Solution: Composition per 55.0mL:

Preparation of Titanium (III) Citrate Solution: Add components to distilled/deionized water and bring volume to 55.0mL. Mix thoroughly. Filter sterilize. Sparge with 100% N2.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except agar solution and titanium citrate solution, to distilled/deionized water and bring volume to 484.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically and anaerobically add 55.0mL of sterile titanium citrate solution to 500.0mL of sterile agar solution. Add the agar/titanium/citrate solution to the rest of the medium. Mix thoroughly. Aseptically and anaerobically, under an atmosphere of 80% H2 + 20% CO2, distribute into sterile Petri dishes, sterile tubes, or sterile bottles.

Use: For the cultivation of Methanobacterium species for transduction.

Methanobacterium Transduction Medium Composition per liter: Basal salts solution ................................................................100.0mL Na2CO3 solution ......................................................................24.0mL Trace elements solution ...........................................................10.0mL L-Cysteine/Na2S solution.........................................................10.0mL

Basal Salts Solution: Composition per liter: KH2PO4....................................................................................... 68.0g NH4Cl ......................................................................................... 21.2g Resazurin ................................................................................. 10.0mg

Preparation of Basal Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Trace Elements Solution: Composition per liter:

Na2CO3 Solution: Composition per liter:

Nitrilotriacetic acid ..................................................................... 9.55g MgCl2·7H2O ............................................................................... 4.06g FeCl2·7H2O ................................................................................. 0.98g NaWO4·6H2O ........................................................................... 0.264g NiCl2·6H2O ............................................................................... 0.118g Na2Se2O3 ...................................................................................... 0.1g NaMoO4·2H2O.......................................................................... 0.024g CoCl2·6H2O .............................................................................. 0.023g

Na2CO3 ....................................................................................... 10.6g

Preparation of Trace Elements Solution: Add nitrilotriacetic

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2.

acid to 500.0mL of distilled/deionized water. Adjust pH to 7.0 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Sparge with 100% N2.

L-Cysteine/Na2S Solution: Composition per 50.0mL:

Na2CO3 Solution: Composition per liter:

L-Cysteine·HCl.............................................................................. 5.0g Na2S·9H2O .................................................................................... 5.0g

Na2CO3 ....................................................................................... 10.6g

Preparation of L-Cysteine/Na2S Solution: Gently heat and

ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2.

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 7.0 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Sparge with 100% N2.

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

Methanobacterium II Medium

1083

L-Cysteine/Na2S Solution: Composition per 50.0mL:

L-Cysteine·HCl.............................................................................. 5.0g Na2S·9H2O .................................................................................... 5.0g

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2.

Preparation of L-Cysteine/Na2S Solution: Gently heat and bring 50.0mL of distilled/deionized water to boiling. Add Lcysteine·HCl and Na2S·9H2O. Mix thoroughly. Cool while sparging with 100% N2. Bring volume to 50.0mL with distilled/deionized water. Sparge with 100% N2. Adjust pH to 10.0 with 3N NaOH. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Anaerobically distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Prior to inoculation, pressurize to 200kPa with an atmosphere of 80% H2 + 20% CO2.

Use: For the cultivation of Methanobacterium species for transduction.

Methanobacterium wolfei Medium Composition per liter: Na2CO3 ......................................................................................... 4.0g Sodium acetate·3H2O.................................................................... 1.0g L-Cysteine·HCl.............................................................................. 0.5g NaCl .............................................................................................. 0.5g Na2S·9H2O .................................................................................... 0.5g K2HPO4 ....................................................................................... 0.26g KH2PO4 ....................................................................................... 0.26g (NH4)2SO4 ................................................................................... 0.26g MgSO4·7H2O ................................................................................ 0.1g CaCl2·2H2O................................................................................. 0.07g NaWO4 ....................................................................................... 2.6mg FeSO4 ......................................................................................... 2.0mg Resazurin ................................................................................... 1.0mg Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ....................................................................... 9.5g MgCl2 .......................................................................................... 4.06g FeCl2 ........................................................................................... 0.99g NiCl2·6H2O ................................................................................. 0.12g Na2MoO4................................................................................... 0.024g CoCl2·6H2O .............................................................................. 0.023g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Mix thoroughly. Sparge with 100% N2. Adjust pH to 7.0 with 10N NaOH. Vitamin Solution: Composition per liter: Calcium DL-pantothenate.............................................................. 5.0g Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Methanobacterium wolfei.

Methanobacterium II Medium (DSMZ Medium 825) Composition per 1050.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g Sodium acetate.............................................................................. 0.5g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.2g CaCl2·2H2O .................................................................................. 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution.....................................................................40.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg

1084

Methanobacterium II Medium

Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2 gas mixture. Add components, except NaHCO3 solution and Na2S·9H2O solution, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Dispense 5.0mL aliquots into Hungate tubes under 80% H2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution and 0.05mL sterile Na2S·9H2O solution. The pH should be 7.0. After inoculation, pressurize culture vessels to 2 bar 80% H2 + 20% CO2 overpressure.

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Use: For the cultivation of Methanobacterium formicicum, Methanosarcina barkeri, Methanosarcina mazei=Methanococcus mazei (Methanosarcina frisia), Methanobacterium bryantii, and Methanobacterium oryzae (Methanobacterium sp.).

Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Methanobacterium II Medium (DSMZ Medium 825)

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize.

Composition per 1060.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g Sodium acetate .............................................................................. 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.2g CaCl2·2H2O................................................................................... 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................40.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol solution ....................................................................10.0mL pH 7.0 ± 0.2 at 25°C Methanol Solution: Composition per 100.0mL: Methanol ..................................................................................15.0mL © 2010 by Taylor and Francis Group, LLC

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture. Add components, except methanol solution, NaHCO3 solution, and Na2S·9H2O solution, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Dispense 5.0mL aliquots into Hungate tubes under 80% H2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to

Methanobacterium II Medium

use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution, 0.05mL sterile Na2S·9H2O solution, and 0.05mL sterile methanol solution. The pH should be 7.0.

Use: For the cultivation of Methanosarcina barkeri DSM 10131 and Methanosarcina mazei=Methanococcus mazei (Methanosarcina frisia) DSM 10132.

Methanobacterium II Medium (DSMZ Medium 825) Composition per 1065.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g Sodium acetate .............................................................................. 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.2g CaCl2·2H2O................................................................................... 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................40.0mL Na-formate solution .................................................................15.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0 ± 0.2 at 25°C Na-Formate Solution: Composition per 10.0mL: Na-formate .................................................................................... 5.0g

Preparation of Na-Formate Solution: Add Na-formate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

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D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ...................................................................................... 10.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture. Add components, except Na-formate solution, NaHCO3 solution, and Na2S·9H2O solution, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Dispense 5.0mL aliquots into Hungate tubes under 80% H2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution, 0.05mL sterile Na2S·9H2O solution, and 0.075mL sterile Na-formate solution. The pH should be 7.0.

Use: For the cultivation of Methanobacterium formicicum DSM 10111.

Methanobacterium II Medium (DSMZ Medium 825) Composition per 1075.0mL: Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.6g Cysteine-HCl·H2O ........................................................................ 0.5g Sodium acetate.............................................................................. 0.5g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.2g CaCl2·2H2O .................................................................................. 0.1g KCl................................................................................................ 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution.....................................................................40.0mL Na-formate solution .................................................................15.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trypticase™ solution...............................................................10.0mL pH 7.0 ± 0.2 at 25°C Trypticase™ Solution: Composition per 10.0mL: Trypticase™.................................................................................. 1.0g

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Methanobrevibacter curvatus Medium

Preparation of Trypticase™ Solution: Add Trypticase™ to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Na-Formate Solution: Composition per 10.0mL: Na-formate .................................................................................... 5.0g

Preparation of Na-Formate Solution: Add Na-formate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

NaHCO3 Solution: Composition per 100.0mL:

clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture. Add components, except Trypticase™ solution, Na-formate solution, NaHCO3 solution, and Na2S·9H2O solution, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Dispense 5.0mL aliquots into Hungate tubes under 80% H2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution, 0.05mL sterile Trypticase™ solution, 0.05mL sterile Na2S·9H2O solution, and 0.075mL sterile Na-formate solution. The pH should be 7.0.

Use: For the cultivation of Methanobacterium oryzae DSM 11106.

Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1010.0mL: NaCl.............................................................................................. 1.0g KCl............................................................................................... 0.5g Casamino acids ............................................................................. 0.5g Yeast extract.................................................................................. 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ........................................................................................... 0.3g KH2PO4......................................................................................... 0.2g Na2SO4 ........................................................................................ 0.15g CaCl2·2H2O .................................................................................. 0.1g Resazurin ................................................................................... 0.5mg NaHCO3 solution .....................................................................40.0mL Dithionite solution ...................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL Selenite-tungstate solution.........................................................1.0mL Seven vitamin solution ..............................................................1.0mL pH 7.4 ± 0.2 at 25°C NaHCO3 Solution: Composition per 40.0mL: NaHCO3 ........................................................................................ 5.8g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Dithionite Solution Composition per 10.0mL: Na-dithionite .............................................................................. 2.0mg

Preparation of Dithionite Solution: Add Na-dithionite to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

NaHCO3 ...................................................................................... 10.0g

Trace Elements Solution SL-10: Composition per liter:

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Auto-

FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg

Methanobrevibacter curvatus Medium

MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

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NaHCO3 Solution: Composition per 40.0mL: NaHCO3 ........................................................................................ 5.8g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution: Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Dithionite Solution: Composition per 10.0mL: Na-dithionite .............................................................................. 2.0mg

Preparation of Dithionite Solution: Add Na-dithionite to dis-

Preparation of Seven Vitamin Solution: Add components to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

tilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-10: Composition per liter:

Preparation of Medium: Prepare and dispense medium under 80%

FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

H2 + 20% CO2 gas atmosphere. Add components, except NaHCO3 solution, seven vitamin solution, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 947.0mL. Mix thoroughly. Adjust pH to 7.6. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 40.0mL NaHCO3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vitamin solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.6. Prior to inoculation add dithionite solution (0.1mL per 10mL medium) as reductant.

Use: For the cultivation of Methanobrevibacter curvatus.

Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1022.0mL: NaCl .............................................................................................. 1.0g KCl............................................................................................... 0.5g Casamino acids ............................................................................. 0.5g Yeast extract.................................................................................. 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ........................................................................................... 0.3g KH2PO4 ......................................................................................... 0.2g Na2SO4 ........................................................................................ 0.15g CaCl2·2H2O................................................................................... 0.1g Resazurin ................................................................................... 0.5mg Rumen fluid, bovine, clarified ..................................................400mL NaHCO3 solution .....................................................................40.0mL Dithionite solution ...................................................................10.0mL MOPS buffer............................................................................10.0mL Nutrient supplement solution.....................................................2.0mL Trace elements solution SL-10 ..................................................1.0mL Selenite-tungstate solution.........................................................1.0mL Seven vitamin solution...............................................................1.0mL pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. Nutrient Supplement Solution: Composition per liter: Pancreatic digest of gelatin........................................................... 5.0g Beef extract................................................................................... 3.0g

Preparation of Nutrient Supplement Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room tempterature.

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Methanobrevibacter curvatus Medium

MOPS Buffer: Composition per 10.0mL: MOPS [3-(N-morpholino) propane sulfonic acid] .......................................................................... 2.1g Na-acetate ..................................................................................... 0.3g EDTA ............................................................................................ 0.1g

Preparation of MOPS Buffer: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Adjust to pH 7.2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2 gas atmosphere. Add components, except clarified bovine rumen fluid, MOPS buffer, nutrient supplement solution, NaHCO3 solution, seven vitamin solution, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 547.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 400.0mL sterile clarified bovine rumen fluid, 10.0mL MOPS buffer, 2.0mL nutrient supplement solution, 40.0mL NaHCO3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vitamin solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.2. Prior to inoculation add dithionite solution (0.1mL per 10mL medium) as reductant.

Use: For the cultivation of Methanobrevibacter curvatus DSM 11111 (strain RFM-2).

Methanobrevibacter curvatus Medium (DSMZ Medium 734) Composition per 1020.0mL: NaCl .............................................................................................. 1.0g KCl............................................................................................... 0.5g Casamino acids ............................................................................. 0.5g Yeast extract.................................................................................. 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ........................................................................................... 0.3g KH2PO4 ......................................................................................... 0.2g Na2SO4 ........................................................................................ 0.15g CaCl2·2H2O................................................................................... 0.1g Resazurin ................................................................................... 0.5mg Rumen fluid, bovine, clarified ..................................................200mL NaHCO3 solution .....................................................................40.0mL Dithionite solution ...................................................................10.0mL MOPS buffer ............................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL Selenite-tungstate solution .........................................................1.0mL Seven vitamin solution...............................................................1.0mL pH 7.7 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 40.0mL: NaHCO3 ........................................................................................ 5.8g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Selenite-Tungstate Solution: Composition per liter: NaOH ............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Dithionite Solution: Composition per 10.0mL: Na-dithionite .............................................................................. 2.0mg

Preparation of Dithionite Solution: Add Na-dithionite to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. MOPS Buffer: Composition per 10.0mL: MOPS [3-(N-morpholino) propane sulfonic acid]........................ 2.1g Na-acetate ..................................................................................... 0.3g EDTA ............................................................................................ 0.1g

Preparation of MOPS Buffer: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Adjust to pH 7.7. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2 gas atmosphere. Add components, except clarified bovine rumen fluid, MOPS buffer, NaHCO3 solution, seven vitamin solution, selenite-tungstate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 747.0mL. Mix thoroughly. Adjust pH to 7.7. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 200.0mL sterile clarified bovine rumen fluid, 10.0mL MOPS buffer, 240.0mL NaHCO3 solution, 1.0mL selenite-tungstate solution, 1.0mL seven vitamin solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. Adjust pH to 7.7. Prior to inoculation add dithionite solution (0.1mL per 10mL medium) as reductant.

Methanocalculus pumilus Medium Use: For the cultivation of Methanobrevibacter curvatus DSM 11111 DSM 11139 (strain RFM-1)

Methanocalculus halotolerans Medium (DSMZ Medium 905) Composition per 1010.0mL: NaCl ............................................................................................ 50.0g NH4Cl ........................................................................................... 1.0g Na-acetate ..................................................................................... 0.5g Yeast extract.................................................................................. 0.5g Trypticase™.................................................................................. 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g KCl.............................................................................................. 0.17g Resazurin ................................................................................... 0.5mg Magnesium chloride solution...................................................30.0mL NaHCO3 solution .....................................................................20.0mL Trace elements solution ...........................................................10.0mL Calcium chloride solution ........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL L-Cysteine solution ..................................................................10.0mL pH 7.2-7.6 at 25°C Magnesium Chloride Solution: Composition per 30.0mL: MgCl2·6H2O.................................................................................. 3.2g

Preparation

of Magnesium Chloride Solution: Add MgCl2·6H2O to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Calcium Chloride Solution: Composition per 10.0mL: CaCl2·2H2O................................................................................... 0.6g

Preparation of Calcium Chloride Solution: Add CaCl2·2H2O

to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Solution: Composition per 10.0mL:

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under sparge with 80% N2 + 20% CO2. Add components, except NaHCO3 solution, magnesium chloride solution, calcium chloride solution, Lcysteine-HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to room temperature while sparging with 80% N2 + 20% CO2. Add 10.0mL L-cysteine-HCl·H2O solution and 20.0mL NaHCO3 solution. Mix thoroughly. Adjust pH to 7.5. Distribute into anaerobic tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add per liter, 30.0mL magnesium chloride solution, 10.0mL calcium chloride solution, and 10.0mL Na2S·9H2O. Mix thoroughly. After inoculation pressurize vessels with 80% H2 + 20% CO2 gas mixture to 1 bar overpressure and add sulfide from a sterile, anaerobic stock solution. The final pH of the medium should be 7.2–7.6. Use: For the cultivation of Methanocalculus halotolerans.

Methanocalculus pumilus Medium (DSMZ Medium 892)

L-Cysteine

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

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Composition per 1080.0mL: NaCl............................................................................................ 10.0g Yeast extract.................................................................................. 2.0g Trypticase™.................................................................................. 2.0g NH4Cl ........................................................................................... 0.9g K2HPO4......................................................................................... 0.4g MgCl2·6H2O ............................................................................... 0.36g Resazurin ................................................................................... 0.5mg Na2CO3 solution.......................................................................50.0mL Na2S·9H2O solution .................................................................15.0mL L-Cysteine-HCl·H2O solution ..................................................15.0mL Vitamin solution.......................................................................10.0mL Trace elements solution ...........................................................10.0mL

NaHCO3 Solution: Composition per 20.0mL:

L-Cysteine

NaHCO3 ........................................................................................ 2.0g

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Must be prepared freshly.

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

Solution: Composition per 15.0mL:

distilled/deionized water and bring volume to 15.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

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Methanococcoides Medium

Na2CO3 Solution: Composition per 50.0mL:

period to 7.3–7.5. After inoculation use atmosphere of 80% H2 + 20% CO2 to 1.5 bar overpressure.

Na2CO3 ......................................................................................... 5.0g

Use: For the cultivation of Methanocalculus pumilus.

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize.

Na2S·9H2O Solution: Composition per 15mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 15.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

tion, Na2S·9H2O solution, and L-cysteine solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2 for 30 min. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically distribute 5.0mL aliquots into Hungate tubes under 100% N2. Aseptically and anaerobically add per 5.0mL medium 0.25mL Na2CO3 solution, 0.075mL Na2S·9H2O solution, and 0.075mL L-cysteine solution. Mix thoroughly. Replace N2 atmosphere with atmosphere of 80% H2 + 20% CO2. Repeat atmosphere replacement several times with overpressurization. The initial pH of 9.0 will decrease over a 30 min © 2010 by Taylor and Francis Group, LLC

Methanococcal Complex Medium See: Methanococcus McC Medium

Methanococcoides Medium Composition per liter: NaCl............................................................................................ 18.0g NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O ................................................................................. 4.0g MgSO4·7H2O ............................................................................. 3.45g Trimethylamine·HCl ..................................................................... 3.0g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate.............................................................................. 1.0g L-Cysteine·HCl ............................................................................. 0.5g Na2S·9H2O.................................................................................... 0.5g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0. Add distilled/deionized water to 1.0L. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Methanococcus jannaschii Medium Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thoroughly. Preparation of Medium: Prepare the medium anaerobically under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Methanococcoides methylutens.

Methanococcus deltae Medium Composition per liter: NaCl ............................................................................................ 35.0g NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O.................................................................................. 4.0g NH4Cl ........................................................................................... 2.7g Sodium acetate .............................................................................. 2.5g L-Cysteine·HCl.............................................................................. 0.3g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g Na2S·9H2O .................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.13g Resazurin ................................................................................... 1.0mg (NH4)2SO4 .................................................................................. 0.3mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution...........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg © 2010 by Taylor and Francis Group, LLC

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Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thoroughly. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl ............................................................................. 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except L-cysteine·HCl solution and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile Lcysteine·HCl solution and sterile Na2S·9H2O solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanococcus deltae.

Methanococcus jannaschii Medium Composition per liter: NaCl............................................................................................ 30.0g MgSO4·7H2O .............................................................................. 3.40g MgCl2·2H2O ................................................................................. 2.7g NaHCO3 ........................................................................................ 1.0g Na2S·9H2O.................................................................................... 0.5g KCl.............................................................................................. 0.33g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·6H2O ................................................................ 0.01g Resazurin ................................................................................... 1.0mg Na2SeO3·5H2O........................................................................... 0.5mg NiCl2·6H2O................................................................................ 0.5mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.0 ± 0.2 at 25°C

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Methanococcus jannaschii Medium

FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

CaCl2·2H2O ................................................................................ 0.14g KCl.............................................................................................. 0.33g Minerals solution .....................................................................10.0mL Na2S2O3 solution......................................................................10.0mL β-Mercaptoethanol solution.....................................................10.0mL SeO2 solution .............................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Minerals Solution: Composition per liter:

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thoroughly.

Nitrilotriacetic acid ....................................................................... 4.5g FeCl2·4H2O ................................................................................... 0.4g CoCl2·2H2O ................................................................................ 0.17g MnCl2·4H2O ................................................................................. 0.1g ZnCl2 ............................................................................................. 0.1g NaMoO4·6H2O......................................................................... 36.0mg CaCl2·H2O ............................................................................... 27.0mg

Preparation of Minerals Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Na2S2O3 Solution: Composition per 10.0mL: Na2S2O3·5H2O ............................................................................ 0.63g

Preparation of Na2S2O3 Solution: Add Na2S2O3·5H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

L-Cysteine·HCl

β-Mercaptoethanol Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.5g

Preparation of β-Mercaptoethanol Solution: Add β-mercapto-

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

ethanol to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL:

SeO2 Solution: Composition per 100.0mL:

Solution: Composition per 10.0mL:

β-Mercaptoethanol...................................................................... 0.39g

Na2S·9H2O .................................................................................... 0.3g

SeO2 .......................................................................................... 0.011g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

Preparation of SeO2 Solution: Add SeO2 to distilled/deionized

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Prepare and dispense medium under

H2 + 20% CO2. Add components, except L-cysteine·HCl solution and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L-cysteine·HCl solution and sterile Na2S·9H2O solution into individual tubes containing medium.

100% N2. Add components, except Na2S2O3 solution and β-mercaptoethanol solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 100% N2. Adjust pH to 7.0 with KOH. Anaerobically distribute 100.0mL volumes into anaerobic bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 1.0mL of sterile Na2S2O3 solution and 1.0mL of sterile β-mercaptoethanol solution to each bottle. Mix thoroughly. Prior to inoculation, flush each bottle with 80% H2 + 20% CO2.

Use: For the cultivation and maintenance of Methanococcus species.

Use: For the cultivation of Methanococcus jannaschii.

Preparation of Medium: Prepare and dispense medium under 80%

Methanococcus jannaschii Medium Composition per 1020.0mL: PIPES (piperazine-N,N´bis[2-ethanesulfonic acid]) buffer ...................................... 15.12g MgCl2·6H2O.................................................................................. 4.3g MgSO4·7H2O ................................................................................ 3.4g NaCl .............................................................................................. 3.0g NH4Cl ......................................................................................... 0.25g K2HPO4 ....................................................................................... 0.14g © 2010 by Taylor and Francis Group, LLC

Methanococcus McC Medium Composition per 1100.0mL: NaHCO3 ........................................................................................ 5.0g Yeast extract.................................................................................. 2.0g L-Cysteine·HCl·H2O ..................................................................... 0.5g General salts solution.............................................................500.0mL NaCl solution ...........................................................................75.0mL Na2S·9H2O solution .................................................................20.0mL K2HPO4 solution......................................................................10.0mL

Methanococcus McN Medium

Trace minerals solution............................................................10.0mL Sodium acetate solution ...........................................................10.0mL Iron stock solution .....................................................................5.0mL Resazurin solution......................................................................1.0mL

General Salts Solution: Composition per liter: MgSO4·7H2O ................................................................................ 6.9g MgCl2·6H2O.................................................................................. 5.5g NH4Cl ........................................................................................... 1.0g KCl.............................................................................................. 0.67g CaCl2·2H2O................................................................................. 0.28g

Preparation of General Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

NaCl Solution: Composition per 100.0mL: NaCl ............................................................................................ 29.3g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Na2S·9H2O Solution: Composition per 100.0mL: NaOH ....................................................................................... 1 pellet Na2S·9H2O .................................................................................... 2.5g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

K2HPO4 Solution: Composition per 100.0mL: K2HPO4 ......................................................................................... 1.4g

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Preparation of Sodium Acetate Solution: Add sodium acetate·3H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Iron Stock Solution: Composition per 100.0mL: Fe(NH4)2(SO4)2·6H2O .................................................................. 0.2g

Preparation of Iron Stock Solution: Add Fe(NH4)2(SO4)2·6H2O

to 5.0mL of distilled H2O containing 2 drops of concentrated HCl. Mix thoroughly. When the Fe(NH4)2(SO4)2·6H2O has dissolved, bring the volume to 100.0mL with distilled/deionized water.

Resazurin Solution: Composition per 10.0mL: Resazurin ................................................................................. 10.0mg

Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except NaHCO3 and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1080.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add NaHCO3. Mix thoroughly. Anaerobically distribute 9.8mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically and anaerobically add 0.2mL of sterile Na2S·9H2O solution to each tube. Mix thoroughly.

Use: For the cultivation of Methanococcus species.

Methanococcus McN Medium Composition per 1100.0mL: NaHCO3 ........................................................................................ 5.0g L-Cysteine·HCl·H2O ..................................................................... 0.5g General salts solution.............................................................500.0mL NaCl solution ...........................................................................75.0mL Na2S·9H2O solution .................................................................20.0mL K2HPO4 solution......................................................................10.0mL Trace minerals solution............................................................10.0mL Iron stock solution .....................................................................5.0mL Resazurin solution .....................................................................1.0mL

Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/de-

General Salts Solution: Composition per liter:

Trace Minerals Solution: Composition per liter:

MgSO4·7H2O ................................................................................ 6.9g MgCl2·6H2O ................................................................................. 5.5g NH4Cl ........................................................................................... 1.0g KCl.............................................................................................. 0.67g CaCl2·2H2O ................................................................................ 0.28g

ionized water and bring volume to 100.0mL. Mix thoroughly.

Nitrilotriacetic acid ....................................................................... 1.5g Na2WO4·2H2O .............................................................................. 1.0g Fe(NH4)2(SO4)2·6H2O .................................................................. 0.2g Na2SeO3 ........................................................................................ 0.2g Na2MoO4·2H2O ............................................................................ 0.1g Mn4·2H2O ..................................................................................... 0.1g Zn4·7H2O ...................................................................................... 0.1g NiCl2·7H2O ............................................................................... 0.025g CuSO4·5H2O ............................................................................... 0.01g

Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0.

Preparation of General Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

NaCl Solution: Composition per 100.0mL: NaCl............................................................................................ 29.3g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................... 2.5g NaOH....................................................................................... 1 pellet

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

tilled/deionized water to boiling. Cool to room temperature while

1094

Methanococcus McNail Medium

sparging with 100%N2. Dissolve 1 pellet of NaOH in the anaerobic water. Weigh out a little more than 2.5g of Na2S·9H2O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on paper towels or filter paper. Add 2.5g of washed Na2S·9H2O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Pressurize to 60kPa with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

K2HPO4 Solution: Composition per 100.0mL: K2HPO4 ......................................................................................... 1.4g

Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid ....................................................................... 1.5g Na2WO4·2H2O .............................................................................. 1.0g Fe(NH4)2(SO4)2·6H2O .................................................................. 0.2g Na2SeO3 ........................................................................................ 0.2g Na2MoO4·2H2O ............................................................................ 0.1g Mn4·2H2O ..................................................................................... 0.1g Zn4·7H2O ...................................................................................... 0.1g NiCl2·7H2O ............................................................................... 0.025g CuSO4·5H2O ............................................................................... 0.01g

Iron Stock Solution: Composition per 100.0mL: Fe(NH4)2(SO4)2·6H2O .................................................................. 0.2g

Preparation of Iron Stock Solution: Add Fe(NH4)2(SO4)2·6H2O to 5.0mL of distilled H2O containing 2 drops of concentrated HCl. Mix thoroughly. When the Fe(NH4)2(SO4)2·6H2O has dissolved, bring the volume to 100.0mL with distilled/deionized water.

Resazurin Solution: Composition per 10.0mL: Resazurin ................................................................................. 10.0mg

Preparation of Resazurin Solution: Add resazurin to distilled/

NaCl solution ...........................................................................75.0mL Na2S·9H2O solution .................................................................20.0mL K2HPO4 solution......................................................................10.0mL Trace minerals solution............................................................10.0mL Sodium acetate solution...........................................................10.0mL Pantoyllactone solution............................................................10.0mL Iron stock solution .....................................................................5.0mL Resazurin solution .....................................................................1.0mL

General Salts Solution: Composition per liter: MgSO4·7H2O ................................................................................ 6.9g MgCl2·6H2O ................................................................................. 5.5g NH4Cl ........................................................................................... 1.0g KCl.............................................................................................. 0.67g CaCl2·2H2O ................................................................................ 0.28g

Preparation of General Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

NaCl Solution: Composition per 100.0mL: NaCl............................................................................................ 29.3g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Na2S·9H2O Solution: Composition per 100.0mL: NaOH....................................................................................... 1 pellet Na2S·9H2O.................................................................................... 2.5g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

K2HPO4 Solution: Composition per 100.0mL:

deionized water and bring volume to 10.0mL. Mix thoroughly.

K2HPO4......................................................................................... 1.4g

Preparation of Medium: Prepare and dispense medium under 80%

Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

H2 + 20% CO2. Add components, except NaHCO3 and Na2S·9H2O solution, to distilled/deionized water and bring volume to 1080.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add NaHCO3. Mix thoroughly. Anaerobically distribute 9.8mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically and anaerobically add 0.2mL of sterile Na2S·9H2O solution to each tube. Mix thoroughly.

Use: For the cultivation of Methanococcus species.

Methanococcus McNail Medium Composition per 1100.0mL: NaHCO3 ........................................................................................ 5.0g L-Leucine ...................................................................................... 1.0g L-Isoleucine................................................................................... 0.5g L-Cysteine·HCl·H2O ..................................................................... 0.5g General salts solution.............................................................500.0mL © 2010 by Taylor and Francis Group, LLC

Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid ....................................................................... 1.5g Na2WO4·2H2O .............................................................................. 1.0g Fe(NH4)2(SO4)2·6H2O.................................................................. 0.2g Na2SeO3 ........................................................................................ 0.2g Na2MoO4·2H2O ............................................................................ 0.1g Mn4·2H2O ..................................................................................... 0.1g Zn4·7H2O ...................................................................................... 0.1g NiCl2·7H2O ............................................................................... 0.025g CuSO4·5H2O ............................................................................... 0.01g

Methanococcus sp. Medium

1095

Sodium Acetate Solution: Composition per 100.0mL:

Trace Minerals Stock Solution: Composition per liter:

Sodium acetate·3H2O.................................................................. 13.6g

Nitrilotriacetic acid ....................................................................... 1.5g MnSO4·H2O .................................................................................. 0.5g CoCl2 ............................................................................................ 0.1g ZnSO4 ........................................................................................... 0.1g AIK(SO4)2·12H2O ...................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g NiCl2 ........................................................................................... 0.01g

Preparation of Sodium Acetate Solution: Add 13.6g of sodium acetate·3H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Pantoyllactone Solution: Composition per 100.0mL: Pantoyllactone........................................................................... 0.013g

Preparation of Pantoyllactone Solution: Add pantoyllactone to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Iron Stock Solution: Composition per 100.0mL: Fe(NH4)2(SO4)2·6H2O .................................................................. 0.2g

Resazurin Solution: Composition per 10.0mL: Resazurin ................................................................................. 10.0mg

Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly.

Use: For the cultivation of Methanococcus species.

Methanococcus Medium Composition per liter: NaCl ............................................................................................ 18.0g Mg2SO4·7H2O............................................................................. 3.45g MgCl2·2H2O................................................................................ 2.75g Pancreatic digest of casein ............................................................ 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate .............................................................................. 1.0g L-Cysteine·HCl.............................................................................. 0.5g Na2S·9H2O .................................................................................... 0.5g NH4HCO3 ..................................................................................... 0.5g KCl............................................................................................ 0.335g NH4Cl ....................................................................................... 0.225g CaCl2·2H2O................................................................................. 0.14g KH2PO4 ....................................................................................... 0.14g Calcium DL-pantothenate........................................................... 5.0mg Na2SeO3 ..................................................................................... 2.0mg FeSO4·2H2O............................................................................... 1.0mg Resazurin ................................................................................... 1.0mg Trace minerals stock solution .....................................................10mL pH 6.5 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Trace Minerals Stock Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 6.8. Preparation of Medium: Prepare medium anaerobically under 80% N2 + 20% CO2. Add components, except Na2CO3, Lcysteine·HCl, and Na2S·9H2O, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Cool while sparging with 80% N2 + 20% CO2. Add Na2CO3, Lcysteine·HCl, and Na2S·9H2O. Dispense into tubes, bottles, or flasks under an atmosphere of 80% H2 + 20% CO2. Seal with butyl rubber stoppers secured with aluminum crimp seals. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Pressurize the head space to 69kPA with 80% H2 + 20% CO2.

Use: For the cultivation of Methanococcus species.

Methanococcus sp. Medium (DSMZ Medium 141a) Composition per 1040.0mL: NaCl............................................................................................ 18.0g MgCl2·6H2O ................................................................................. 4.0g MgSO4·7H2O .............................................................................. 3.45g Na-acetate ..................................................................................... 1.0g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine solution ..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g

1096

Methanococcus vannielii Medium

CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Solution A: Composition per 500.0mL:

Preparation of Trace Elements Solution: Add nitrilotriacetic

Sodium formate .......................................................................... 10.0g Phenol Red................................................................................. 3.0mg Methylene Blue.......................................................................... 2.0mg

acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly.

Preparation of Solution A: Add components to distilled/deionized

Vitamin Solution: Composition per liter:

water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Inorganic Salts Solution: Composition per 500.0mL:

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

L-Cysteine

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Methanococcus sp.

Methanococcus vannielii Medium Composition per 1020.0mL: Solution A ..............................................................................500.0mL Inorganic salts solution ..........................................................500.0mL Na2S·9H2O solution .................................................................10.0mL Na2CO3 solution.......................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

K2HPO4·3H2O ............................................................................ 1.45g NH4Cl ........................................................................................... 1.0g KH2PO4....................................................................................... 0.75g MgCl2·6H2O ................................................................................. 0.2g Nitrilotriacetic acid ..................................................................... 0.04g CaCl2·2H2O ................................................................................ 0.02g FeCl2·4H2O ................................................................................ 3.6mg CoCl2·6H2O ............................................................................... 1.5mg MnCl2·4H2O.............................................................................. 0.9mg ZnCl2 .......................................................................................... 0.9mg H3BO2 ...................................................................................... 0.17mg Na2MoO4·2H2O ....................................................................... 0.09mg

Preparation of Inorganic Salts Solution: Add nitrilotriacetic acid to 250.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Readjust pH to 7.2 with H2SO4 or KOH. Add distilled/deionized water to 500.0mL. Filter sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 2.5g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Prepare and distribute medium anaerobically under 80% N2 + 20% CO2. Aseptically and anaerobically combine 500.0mL of sterile inorganic salts solution, 500.0mL of sterile solution A, 10.0mL of sterile Na2S·9H2O solution, and 10.0mL of sterile Na2CO3 solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the isolation and cultivation of Methanococcus vannielii from marine mud.

Methanococcus vannielii Medium Composition per liter: Sodium formate .......................................................................... 15.0g K2HPO4....................................................................................... 3.48g CoCl2·6H2O ................................................................................ 2.38g NH4Cl ........................................................................................... 1.0g L-Cysteine·HCl·H2O ..................................................................... 0.3g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 0.01g FeSO4·7H2O................................................................................ 0.01g

Methanocorpusculum Medium

MnSO4·H2O ............................................................................... 7.5mg Na2MoO4·2H2O ......................................................................... 7.5mg Na2SeO3 ..................................................................................... 1.7mg Na2S·9H2O solution .................................................................10.0mL

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.15g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

1097

Nicotinic acid............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.5mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Prepare and distribute medium anaerobi-

L-Cysteine/Na2S Solution: Composition per liter:

cally under 100% N2. Add components, except Na2S·9H2O solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until resazurin turns colorless, indicating reduction. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Preparation of L-Cysteine/Na2S Solution: Add L-cysteine·HCl and Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.

Use: For the cultivation of Methanococcus vannielii.

Methanococcus voltae BD Medium Composition 1003.0mL: L-Leucine .................................................................................... 50.0g

NaCl ............................................................................................ 18.0g Sodium panthothenate................................................................... 5.0g MgSO4·7H2O .............................................................................. 3.48g MgCl2·6H2O................................................................................ 2.75g Sodium acetate·3H2O.................................................................... 1.0g KCl.............................................................................................. 0.34g NH4Cl ......................................................................................... 0.26g CaCl2·2H2O................................................................................. 0.14g K2PO4 .......................................................................................... 0.14g L-Isoleucine................................................................................... 0.1g L-Cysteine/Na2S solution.........................................................17.5mL Trace minerals solution............................................................10.0mL Vitamin solution.......................................................................10.0mL Na2CO3 solution.........................................................................6.0mL FeSO4·7H2O solution.................................................................4.5mL

Trace Minerals Solution: Composition per liter: Nitrilotriacetic acid ....................................................................... 1.5g MnSO4·7H2O ................................................................................ 0.5g NiCl2·6H2O ................................................................................. 0.12g CoCl2·6H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g Resazurin ...................................................................................... 0.1g ZnSO4·7H2O ................................................................................. 0.1g AIK(SO4)2·5H2O......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g

Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg © 2010 by Taylor and Francis Group, LLC

L-Cysteine·HCl ........................................................................... 1.25g Na2S·9H2O.................................................................................. 1.25g

Na2CO3 Solution: Composition per 100.0mL: Na2CO3 ......................................................................................... 1.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

FeSO4·7H2O Solution: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.1g

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Sparge with 100% N2. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except L-cysteine/Na2S solution, Na2CO3 solution, and FeSO4·7H2O solution, to distilled/deionized water and bring volume to 975.0mL. Mix thoroughly. Gently heat and bring to boiling while sparging with 80% H2 + 20% CO2. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add 17.5mL of L-cysteine/Na2S·9H2O solution and 6.0mL of Na2CO3 solution. Anaerobically dispense into tubes or bottles in 10.0mL aliquots under an atmosphere of 80% H2 + 20% CO2. Seal with butyl rubber stoppers secured with aluminum crimp seals. Autoclave for 15 min at 15 psi pressure–121°C. Immediately prior to inoculation, aseptically and anaerobically add 0.05mL of sterile FeSO4·7H2O solution to each tube.

Use: For the cultivation of Methanococcus voltae.

Methanocorpusculum Medium (DSMZ Medium 279) Composition per liter: Na-acetate ..................................................................................... 4.0g NaHCO3 ........................................................................................ 4.0g Na-formate.................................................................................... 2.0g Yeast extract.................................................................................. 1.0g KH2PO4......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.4g NaCl.............................................................................................. 0.4g NH4Cl ........................................................................................... 0.4g CaCl2·2H2O ................................................................................ 0.05g FeSO4·7H2O.............................................................................. 0.002g

1098

Methanoculleus olentangyi Medium

Resazurin .................................................................................. 0.001g NiCl2·6H2O .............................................................................. 24.0mg Sludge fluid..............................................................................50.0mL Fatty acid mixture ....................................................................20.0mL L-Cysteine solution ..................................................................10.0mL Na2S·9H2O ...............................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.7–7.0

Sludge Fluid: Composition per 500.0mL: Yeast extract.................................................................................. 2.0g Sludge ....................................................................................500.0mL

Preparation of Sludge Fluid: Add yeast extract to sludge from an anaerobic digester. Gas with nitrogen gas for a few minutes. Incubate at 37°C for 24 hrs. Centrifuge the sludge at 13,000 x g. Autoclave for 15 min at 15 psi pressure–121°C. Place the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at room temperature in the dark.

Fatty Acid Mixture: Composition per 20.0mL: Valeric acid ................................................................................... 0.5g Isovaleric acid ............................................................................... 0.5g α-Methylbutyric acid .................................................................... 0.5g Isobutyric acid............................................................................... 0.5g Distilled water..........................................................................20.0mL

Preparation of Fatty Acid Mixture: Add components to 20.0mL distilled/deionized water. Mix thoroughly.

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl. L-Cysteine

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2 gas atmosphere. Add components, except L-cysteine solution, Na2S·9H2O solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Adjust pH to 6.8. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL Lcysteine solution, 10.0mL Na2S·9H2O solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure. Alternately, the medium without Lcysteine solution, Na2S·9H2O solution, and trace elements solution SL10 can be distributed to tubes anaerobically prior to autoclaving. After autoclaving in tubes the appropriate volumes of the individual solutions can be injected through the stoppers so that the final concentrations of the medium are achieved.

Use: For the cultivation of Methanococcoides spp. and Methanolobus bombayensis.

Methanoculleus olentangyi Medium Composition per liter: NaHCO3 ........................................................................................ 5.0g NH4Cl ........................................................................................... 2.7g Sodium acetate.............................................................................. 2.5g NaCl............................................................................................ 0.61g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.13g Resazurin ................................................................................... 1.0mg (NH4)2SO4 ................................................................................. 0.3mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O ............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Methanogen Medium, Zeikus

Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.3g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL:

1099

Na2S·9H2O solution...................................................................3.0mL Na2CO3 solution ........................................................................3.0mL

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.1g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 0.5g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Prepare and distribute medium anaerobi-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

cally under 100% N2. Add components, except Na2S·9H2O solution and Na2CO3 solution, to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Aseptically add 3.0mL of sterile Na2S·9H2O solution and 3.0mL of sterile Na2CO3 solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Preparation of Medium: Prepare and dispense medium under 80%

Use: For the cultivation and enrichment of acetate-utilizing methano-

genic bacteria.

Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

Use: For the cultivation and maintenance of Methanoculleus olentangyi.

Methanogen Enrichment Medium, Barker Composition per liter: CaCO3 ......................................................................................... 20.0g NH4Cl ........................................................................................... 1.0g K2HPO4·3H2O............................................................................... 0.4g MgCl2·6H2O.................................................................................. 0.1g Methanol ..................................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol and CaCO3, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add filter-sterilized methanol solution. Mix thoroughly. Add 1.0g of CaCO3 to each of 50.0mL screw-capped bottles. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Fill each bottle to capacity with enrichment medium. Use: For the cultivation of methanogenic bacteria.

Methanogen Medium Composition per 106.0mL: CaCO3 ......................................................................................... 10.0g Calcium acetate............................................................................. 2.0g NH4Cl ........................................................................................... 0.1g K2HPO4·3H2O............................................................................. 0.04g MgCl2·6H2O................................................................................ 0.01g © 2010 by Taylor and Francis Group, LLC

Methanogen Medium, Zeikus Composition per 1010.0mL: Inorganic salts solution ..........................................................500.0mL Vitamin solution.....................................................................500.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Inorganic Salts Solution: Composition per 500.0mL: K2HPO4·3H2O ............................................................................ 1.45g NH4Cl ........................................................................................... 1.0g KH2PO4....................................................................................... 0.75g MgCl2·6H2O ................................................................................. 0.2g Nitrilotriacetic acid ..................................................................... 0.04g CaCl2·2H2O ................................................................................ 0.02g FeCl2·4H2O................................................................................ 3.6mg CoCl2·6H2O ............................................................................... 1.5mg MnCl2·4H2O.............................................................................. 0.9mg ZnCl2 .......................................................................................... 0.9mg H3BO2 ...................................................................................... 0.17mg Na2MoO4·2H2O ......................................................................... .09mg

Vitamin Solution: Composition per 500.0mL: Pyridoxine·HCl .......................................................................... 1.0mg p-Aminobenzoic acid................................................................. 0.5mg Ca-D-pantothenate...................................................................... 0.5mg Nicotinic acid............................................................................. 0.5mg Riboflavin .................................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.5mg Thioctic acid .............................................................................. 0.5mg

1100

Methanogenium aggregans Medium

Biotin ......................................................................................... 0.2mg Folic acid.................................................................................... 0.2mg Vitamin B12 .............................................................................. 0.01mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Filter sterilize. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and distribute medium anaerobically under 95% N2 + 5% CO2. Aseptically and anaerobically combine 500.0mL of sterile inorganic salts solution, 500.0mL of sterile vitamin solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of methanogenic bacteria.

Methanogenium aggregans Medium (DSMZ Medium 321) Composition per liter: Na-formate .................................................................................... 5.0g Na2CO3 ......................................................................................... 1.5g Na-acetate ..................................................................................... 1.0g Trypticase™.................................................................................. 1.0g Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g K2HPO4·3H2O............................................................................... 0.4g MgCl2·6H2O.................................................................................. 0.4g Resazurin ................................................................................... 1.0mg Mineral solution .......................................................................50.0mL Cysteine solution......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution ...........................................................10.0mL Sludge fluid................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Mineral Solution: Composition per liter: NaCl ............................................................................................ 12.0g KH2PO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.6g CaCl2·2H2O................................................................................. 0.16g

H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 0.2g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.2g

Preparation of Sludge Fluid: Add yeast extract to sludge from an anaerobic digester. Gas with nitrogen gas for a few minutes. Incubate at 37°C for 24 hours. Centrifuge the sludge at 13,000 x g. Autoclave for 15 min at 15 psi pressure–121°C. Place the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at room temperature in the dark.

Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% H2 + 20% CO2 gas mixture. Add components, except sludge fluid, cysteine solution, and Na2S·9H2O solution, to distilled/ deionized water and bring volume to 975.0mL. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C while sparging with 80% H2 + 20% CO2. Aseptically and anaerobically add 5.0mL sterile sludge fluid, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of Methanocorpusculum aggregans (Methanogenium aggregans).

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Methanogenium aggregans Medium Composition per liter: Sodium formate ............................................................................ 5.0g NH4Cl ........................................................................................... 1.0g Sodium acetate.............................................................................. 1.0g Trypticase™.................................................................................. 1.0g Yeast extract.................................................................................. 1.0g K2HPO4·3H2O .............................................................................. 0.4g MgCl2·6H2O ................................................................................. 0.4g Resazurin ................................................................................... 1.0mg Mineral solution.......................................................................50.0mL Trace elements solution ...........................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL

Methanogenium Alcohol Medium

1101

Na2S·9H2O solution .................................................................10.0mL Na2CO3 solution.......................................................................10.0mL Sludge fluid................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

few minutes. Incubate at 37°C for 24 hr. Centrifuge the sludge at 13,000 × g for 15 min. Decant the clear supernatant solution. Sparge with 100% N2 for a few minutes. Store in screw-capped bottles at room temperature in the dark.

Mineral Solution: Composition per liter:

Preparation of Medium: Prepare and dispense medium under 80%

NaCl ............................................................................................ 12.0g KH2PO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.6g CaCl2·2H2O................................................................................. 0.16g

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.2g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 1.5g

H2 + 20% CO2. Add components, except L-cysteine·HCl solution, Na2S·9H2O solution, and Na2CO3 solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Adjust pH to 6.8. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution, 10.0mL of sterile Na2S·9H2O solution, and 10.0mL of sterile Na2CO3 solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L-cysteine·HCl solution, sterile Na2S·9H2O solution, and sterile Na2CO3 solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanocorpusculum aggregans.

Methanogenium Alcohol Medium Composition per 1003.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.5g NH4Cl ........................................................................................... 0.4g Sodium acetate·3H2O.................................................................... 0.4g KH2PO4......................................................................................... 0.2g CaCl2·2H2O .................................................................................. 0.1g NaHCO3 solution.....................................................................60.0mL 2-Propanol..................................................................................5.0mL Na2S·9H2O solution...................................................................3.0mL Cyanocobalamin solution ..........................................................1.0mL Selenite-molybdate-tungstate solution.......................................1.0mL Thiamine solution ......................................................................1.0mL Trace elements solution .............................................................1.0mL Vitamin solution.........................................................................1.0mL

Trace Elements Solution: Composition per 100.0mL: FeSO4·7H2O......................................................................... 1400.0mg ZnSO4·7H2O .......................................................................... 145.0mg CoCl2·6H2O ........................................................................... 120.0mg MnCl2·4H2O .......................................................................... 100.0mg NiCl2·6H2O.............................................................................. 50.0mg H3BO3 ........................................................................................ 6.0mg CuSO4·5H2O.............................................................................. 3.0mg HCl (25%,w/v)...........................................................................8.0mL

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

Selenite-Molybdate-Tungstate Solution: Composition per liter:

Sludge Fluid: Composition per 100.0mL:

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Yeast extract.................................................................................. 0.4g Sludge ....................................................................................100.0mL

Preparation of Selenite-Molybdate-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

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Methanogenium bourgense Medium

NaHCO3 Solution: Composition per liter:

lution, and 1.0mL of sterile vitamin solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

NaHCO3 ...................................................................................... 84.0g

Use: For the cultivation of Methanogenium species.

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 2.5g NaOH ....................................................................................... 1 pellet

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

Preparation of 2-Propanol: Filter sterilize 10.0mL of 2-propanol.

Methanogenium bourgense Medium (DSMZ Medium 322) Composition per liter: Na-formate.................................................................................... 5.0g Na2CO3 ......................................................................................... 1.5g Na-acetate ..................................................................................... 1.0g Trypticase™ peptone .................................................................... 1.0g Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g K2HPO4·3H2O .............................................................................. 0.4g MgCl2·6H2O ................................................................................. 0.1g Resazurin ................................................................................... 1.0mg Cysteine solution .....................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.0–7.0 at 25°C

Cysteine Solution: Composition per 10.0mL:

Sparge with 100% N2.

L-Cysteine·HCl·H2O ..................................................................... 0.5g

Vitamin Solution: Composition per liter:

Sodium 2-mercaptoethanesulfonate............................................ 0.25g Pyridoxine·HCl ........................................................................... 0.15g Calcium pantothenate ................................................................... 0.1g Nicotinic acid ................................................................................ 0.1g p-Aminobenzoic acid ............................................................... 40.0mg Biotin ....................................................................................... 10.0mg Potassium phosphate buffer (25mM solution, pH 7.0) .................1.0L

Preparation of Vitamin Solution: Combine components. Mix thoroughly. Filter sterilize. Sparge with 100% N2.

Thiamine Solution: Composition per liter: Thiamine·HCl ............................................................................... 0.1g Sodium phosphate buffer (0.1M solution, pH 3.6) .......................1.0L

Preparation of Thiamine Solution: Combine components. Mix thoroughly. Filter sterilize. Sparge with 100% N2.

Cyanocobalamin Solution: Composition per liter: Cyanocobalamin ...................................................................... 50.0mg

Preparation of Cyanocobalamin Solution: Add cyanocobalamin to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N2.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.2g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% H2 + 20% CO2 gas mixture. Add components, except cysteine solution and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C while sparging with 80% H2 + 20% CO2. Aseptically and anaerobically add 10.0mL of sterile cysteine solution and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of Methanoculleus bourgensis=Methanogenium bourgense.

Methanogenium bourgense Medium Composition per liter: Sodium formate ............................................................................ 5.0g Sodium acetate.............................................................................. 1.0g NH4Cl ........................................................................................... 1.0g Trypticase™.................................................................................. 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl ............................................................................. 0.5g K2HPO4·3H2O .............................................................................. 0.4g MgCl2·6H2O ................................................................................. 0.1g Resazurin ................................................................................... 1.0mg

Methanogenium Medium

Na2CO3 solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.7 ± 0.2 at 25°C

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Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deion-

CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Na2S·9H2O Solution: Composition per 10.0mL:

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 1.5g ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O .................................................................................... 0.2g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except Na2CO3 solution and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 67. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile Na2CO3 solution and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile Na2CO3 solution and sterile Na2S·9H2O solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanogenium bourgense.

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

Preparation of Medium: Prepare and dispense medium under 80%

Methanogenium CV Medium Composition per liter: NaCl ............................................................................................ 18.0g Isopropanol ................................................................................... 7.5g NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O.................................................................................. 4.0g MgSO4·7H2O ............................................................................. 3.45g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate .............................................................................. 1.0g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O................................................................................. 0.14g K2HPO4 ....................................................................................... 0.14g L-Cysteine·HCl.............................................................................. 0.5g Na2S·9H2O .................................................................................... 0.4g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Na2WoO4·2H2O ....................................................................... 0.03mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 6.8–7.0 at 25°C

H2 + 20% CO2. Add components, except vitamin solution, to distilled/ deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 6.8–7.0. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile vitamin solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile vitamin solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanogenium organophilum.

Methanogenium Medium Composition per liter: NaCl............................................................................................ 18.0g NaHCO3 ........................................................................................ 5.0g MgSO4·7H2O .............................................................................. 3.45g MgCl2·7H2O ............................................................................... 2.75g Yeast extract.................................................................................. 2.0g Pancreatic digest of casein............................................................ 2.0g Sodium acetate.............................................................................. 1.0g L-Cysteine·HCl·H2O ..................................................................... 0.5g Na2S·9H2O.................................................................................... 0.5g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution SL-6 ..................................................10.0mL Wolfe’s vitamin solution..........................................................10.0mL pH 6.8 ± 0.2 at 25°C

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Methanogenium Medium

Trace Elements Solution SL-6: Composition per liter:

Sludge Fluid: Composition per 100.0mL:

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O................................................................................. 0.01g

Sludge ....................................................................................100.0mL Yeast extract.................................................................................. 0.4g

Preparation of Sludge Fluid: To 100.0mL of sludge from an an-

Preparation of Trace Elements Solution SL-6: Add components

aerobic digester, add 0.4g of yeast extract. Sparge with 100% N2 for a few minutes. Incubate at 37°C for 24 hr. Centrifuge the sludge at 13,000 × g for 15 min. Decant the clear supernatant solution. Sparge with 100% N2 for a few minutes. Store in screw-capped bottles at room temperature in the dark.

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Fatty Acid Mixture: Composition per 20.0mL:

Wolfe’s Vitamin Solution: Composition per liter:

Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Cyanocobalamin .....................................................................100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except Wolfe’s vitamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Aseptically add sterile Wolfe’s vitamin solution. Aseptically and anaerobically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Methanococcus frisius, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanoplanus limicola.

Methanogenium Medium Composition per liter: NaHCO3 ........................................................................................ 4.0g Sodium acetate .............................................................................. 4.0g Sodium formate............................................................................. 2.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl.............................................................................. 0.5g KH2PO4 ......................................................................................... 0.5g Na2S·9H2O .................................................................................... 0.5g MgSO4·7H2O ............................................................................... 0.4g NaCl .............................................................................................. 0.4g NH4Cl ........................................................................................... 0.4g CaCl2·2H2O................................................................................. 0.05g NiCl2·6H2O .............................................................................. 24.0mg FeSO4·7H2O.............................................................................. 2.0mg Resazurin ................................................................................... 1.0mg Sludge fluid..............................................................................50.0mL Fatty acid mixture ....................................................................20.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.7 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Fatty Acid Mixture: Add components to distilled/ deionized water and bring volume to 20.0mL. Mix thoroughly. Adjust pH to 7.5 with concentrated NaOH.

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Preparation of Medium: Prepare and dispense medium anaerobically under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Methanobacterium species, Methanococcus species, Methanocorpusculum species, Methanoculleus species, Methanogenium species, Methanoplanus species, and Thermotoga species.

Methanogenium olentangyi Medium (DSMZ Medium 287) Composition per liter: NaHCO3 ........................................................................................ 5.0g NH4Cl ........................................................................................... 2.7g Na-acetate ..................................................................................... 2.5g NaCl............................................................................................ 0.61g K2HPO4......................................................................................... 0.3g KH2PO4......................................................................................... 0.3g (NH4)2SO4 .................................................................................... 0.3g CaCl2·H2O .................................................................................. 0.14g MgSO4·7H2O .............................................................................. 0.13g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL

Methanogenium thermophilicum Medium

Vitamin solution.......................................................................10.0mL L-Cysteine-HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g FeSO4·7H2O.................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 7.0 with KOH.

Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

1105

at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL Lcysteine-HCl·H2O solution, 10.0mL Na2S·9H2O solution, and 10.0mL vitamin solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Methanoculleus olentangyi=Methanogenium olentangyi.

Methanogenium species Medium Composition per liter: NaCl.............................................................................................. 5.0g NaHCO3 ........................................................................................ 4.0g Sodium formate ............................................................................ 2.0g Sodium acetate.............................................................................. 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl ............................................................................. 0.5g KH2PO4......................................................................................... 0.5g Na2S·9H2O.................................................................................... 0.5g MgSO4·7H2O ............................................................................... 0.4g NH4Cl ........................................................................................... 0.4g CaCl2·2H2O ................................................................................ 0.05g FeSO4·7H2O............................................................................... 2.0mg Resazurin ................................................................................... 1.0mg NiCl2·6H2O................................................................................ 2.5mg Trace elements solution SL-10 ..................................................1.0mL pH 6.7 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

L-Cysteine-HCl·H2O

Solution: Composition per 10.0mL:

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

L-Cysteine-HCl·H2O ..................................................................... 0.3g

Preparation of Medium: Prepare and dispense medium anaerobi-

Preparation of L-Cysteine-HCl·H2O Solution: Add L-cyste-

cally under 80% H2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2 gas atmosphere. Add components, except L-cysteineHCl·H2O solution, Na2S·9H2O solution, and vitamin solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Adjust pH to 6.9. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and maintenance of Methanoculleus marisnigri and Methanogenium species.

Methanogenium thermophilicum Medium Composition per liter: NaCl............................................................................................ 11.7g Pancreatic digest of casein............................................................ 6.0g Sodium formate ............................................................................ 5.0g MgCl2·6H2O ................................................................................. 4.0g NaHCO3 ........................................................................................ 4.0g Yeast extract.................................................................................. 2.0g L-Cysteine·HCl ............................................................................. 0.5g Na2S·9H2O.................................................................................. 0.25g K2HPO4....................................................................................... 0.14g

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Methanohalobium Medium Preparation of Trimethylamine·HCl Solution: Add trimethyl-

KH2PO4 ....................................................................................... 0.14g CaCl2·2H2O............................................................................... 0.075g Resazurin ................................................................................... 1.0mg Vitamin solution.......................................................................10.0mL

amine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin Solution: Composition per liter:

Na2S·9H2O Solution: Composition per 10.0mL:

Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Na2S·9H2O.................................................................................... 0.5g

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

Preparation of Medium: Add components, except L-cysteine·HCl and Na2S·9H2O, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Bubble the solution with a stream of oxygen-free 80% N2 + 20% CO2 for 20 min. Adjust the pH to 7.2 with 2M KOH. Add the L-cysteine·HCl and Na2S·9H2O. Mix thoroughly. Tube the medium under oxygen-free 80% N2 + 20% CO2 in either serum tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. After sterilization, the medium may form a precipitate. Allow it to cool and mix thoroughly to bring the precipitate back into solution.

Use: For the cultivation and maintenance of Methanogenium thermophilicum.

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Yeast Extract Solution: Composition per 5.0mL: Yeast extract............................................................................. 50.0mg

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

Methanohalobium Medium Composition per liter:

deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

Trace Elements Solution SL-10: Composition per liter:

NaCl .......................................................................................... 250.0g MgSO4·7H2O ................................................................................ 4.0g CaCl2·2H2O................................................................................. 0.33g KCl.............................................................................................. 0.33g KH2PO4 ....................................................................................... 0.33g MgCl2·6H2O................................................................................ 0.33g NH4Cl ......................................................................................... 0.33g Resazurin ................................................................................... 1.0mg NaHCO3 solution ...................................................................100.0mL Trimethylamine·HCl solution ..................................................20.0mL Na2S·9H2O solution .................................................................10.0mL Yeast extract solution ...............................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution SL-10 ................................................10.0mL Na2CO3 solution...................................................................... variable pH 7.4 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL:

Na2CO3 Solution: Composition per 10.0mL:

NaHCO3 ........................................................................................ 5.0g

Na2CO3 ......................................................................................... 0.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deion-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except NaHCO3 solution, trimethylamine·HCl solution, Na2S·9H2O solution, yeast extract solution, vitamin solution, trace elements solution SL-10, and Na2CO3 solution, to

Methanohalophilus halophilus Medium

distilled/deionized water and bring volume to 840.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 100.0mL of sterile NaHCO3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na2S·9H2O solution, 10.0mL of sterile yeast extract solution, 10.0mL of sterile vitamin solution, and 10.0mL of sterile trace elements solution SL-10. Mix thoroughly. Add a sufficient quantity of sterile Na2CO3 solution to bring the pH to 7.4.

Use: For the cultivation and maintenance of Methanohalobium evestigatus.

Methanohalophilus euhalobius Medium Composition per 1010.0mL: NaCl ............................................................................................ 60.0g MgCl2·6H2O.................................................................................. 2.4g Yeast extract.................................................................................. 2.0g KCl................................................................................................ 1.0g Disodium EDTA ........................................................................... 0.5g NH4Cl ........................................................................................... 0.5g KH2PO4 ......................................................................................... 0.4g Resazurin ................................................................................... 0.5mg Trimethylamine solution ..........................................................50.0mL NaHCO3 solution .....................................................................40.0mL CaCl2 solution ..........................................................................20.0mL Na2S·9H2O solution .................................................................15.0mL L-Cysteine·HCl solution...........................................................15.0mL Wolfe’s mineral solution ..........................................................10.0mL Wolfe’s vitamin solution ............................................................5.0mL pH 6.8–7.0 at 25°C

Trimethylamine Solution: Composition per 50.0mL: Trimethylamine·HCl ................................................................... 10.0g

Preparation of Trimethylamine Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

NaHCO3 Solution: Composition per 40.0mL: NaHCO3 ........................................................................................ 4.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 40.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

CaCl2 Solution: Composition per 20.0mL: CaCl2·2H2O................................................................................... 2.0g

Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/de-

ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 20.0mL:

1107

L-Cysteine·HCl

Solution: Composition per 20.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.6g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoCl2·6H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture. Add components, except trimethylamine solution, NaHCO3 solution, CaCl2 solution solution, L-cysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 860.0mL. Mix thoroughly. Adjust pH to 6.8–7.0. Sparge with 80% N2 + 20% CO2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 50.0mL of sterile trimethylamine solution, 40.0mL of sterile NaHCO3 solution, 20.0mL of sterile CaCl2 solution, 15.0mL of sterile L-cysteine·HCl solution, and 15.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Methanohalophilus euhalobius.

Na2S·9H2O .................................................................................... 0.6g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl. © 2010 by Taylor and Francis Group, LLC

Methanohalophilus halophilus Medium Composition per 1011.0mL: NaCl............................................................................................ 70.0g KCl.............................................................................................. 0.33g

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Methanohalophilus mahii Medium

KH2PO4 ....................................................................................... 0.33g NH4Cl ......................................................................................... 0.33g Yeast extract................................................................................ 0.05g K2SO4 .......................................................................................... 0.01g Resazurin ................................................................................... 1.0mg Methylamine·HCl solution.......................................................20.0mL MgCl2/CaCl2 solution ..............................................................10.0mL NaHCO3 solution .....................................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution...........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

MgCl2/CaCl2 Solution: Composition per 10.0mL: MgCl2·6H2O................................................................................ 0.33g CaCl2·2H2O................................................................................. 0.33g

Preparation of MgCl2/CaCl2 Solution: Add components to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

Methylamine·HCl Solution: Composition per 20.0mL: Methylamine·HCl ......................................................................... 5.0g

Preparation of Methylamine·HCl Solution: Add methylamine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.2g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except MgCl2/CaCl2 solution, NaHCO3 solution, vitamin solution, methylamine·HCl solution, Lcysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 840.0mL. Mix thoroughly. Adjust pH to 6.9–7.0. Sparge with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile MgCl2/CaCl2 solution, 10.0mL of sterile NaHCO3 solution, 10.0mL of sterile vitamin solution, 20.0mL of sterile methylamine·HCl solution, 10.0mL of sterile L-cysteine·HCl solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Use: For the cultivation and maintenance of Methanohalophilus halophilus.

Methanohalophilus mahii Medium Composition per 1010.0mL: NaCl............................................................................................ 87.0g MgCl2·6H2O ................................................................................. 6.0g NaHCO3 ........................................................................................ 4.0g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g KCl................................................................................................ 1.5g NH4Cl ........................................................................................... 1.0g CaCl2·2H2O .................................................................................. 0.4g K2HPO4·3H2O .............................................................................. 0.4g Coenzyme M (mercaptoethane sulfonic acid) .............................. 0.2g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol ....................................................................................4.0mL pH 7.0 ± 0.2 at 25°C

Methanohalophilus zhilinae Medium

CaCl2·2H2O................................................................................... 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O ............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except Na2S·9H2O solution, to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Use: For the cultivation and maintenance of Methanohalophilus mahii.

Methanohalophilus Medium See: Halomethanococcus Medium

Methanohalophilus oregonense Medium Composition per 1010.0mL: NaCl ............................................................................................ 29.0g Trimethylamine·HCl ..................................................................... 2.0g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g MgCl2·6H2O.................................................................................. 1.7g KCl................................................................................................ 1.5g NH4Cl ........................................................................................... 1.0g Resazurin ...................................................................................... 1.0g Coenzyme M (mercaptoethane sulfonic acid) .......................................................................... 0.5g K2HPO4·3H2O............................................................................... 0.4g Na2S·9H2O solution .................................................................10.0mL Trace elements solution ...........................................................10.0mL Na2CO3 solution...................................................................... variable pH 8.5 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g © 2010 by Taylor and Francis Group, LLC

1109

Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 1.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Adjust pH to 8.5 with a sufficient quantity of sterile Na2CO3 solution (approximately 0.1mL per 10.0mL of medium).

Use: For the cultivation and maintenance of Methanohalophilus oregonense.

Methanohalophilus zhilinae Medium Composition per liter: NaCl............................................................................................ 40.0g MgCl2·6H2O ................................................................................. 3.5g MgSO4·7H2O ................................................................................ 3.0g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g KCl................................................................................................ 1.0g NH4Cl ........................................................................................... 1.0g L-Cysteine·HCl ............................................................................. 0.5g K2HPO4......................................................................................... 0.4g Resazurin ................................................................................... 1.0mg Na2SeO3·5H2O........................................................................... 0.1mg Trimethylamine·HCl solution ..................................................20.0mL NaHCO3 solution .....................................................................10.0mL Na2CO3 solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution .............................................................5.0mL pH 9.2 ± 0.2 at 25°C

Trimethylamine·HCl Solution: Composition per 20.0mL: Trimethylamine·HCl ..................................................................... 2.0g

Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

1110

Methanol Agar

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 0.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 2.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O ............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Medium: Prepare and dispense medium under 100% N2. Add components, except trimethylamine·HCl solution, NaHCO3 solution, Na2CO3 solution, L-cysteine·HCl, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Allow to cool to room temperature while sparging with 100% N2. Add L-cysteine·HCl. Distribute medium into tubes and seal. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile NaHCO3 solution, 10.0mL of sterile Na2CO3 solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Check pH.

Use: For the cultivation and maintenance of Methanohalophilus zhilinae.

Methanol Agar (LMG Medium 72) Composition per liter: Agar ............................................................................................ 15.0g K2HPO4 ......................................................................................... 1.2g © 2010 by Taylor and Francis Group, LLC

KH2PO4....................................................................................... 0.62g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g NaCl.............................................................................................. 0.1g CaCl2·2H2O ............................................................................. 34.0mg FeCl3·6H2O ................................................................................ 1.0mg Methanol ..................................................................................10.0mL Trace elements solution .............................................................1.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: ZnSO4·7H2O ............................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 10.0mg H3BO3 ...................................................................................... 10.0mg MnSO4·H2O ............................................................................... 7.0mg CuSO4·5H2O .............................................................................. 5.0mg CoCl2·6H2O ............................................................................... 5.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except methanol, to 990.0mL distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptically add 10.0mL sterile methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Methylobacterium spp., Methylobacillus glycogens, and Methylophilus methylotrophus.

Methanol Ammonium Salts Medium Composition per liter: MgSO4·7H2O ................................................................................ 1.0g NH4Cl ........................................................................................... 0.5g Na2HPO4 ..................................................................................... 0.33g KH2PO4....................................................................................... 0.26g CaCl2 ............................................................................................. 0.2g Ferrous EDTA............................................................................ 5.0mg Na2MoO4·2H2O ......................................................................... 2.0mg FeSO4·7H2O............................................................................ 500.0μg ZnSO4·7H2O ........................................................................... 400.0μg EDTA ...................................................................................... 250.0μg CoCl2·6H2O .............................................................................. 50.0μg MnCl2·4H2O ............................................................................. 20.0μg H3BO4 ....................................................................................... 15.0μg NiCl2·6H2O ............................................................................... 10.0μg Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add Na2HPO4 and KH2PO4 to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. In a separate container, add remaining components, except methanol, to distilled/deionized water and bring volume to 895.0mL. Mix thoroughly. Autoclave both solutions for 15 min at 15 psi pressure–121°C. Cool to room temperature. Filter sterilize methanol. Aseptically add the sterile phosphate solution and the sterile methanol to the cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the maintenance and cultivation of Methylomonas methylotrophus.

Methanol Salts Medium

Methanol Medium (ATCC Medium 436) Composition per liter: Agar ............................................................................................ 15.0g K2HPO4 ......................................................................................... 7.0g (NH4)2SO4 ..................................................................................... 3.0g KH2PO4 ......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g Yeast extract.................................................................................. 0.2g FeSO4·7H2O................................................................................ 0.01g MnSO4·H2O ............................................................................... 8.0mg Biotin ..........................................................................................0.2μg Thiamine·HCl .............................................................................0.2μg Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

1111

cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Achromobacter methanolophila, Methylobacterium rhodesianum, Pseudomonas insueta, and Pseudomonas polysaccharogenes.

Methanol Medium with 1% Peptone Composition per liter:

distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Filter sterilize methanol. Aseptically add the sterile methanol to the cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Agar ............................................................................................ 15.0g Peptone ....................................................................................... 10.0g K2HPO4......................................................................................... 7.0g (NH4)2SO4 .................................................................................... 3.0g KH2PO4......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g Yeast extract.................................................................................. 0.2g FeSO4·7H2O................................................................................ 0.01g MnSO4·H2O ............................................................................... 8.0mg Biotin .......................................................................................... 0.2μg Thiamine·HCl ............................................................................. 0.2μg Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Ancylobacter species,

Preparation of Medium: Add components, except methanol, to dis-

Preparation of Medium: Add components, except methanol, to

Methanomonas methylovora, and Methylobacterium species.

Methanol Medium (ATCC Medium 1096) Composition per liter: NH4NO3 ...................................................................................... 0.75g FeCl3 ......................................................................................... 0.743g Methanol ..................................................................................... 0.45g MgSO4 ........................................................................................ 0.09g KH2PO4 ..................................................................................... 0.044g Na2MoO4·2H2O ......................................................................... 0.1mg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Prepare and dispense medium under 97% N2 + 3% H2. Add components, except methanol, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Filter sterilize methanol. Aseptically add the sterile methanol to the cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Ancylobacter species, Methylobacterium species, and Methanomonas methylovora.

Methanol Medium for Achromobacter Composition per liter: NH4Cl ........................................................................................... 5.0g KH2PO4 ......................................................................................... 2.0g NaCl .............................................................................................. 0.5g MgSO4 .......................................................................................... 0.2g Yeast extract.................................................................................. 0.2g FeSO4 ......................................................................................... 2.0mg MnCl2 ......................................................................................... 2.0mg Methanol ..................................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Filter sterilize methanol. Aseptically add the sterile methanol to the © 2010 by Taylor and Francis Group, LLC

tilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Filter sterilize methanol. Aseptically add the sterile methanol to the cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Methylobacterium species.

Methanol Mineral Salts Medium Composition per liter: Agar ............................................................................................ 20.0g (NH4)2SO4 .................................................................................... 2.0g NH4Cl ........................................................................................... 2.0g (NH4)2HPO4.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g KH2PO4......................................................................................... 1.0g K2HPO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g Fe2SO4·7H2O .............................................................................. 0.01g CaCl2·2H2O ................................................................................ 0.01g Methanol ..................................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Filter sterilize methanol. Aseptically add the sterile methanol to the cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile Petri dishes or sterile tubes. Use: For the cultivation and maintenance of Pseudomonas viscogena.

Methanol Salts Medium Composition per liter: Agar, noble.................................................................................. 20.0g K2HPO4......................................................................................... 1.2g KH2PO4....................................................................................... 0.62g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g

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Methanol Urea Mineral Salts Medium

NaCl .............................................................................................. 0.1g CaCl2·6H2O................................................................................. 0.05g ZnSO4·7H2O .............................................................................70.0μg H3BO3 .......................................................................................10.0μg MnSO4·5H2O ............................................................................10.0μg Na2MoO4·2H2O ........................................................................10.0μg CoCl2·6H2O ................................................................................5.0μg CuSO4·5H2O ...............................................................................5.0μg FeCl3·6H2O ................................................................................ 1.0mg Methanol ....................................................................................1.0mL pH 7.0 ± 0.2 at 25°C

KH2PO4......................................................................................... 1.4g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ............................................................................. 30.0mg Ferric citrate............................................................................. 30.0mg MnCl2·4H2O .............................................................................. 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.4mg Methanol ..................................................................................10.0mL pH 7.1 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 1.0mL of filter-sterilized methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Hyphomicrobium species, Methylobacillus glycogenes, Methylobacterium extorquens, Methylobacterium fujisawaense, Methylobacterium mesophilicum, Methylobacterium organophilum, Methylobacterium radiotolerans, Methylobacterium rhodesianum, Methylobacterium rhodinum, Methylobacterium species, Methylobacterium zatmanii, Methylomonas species, Methylophilus methylotrophus, Methylovorus glucosotrophus, Paracoccus species, Protaminobacter thiaminophaga, and Pseudomonas insueta.

Methanol Urea Mineral Salts Medium Composition per liter: Na2HPO4 ..................................................................................... 2.13g KH2PO4 ....................................................................................... 1.36g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O................................................................................. 0.01g FeSO4·7H2O............................................................................... 5.0mg MnSO4·5H2O ............................................................................. 2.5mg NaMoO4·2H2O........................................................................... 2.5mg Urea solution............................................................................30.0mL Methanol ....................................................................................5.0mL

Urea Solution: Composition per 50.0mL:

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of methanol-utilizing bacteria.

Methanol-Utilizing Bacteria Medium B Composition per liter: Na2HPO4 ....................................................................................... 3.0g (NH4)2SO4 .................................................................................... 3.0g KH2PO4......................................................................................... 1.4g Yeast extract.................................................................................. 0.2g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ............................................................................. 30.0mg Ferric citrate............................................................................. 30.0mg MnCl2·4H2O .............................................................................. 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg Methanol ..................................................................................10.0mL pH 7.1 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of methanol-utilizing bacteria.

Methanol-Utilizing Bacteria Medium D Composition per liter:

Preparation of Medium: Add components, except urea solution and methanol, to distilled/deionized water and bring volume to 965.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 30.0mL of sterile urea solution and 5.0mL of sterile methanol. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Na2HPO4 ....................................................................................... 3.0g (NH4)2SO4 .................................................................................... 3.0g KH2PO4......................................................................................... 1.4g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ............................................................................. 30.0mg Ferric citrate............................................................................. 30.0mg MnCl2·4H2O .............................................................................. 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.4mg Methanol ..................................................................................10.0mL pH 9.0 ± 0.2 at 25°C

Use: For the cultivation of Hyphomicrobium vulgare.

Preparation of Medium: Add components to distilled/deionized

Urea............................................................................................. 10.0g

Preparation of Urea Solution: Add urea to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize. Preparation of Methanol: Filter sterilize 5.0mL of methanol using a teflon filter.

water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically adjust pH to 9.0 with filter-sterilized 10% NaCO3 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Paracoccus alcaliphilus.

Methanolobus Medium

Methanol-Utilizing Bacteria Medium D Composition per liter: Na2HPO4 ....................................................................................... 3.0g (NH4)2SO4 ..................................................................................... 3.0g KH2PO4 ......................................................................................... 1.4g Yeast extract.................................................................................. 0.2g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O.............................................................................. 30.0mg Ferric citrate ............................................................................. 30.0mg MnCl2·4H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg Methanol ..................................................................................10.0mL pH 9.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically adjust pH to 9.0 with filter-sterilized 10% NaCO3 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Paracoccus alcaliphilus.

Methanol-Utilizing Bacteria Medium E Composition per 1010.0mL: NaCl ............................................................................................ 30.0g Na2HPO4 ....................................................................................... 3.0g (NH4)2SO4 ..................................................................................... 3.0g KH2PO4 ......................................................................................... 1.4g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O.............................................................................. 30.0mg Ferric citrate ............................................................................. 30.0mg MnCl2·4H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.4mg Methanol ..................................................................................10.0mL Vitamin B12 ...............................................................................10.0μg pH 9.0 ± 0.2 at 25°C

1113

Methanol ..................................................................................10.0mL Vitamin B12 ............................................................................... 10.0μg pH 9.0 ± 0.2 at 25°C

Use: For the cultivation of Methylophaga marina and Methylophaga thalassica.

Methanolobus Medium Composition per liter: NaCl............................................................................................ 18.0g NaHCO3 ........................................................................................ 5.0g MgSO4·7H2O .............................................................................. 3.45g MgCl2·6H2O ............................................................................... 2.75g L-Cysteine·HCl·H2O ..................................................................... 0.5g Na2S·9H2O.................................................................................... 0.5g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·6H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Wolfe’s mineral solution..........................................................10.0mL Wolfe’s vitamin solution..........................................................10.0mL Methanol ....................................................................................5.0mL pH 6.5 ± 0.2 at 25°C

Wolfe’s Mineral Solution: Composition per liter:

MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Use: For the cultivation of Methylophaga marina and Methylophaga

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic

Preparation of Medium: Add components to distilled/deionized

thalassica.

Methanol-Utilizing Bacteria Medium E Composition per 1010.0mL: NaCl ............................................................................................ 30.0g Na2HPO4 ....................................................................................... 3.0g (NH4)2SO4 ..................................................................................... 3.0g KH2PO4 ......................................................................................... 1.4g Yeast extract.................................................................................. 0.2g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O.............................................................................. 30.0mg Ferric citrate ............................................................................. 30.0mg MnCl2·4H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg © 2010 by Taylor and Francis Group, LLC

acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L.

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Cyanocobalamin ..................................................................... 100.0μg

1114

Methanolobus 2 Medium

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except methanol and Wolfe’s vitamin solution, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Aseptically add sterile Wolfe’s vitamin solution and sterile methanol. Adjust pH to 6.5. Aseptically and anaerobically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Methanolobus siciliae and Methanolobus tindarius.

Methanolobus 2 Medium

Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

Methanol Solution: Composition per 5.0mL: Methanol ....................................................................................5.0mL

Composition per liter:

Preparation of Methanol Solution: Sparge 5.0mL of methanol

NaCl ............................................................................................ 18.0g NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O.................................................................................. 4.0g MgSO4·7H2O ............................................................................. 3.45g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate .............................................................................. 1.0g L-Cysteine·HCl.............................................................................. 0.5g Na2S·9H2O .................................................................................... 0.4g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O................................................................................. 0.14g K2HPO4 ....................................................................................... 0.14g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Na2WoO4·2H2O ....................................................................... 0.03mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol solution ......................................................................5.0mL pH 6.8–7.0 at 25°C

with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except vitamin solution, to distilled/ deionized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 6.9–7.0. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile vitamin solution and 10.0mL of sterile methanol to each liter of medium or, using a syringe, inject the appropriate amount of sterile vitamin solution and sterile methanol into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanolobus siciliae, Methanolobus vulcani, and Methanosarcina species.

Methanolobus taylorii Medium (DSMZ Medium 490a) Composition per liter: NaCl............................................................................................ 29.0g Yeast extract.................................................................................. 2.0g Trypticase™ peptone .................................................................... 2.0g MgCl2·6H2O ................................................................................. 1.7g KCl................................................................................................ 1.5g NH4Cl ........................................................................................... 1.0g Resazurin ...................................................................................... 1.0g Mercaptoethanesulfonic acid (coenzyme M)................................ 0.5g K2HPO4·3H2O .............................................................................. 0.4g Trace elements solution ...........................................................10.0mL Trimethylamine·HCl solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL NaHCO3 solution .....................................................................10.0mL pH 8.5 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g

Methanomicrobium mobile Medium

CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Sparge with N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.

Trimethylamine·HCl Solution: Composition per 10.0mL: Trimethylamine·HCl ..................................................................... 2.0g

Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Should be prepared freshly. Preparation of Medium: Prepare and dispense medium under an oxygen-free 100% N2 gas mixture. Add components, except trimethylamine·HCl solution, NaHCO3 solution, and Na2S·9H2O solution, to 970.0mL distilled/deionized water. Mix thoroughly. Sparge for 20 min with 100% N2. Adjust pH to 8.0. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically and anaerobically add 10.0mL sterile trimethylamine·HCl solution, and 10.0mL Na2S·9H2O solution. Adjust pH to 8.5 using sterile NaHCO3 solution, approximately 10.0mL per liter medium. Aseptically and anaerobically distribute to sterile tubes or flasks under 100% N2.

Use: For the cultivation of Methanolobus taylorii.

Methanomicrobium Medium Composition per liter: NaHCO3 ........................................................................................ 2.0g Yeast extract.................................................................................. 1.0g Pancreatic digest of casein ............................................................ 1.0g NaCl .............................................................................................. 0.6g L-Cysteine·HCl·H2O...................................................................... 0.5g Na2S·9H2O .................................................................................... 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g (NH4)2SO4 ..................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.13g CaCl2·2H2O................................................................................ 8.0mg FeSO4·7H2O............................................................................... 2.0mg Rumen fluid, clarified ............................................................300.0mL Fatty acid mixture ....................................................................20.0mL Wolfe’s mineral solution ..........................................................10.0mL © 2010 by Taylor and Francis Group, LLC

1115

Wolfe’s vitamin solution..........................................................10.0mL Resazurin (0.1% solution) .........................................................1.0mL pH 6.5 ± 0.2 at 25°C

Fatty Acid Mixture: Composition per liter: Valeric acid ................................................................................0.7mL Isovaleric acid............................................................................0.7mL α-Methylbutyric acid.................................................................0.5mL Isobutyric acid ...........................................................................0.5mL

Preparation of Fatty Acid Mixture: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Cyanocobalamin ..................................................................... 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Distribute into tubes or flasks under 80% N2 + 20% CO2. Cap with rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Methanomicrobium mobile.

Methanomicrobium mobile Medium (DSMZ Medium 161) Composition per liter: NaHCO3 ........................................................................................ 2.0g Yeast extract.................................................................................. 1.0g Trypticase™.................................................................................. 1.0g NaCl.............................................................................................. 0.6g

1116

Methanomicrobium paynteri Medium

Cysteine-HCl·H2O ........................................................................ 0.5g Na2S·9H2O .................................................................................... 0.5g K2HPO4 ......................................................................................... 0.3g KH2PO4 ......................................................................................... 0.3g (NH4)2SO4 ..................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.13g CaCl2·2H2O............................................................................... 0.008g FeSO4·7H2O.............................................................................. 0.002g Resazurin .................................................................................. 0.001g Rumen fluid, clarified ............................................................300.0mL Fatty acid mixture ....................................................................20.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% H2 + 20% CO2 gas mixture. Add components, except vitamin solution, to 990.0mL distilled/deionized water. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Adjust pH to 6.5 with concentrated NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C while sparging with 80% H2 + 20% CO2. Aseptically and anaerobically add 10.0mL sterile vitamin solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks. Alternately the medium can be distributed to tubes under anaerobic conditions and autoclaved in tubes prior to addition of vitamin solution. Appropriate amounts of the vitamin solution can then be added to each tube by syringes. After inoculation, pressurize culture vessels to 2 bar 80% H2 + 20% CO2 overpressure.

Use: For the cultivation of Methanomicrobium mobile.

Methanomicrobium paynteri Medium Composition per liter:

3-(N-morpholino) propane sulfonic acid (MOPS buffer) .............................................. 20.93g NaCl............................................................................................ 6.31g NaHCO3 ........................................................................................ 5.0g Sodium acetate·3H2O.................................................................. 4.14g MgSO4·7H2O .............................................................................. 3.40g MgCl2·2H2O ............................................................................... 2.75g NH4Cl ........................................................................................... 1.5g KCl.............................................................................................. 0.34g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·6H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0 ± 0.2 at 25°C

Vitamin Solution: Composition per liter:

Trace Elements Solution: Composition per liter:

Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

MgSO4·7H2O ............................................................................... 3.0g NaCl.............................................................................................. 1.0g Nitrilotriacetic acid ....................................................................... 1.5g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O ............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Fatty Acid Mixture: Composition per 20.0mL: Valeric acid ................................................................................... 0.5g Isovaleric acid ............................................................................... 0.5g α-Methylbutyric acid .................................................................... 0.5g Isobutyric acid............................................................................... 0.5g Distilled water..........................................................................20.0mL

Methanomicrococcus Medium

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except MOPS buffer, L-cysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Sparge with O2-free 80% H2 + 20% CO2. In a separate flask, add MOPS buffer to distilled/deionized water and bring volume to 90.0mL. Adjust pH to 7.0 with 2M KOH. Add the 90.0mL of MOPS solution to the 890.0mL of medium and continue sparging with O2-free 80% H2 + 20% CO2. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile L-cysteine·HCl solution and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile L-cysteine·HCl solution and sterile Na2S·9H2O solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanomicrobium paynteri.

Methanomicrococcus Medium (DSMZ Medium 120b) Composition 1112.0mL: NaCl ............................................................................................ 2.25g Yeast extract.................................................................................. 2.0g Casitone ........................................................................................ 2.0g NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g K2HPO4 ..................................................................................... 0.348g CaCl2·2H2O................................................................................. 0.25g KH2PO4 ..................................................................................... 0.227g FeSO4·7H2O.............................................................................. 0.002g Resazurin .................................................................................. 0.001g NaHCO3 solution .....................................................................40.0mL Methanol solution ....................................................................20.0mL L-Cysteine-HCl·H2O solution ..................................................15.0mL Na2S·9H2O solution .................................................................15.0mL Na-acetate solution ..................................................................10.0mL Vitamin solution.......................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

1117

Coenzyme M solution................................................................1.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Vitamin Solution: Composition per liter: Pyridoxamine-HCl................................................................... 10.0mg Pantothenic acid......................................................................... 5.0mg Riboflavin .................................................................................. 5.0mg Alpha-lipoic acid ....................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C. Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................... 3.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl. Methanol Solution: Composition per 100.0mL: Methanol ..................................................................................50.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na-Acetate Solution: Composition per 100.0mL: Na-acetate ................................................................................... 25.0g

1118

Methanomonas Autotrophic Medium

Preparation of Na-Acetate Solution: Add Na-acetate to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Coenzyme M Solution: Composition per 10.0mL: Coenzyme M................................................................................. 0.1g

Preparation of Coenzyme M Solution: Add coenzyme M to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. L-Cysteine

Solution: Composition per 100.0mL:

L-Cysteine·HCl·H2O ..................................................................... 3.0g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, Na-acetate solution, coenzyme M solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL vitamin solution, 40.0mL NaHCO3 solution, 20.0mL methanol solution, 15.0mL L-cysteineHCl·H2O solution, 15.0mL Na2S·9H2O solution, 10.0mL Na-acetate solution, 1.0mL coenzyme M solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Methanomicrococcus blatticola.

Methanomonas Autotrophic Medium Composition per liter: NaNO3........................................................................................... 2.0g Na2HPO4 ..................................................................................... 0.21g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.09g KCl.............................................................................................. 0.04g CaCl2 ......................................................................................... 0.015g FeSO4·7H2O............................................................................... 1.0mg ZnSO4·7H2O .............................................................................. 0.3mg CuSO4·5H2O .............................................................................. 0.2mg H3BO3 ...................................................................................... 0.06mg MnSO4·H2O ............................................................................. 0.03mg MoO3...................................................................................... 0.015mg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until dissolved. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the autotrophic cultivation of Methanomonas species.

Methanopyrus Medium Composition per liter: NaCl .......................................................................................... 11.80g MgCl2·6H2O................................................................................ 4.50g MgSO4·7H2O .............................................................................. 1.75g Na2SO4 ........................................................................................ 0.81g © 2010 by Taylor and Francis Group, LLC

CaCl2·2H2O ................................................................................ 0.78g KCl.............................................................................................. 0.30g KH2PO4....................................................................................... 0.09g K2HPO4·3H2O ............................................................................ 0.07g Na2WO4·2H2O ........................................................................... 2.0mg (NH4)2Fe (SO4)2·6H2O .............................................................. 2.0mg (NH4)2Ni(SO4)2 ......................................................................... 2.0mg Resazurin ................................................................................... 0.2mg Marine trace elements..............................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL NaHCO3 solution .....................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.5 ± 0.2 at 25°C

Marine Trace Elements: Composition per liter: NaBr.............................................................................................. 4.0g SrCl2·6H2O ................................................................................... 1.8g H3BO3 ........................................................................................... 1.3g Sodium silicate....................................................................... 100.0mg NaF .......................................................................................... 60.0mg KNO3 ....................................................................................... 40.0mg KI ............................................................................................. 1.25mg Na2HPO4·3H2O........................................................................ 0.25mg

Preparation of Marine Trace Elements: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Methanosarcina acetivorans Medium Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% H2 + 20% CO2.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 0.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except vitamin solution, NaHCO3 solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile vitamin solution, 10.0mL of sterile NaHCO3 solution, and 10.0mL of sterile Na2S·9H2O solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile vitamin solution, sterile NaHCO3 solution, and sterile Na2S·9H2O solution into individual tubes containing medium. Check that final pH of medium is 6.5.

Use: For the cultivation and maintenance of Methanopyrus kandleri.

Methanosarcina acetivorans Medium Composition per liter: NaCl ............................................................................................ 23.4g Agar ............................................................................................ 10.0g MgSO4 .......................................................................................... 6.3g Na2CO3 ......................................................................................... 5.0g Trimethylamine·HCl ..................................................................... 3.0g Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g KCl................................................................................................ 0.8g Na2HPO4 ....................................................................................... 0.6g L-Cysteine·HCl·H2O.................................................................... 0.25g Na2S·9H2O .................................................................................. 0.25g CaCl2·2H2O................................................................................. 0.14g Resazurin ................................................................................... 1.0mg Wolfe’s mineral solution ..........................................................10.0mL pH 7.2 ± 0.2 at 25°C

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g © 2010 by Taylor and Francis Group, LLC

1119

H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Medium: Add components, except Na2S·9H2O, to glass-distilled water and bring volume to 990.0mL. Mix thoroughly. Methanol or methylamine·HCl may be substituted for the trimethylamine·HCl at a concentration of 50 mM. Heat gently and bring to boiling. Adjust pH to 7.2 with 6N HCl. Autoclave for 5 min at 10 psi pressure–115°C. Cool to 50°C under 80% N2 + 20% CO2. If a large precipitate is present, add a small amount of HCl and mix thoroughly. Add Na2S·9H2O. Mix thoroughly. Distribute into tubes under 80% N2 + 20% CO2. Cap with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. A precipitate will form but resolubilizes as the medium cools. Invert tubes as they are cooling to facilitate resolubilization. Allow tubes to cool in a slanted position. Use: For the cultivation and maintenance of Methanococcoides methylutens and Methanosarcina acetivorans.

Methanosarcina acetivorans Medium Composition per 1010.0mL: NaCl............................................................................................ 23.4g MgSO4·7H2O .............................................................................. 9.45g Na2CO3 ......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g CaCl2·2H2O ................................................................................ 0.14g KCl................................................................................................ 0.1g Na2HPO4 ....................................................................................... 0.6g L-Cysteine·HCl·H2O ................................................................ 0.025g Na2S·9H2O ............................................................................... 0.025g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ..................................................................... 12.8g FeCl3·6H2O................................................................................. 1.35g NaCl.............................................................................................. 1.0g NiCl2·6H2O................................................................................. 0.12g MnCl2·4H2O ................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g ZnCl2 ............................................................................................. 0.1g Na2SeO3·5H2O.......................................................................... 0.026g CuCl2·2H2O .............................................................................. 0.025g CoCl2·6H2O .............................................................................. 0.024g Na2MoO4·2H2O ........................................................................ 0.024g H3BO3 ......................................................................................... 0.01g

1120

Methanosarcina barkeri Medium

Sparge under 80% N2 + 20% CO2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Methanosarcina acetivorans.

Methanosarcina barkeri Medium Composition per liter:

Preparation of L-Cysteine·HCl·H2O Solution: Bring 100.0mL of distilled/deionized water to boiling. Cool to room temperature while sparging with 100% N2. Add L-cysteine·HCl·H2O to the 100.0mL of anaerobic water. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Autoclave for 20 min at 15 psi pressure–121°C.

NaHCO3 ........................................................................................ 2.5g NaCl ............................................................................................ 0.46g Yeast extract................................................................................ 0.24g KH2PO4 ....................................................................................... 0.23g K2HPO4 ....................................................................................... 0.23g (NH4)2SO4 ................................................................................... 0.23g MgCl2·6H2O................................................................................ 0.09g CaCl2·2H2O................................................................................. 0.06g NiCl2·6H2O ................................................................................ 2.0mg Methanol ..................................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Resazurin (0.01% solution)........................................................1.0mL

tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N2. Dissolve 1 pellet of NaOH in the anaerobic water. Weigh out a little more than 2.5g of Na2S·9H2O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on paper towels or filter paper. Weigh out 2.5g of washed Na2S·9H2O crystals. Add to the 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Pressurize to 60kPa with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

Preparation of Methanol: Filter sterilize 10.0mL of methanol.

Preparation of Medium: Prepare and dispense medium under 80%

Trace Elements Solution: Composition per liter:

N2 + 20% CO2. Add components, except methanol, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Anaerobically distribute 9.7mL volumes into anaerobic tubes. Autoclave for 20 min at 15 psi pressure–121°C. Aseptically and anaerobically add 0.1mL of sterile methanol, 0.1mL of sterile L-cysteine·HCl·H2O solution, and 0.1mL of sterile Na2S·9H2O solution to each tube. Mix thoroughly.

MgSO4·5H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g NaS4·SeO3·5H2O........................................................................... 0.3g NiCl2·6H2O ................................................................................. 0.25g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·7H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g KAI(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Mix thoroughly. Add distilled/deionized water to 1.0L. Adjust pH to 6.8. Vitamin Solution: Composition per liter: Calcium DL-pantothenate.............................................................. 5.0g Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid ................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. L-Cysteine·HCl·H2O Solution: Composition per 100.0mL: L-Cysteine·HCl·H2O ..................................................................... 2.5g © 2010 by Taylor and Francis Group, LLC

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

Use: For the cultivation of Methanosarcina barkeri.

Methanosarcina BCYT Medium (DSMZ Medium 318) Composition per liter: KHCO3.......................................................................................... 2.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.6g Yeast extract.................................................................................. 0.5g Trypticase™.................................................................................. 0.5g KH2PO4......................................................................................... 0.3g MgCl2·6H2O ................................................................................. 0.1g CaCl2·2H2O ................................................................................ 0.08g Resazurin ................................................................................... 1.0mg Cysteine solution .....................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Methanosarcina DPB Medium

Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Preparation of Medium: Add components, except methanol, vitamin solution, cysteine solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Sparge with 100% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C while sparging with 100% CO2. Aseptically and anaerobically add 5.0mL filter sterilized methanol, 10.0mL vitamin solution, 10.0mL cysteine solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

1121

L-Cysteine·HCl·H2O ................................................................... 0.27g

CaCl2·2H2O ................................................................................ 0.06g Fe(NH4)2(SO4)2 .......................................................................... 0.01g Antifoam C ..............................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution...................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Source: Antifoam C is available from Sigma Chemical Co. Trace Elements Solution: Composition per liter: MgSO4·5H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g NaS4·SeO3·5H2O .......................................................................... 0.3g NiCl2·6H2O................................................................................. 0.25g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·7H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g KAI(SO4)2·12H2O ...................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Mix thoroughly. Add distilled/deionized water to 1.0L. Adjust pH to 6.8. Vitamin Solution: Composition per liter: Calcium DL-pantothenate.............................................................. 5.0g Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O.................................................................................. 25.0g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

Composition per 1001.0mL:

Sodium acetate·3H2O.................................................................... 4.1g NH4Cl ........................................................................................... 1.4g K2HPO4 ......................................................................................... 1.3g KH2PO4 ......................................................................................... 1.3g MgSO4 .......................................................................................... 0.5g NaCl .............................................................................................. 0.5g

Use: For the cultivation of Methanosarcina spp.

Methanosarcina DPB Medium

1122

Methanosarcina frisia Medium

dium. Repeat the addition of 1.0mL of sterile Na2S·9H2O solution per liter of medium every 48 hr during growth.

Use: For the cultivation of Methanosarcina species.

Methanosarcina frisia Medium Composition per liter: NaCl ............................................................................................ 18.0g NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O.................................................................................. 4.0g MgSO4·7H2O ............................................................................. 3.45g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate .............................................................................. 1.0g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O................................................................................. 0.14g K2HPO4 ....................................................................................... 0.14g L-Cysteine·HCl.............................................................................. 0.5g Na2S·9H2O .................................................................................... 0.5g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol solution ......................................................................2.0mL pH 6.8–7.0 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g NiCl2·6H2O ............................................................................... 0.025g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic

Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2.

Methanol Solution: Composition per 10.0mL: Methanol ..................................................................................10.0mL

Preparation of Methanol Solution: Sparge 10.0mL of methanol with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except methanol solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Anaerobically distribute into tubes or flasks fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically add 10.0mL of sterile methanol solution to each liter of medium or, using a syringe, inject the appropriate amount of sterile methanol solution into individual tubes containing medium.

Use: For the cultivation and maintenance of Methanosarcina frisia.

Methanosarcina Mass-Culturing Medium Composition per liter: NaCl............................................................................................ 0.58g NH4Cl ......................................................................................... 0.53g K2HPO4....................................................................................... 0.44g KH2PO4....................................................................................... 0.35g MgCl2·6H2O ............................................................................... 0.04g CaCl2·2H2O ................................................................................ 0.03g Resazurin .................................................................................. 0.001g Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol ....................................................................................8.0mL Na2S·9H2O solution ...................................................................8.0mL

Trace Elements Solution: Composition per liter: Sodium nitrilotriacetate .............................................................. 1.67g CoCl2·6H2O ................................................................................ 0.18g FeCl2·4H2O ................................................................................. 0.14g MnCl2·4H2O ............................................................................... 0.09g NiCl2·6H2O ................................................................................. 0.09g ZnCl2 ........................................................................................... 0.09g CaCl2 ........................................................................................... 0.06g Na2MoO4·2H2O ........................................................................ 0.046g CuSO4 ....................................................................................... 0.045g

acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Adjust pH to 7.0. Add distilled/deionized water to 1.0L.

Vitamin Solution: Composition per liter:

Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg © 2010 by Taylor and Francis Group, LLC

Calcium DL-pantothenate.............................................................. 5.0g Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg

Methanosarcina mazei Medium Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Methanol: Filter sterilize 10.0mL of methanol. Na2S·9H2O Solution: Composition per 100.0mL: NaOH ....................................................................................... 1 pellet Na2S·9H2O .................................................................................... 2.5g

Preparation of Na2S·9H2O Solution: Bring 100.0mL of dis-

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except methanol, vitamin solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 974.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to room temperature while sparging with 80% H2 + 20% CO2. Anaerobically distribute 9.7mL volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 0.1mL of sterile vitamin solution, 0.08mL of sterile methanol, and 0.08mL of sterile Na2S·9H2O solution to each tube. Mix thoroughly.

Use: For the cultivation of Methanosarcina species.

Methanosarcina mazei Alpha Basal Medium Composition per liter: NaHCO3 ........................................................................................ 4.4g Pancreatic digest of casein ............................................................ 2.0g Yeast extract.................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g Na2S·6H2O .................................................................................... 0.5g K2HPO4 ......................................................................................... 0.4g Sodium acetate·3H2O.................................................................. 0.27g MgCl2·6H2O................................................................................ 0.08g CaCl2·2H2O................................................................................. 0.04g CoCl2·6H2O ............................................................................... 1.5mg FeSO4·7H2O............................................................................... 1.0mg MnCl2·4H2O............................................................................... 1.0mg Resazurin ................................................................................... 1.0mg H3BO4 ........................................................................................ 0.1mg NaMoO4·2H2O........................................................................... 0.1mg ZnCl2 .......................................................................................... 0.1mg pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Prepare and dispense medium under 70% N2 + 30% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Sparge with 70% N2 + 30% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically distribute into sterile tubes or bottles.

1123

Methanosarcina mazei Medium (DSMZ Medium 120) Composition 1112.0mL: NaCl............................................................................................ 2.25g Yeast extract.................................................................................. 2.0g Casitone ........................................................................................ 2.0g NaHCO3 ...................................................................................... 0.85g NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g K2HPO4..................................................................................... 0.348g CaCl2·2H2O ................................................................................ 0.25g KH2PO4..................................................................................... 0.227g FeSO4·7H2O.............................................................................. 0.002g Resazurin .................................................................................. 0.001g Methanol solution ....................................................................20.0mL L-Cysteine-HCl·H2O solution ..................................................15.0mL Na2S·9H2O solution .................................................................15.0mL Na-acetate solution ..................................................................10.0mL Vitamin solution.......................................................................10.0mL NaHCO3 solution.....................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.5–6.8 at 25°C Vitamin Solution: Composition per liter: Pyridoxamine-HCl................................................................... 10.0mg Pantothenic acid......................................................................... 5.0mg Riboflavin .................................................................................. 5.0mg Alpha-lipoic acid ....................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12................................................................................ 0.1mg

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge

1124

Methanosarcina Medium

with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 3.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

L-Cysteine-sulfide reducing agent ...........................................20.0mL Wolfe’s vitamin solution..........................................................10.0mL Methanol ..................................................................................10.0mL Resazurin (0.025% solution) .....................................................4.0mL Trace elements solution SL-6 ....................................................3.0mL pH 6.8 ± 0.2 at 25°C

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.

NaHCO3 Solution: Composition per 20.0mL:

Methanol Solution: Composition per 100.0mL:

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

NaHCO3 ................................................................................. 850.0mg

Methanol ..................................................................................50.0mL

ionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Gas with 100% CO2 for 20 min.

Preparation of Methanol Solution: Add methanol to distilled/de-

L-Cysteine-Sulfide

ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na-Acetate Solution: Composition per 100.0mL: Na-acetate ................................................................................... 25.0g

Solution: Composition per 100.0mL:

L-Cysteine·HCl·H2O ..................................................................... 3.0g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, Na-acetate solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL vitamin solution, 10.0mL NaHCO3 solution, 20.0mL methanol solution, 15.0mL L-cysteine-HCl·H2O solution, 15.0mL Na2S·9H2O solution, 10.0mL Na-acetate solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure.

Use: For the cultivation of Mathanosarcina mazei.

Methanosarcina Medium Composition per liter: Agar ............................................................................................ 20.0g NaCl ............................................................................................ 2.25g Yeast extract.................................................................................. 2.0g Pancreatic digest of casein ............................................................ 2.0g NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g K2HPO4 ....................................................................................... 0.35g CaCl2·2H2O................................................................................. 0.25g KH2PO4 ....................................................................................... 0.23g FeSO4·7H2O............................................................................... 2.0mg NaHCO3 solution .....................................................................20.0mL © 2010 by Taylor and Francis Group, LLC

Reducing Agent: Composition per 20.0mL:

L-Cysteine·HCl·H2O...................................................................... 0.3g Na2S·9H2O.................................................................................... 0.3g

Preparation of L-Cysteine-Sulfide Reducing Agent: Add

L-

Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Thiamine·HCl ............................................................................ 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Calcium pantothenate ................................................................ 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Thioctic acid .............................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Cyanocobalamin ..................................................................... 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Preparation of Medium: Add components, except NaHCO3 solu-

tion, L-cysteine-sulfide reducing agent, Wolfe’s vitamin solution, and methanol, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool under 80% N2 + 20% CO2. Aseptically and anaerobically add the sterile NaHCO3 solution, the sterile L-cysteine-sulfide reducing agent, the sterile Wolfe’s vitamin solution, and filter-sterilized methanol. Mix

Methanosarcina Medium

1125

thoroughly. Adjust pH to 6.8. Aseptically and anaerobically distribute into sterile tubes or flasks.

L-Cysteine·HCl·H2O

Use: For the cultivation and maintenance of Bifidobacterium asteroides,

L-Cysteine·HCl·H2O ................................................................... 0.25g

Methanosarcina barkeri, Methanosarcina species, and Methanosarcina vacuolata.

Methanosarcina Medium (BCYT) Composition per liter: KHCO3 .......................................................................................... 2.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g Pancreatic digest of casein ............................................................ 0.5g Yeast extract.................................................................................. 0.5g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.1g CaCl2·2H2O................................................................................. 0.08g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ..................................................................... 12.8g FeCl3·6H2O ................................................................................. 1.35g NaCl .............................................................................................. 1.0g NiCl2·6H2O ................................................................................. 0.12g MnCl2·4H2O.................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g ZnCl2 ............................................................................................. 0.1g Na2SeO3·5H2O.......................................................................... 0.026g CuCl2·2H2O .............................................................................. 0.025g CoCl2·6H2O .............................................................................. 0.024g Na2MoO4·2H2O ........................................................................ 0.024g H3BO3 ......................................................................................... 0.01g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL of distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2. © 2010 by Taylor and Francis Group, LLC

Solution: Composition per 10.0mL:

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except vitamin solution, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Sparge under 80% N2 + 20% CO2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 20.0mL of sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl·H2O solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile screw-cappped bottles under 80% N2 + 20% CO2.

Use: For the cultivation and maintenance of Methanosarcina mazei and Methanosarcina thermophila.

Methanosarcina Medium Composition per liter: NaCl............................................................................................ 2.25g Pancreatic digest of casein............................................................ 2.0g Yeast extract.................................................................................. 2.0g NaHCO3...................................................................................... 0.85g MgSO4·7H2O................................................................................ 0.5g NH4Cl ........................................................................................... 0.5g K2HPO4 .................................................................................... 0.348g CaCl2·2H2O ................................................................................ 0.25g KH2PO4 .................................................................................... 0.227g FeSO4·7H2O .............................................................................. 2.0mg Resazurin ................................................................................... 1.0mg Methanol solution ....................................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.5–6.8 at 25°C

Methanol Solution: Composition per 10.0mL: Methanol ....................................................................................5.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 10.0mL. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

1126

Methanosarcina MP Medium

Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Trace elements solution ...........................................................10.0mL Na2CO3 solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2.

Mineral Solution: Composition per liter:

L-Cysteine·HCl·H2O

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cyste-

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2.

Preparation of Medium: Add components, except methanol solution, vitamin solution, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Sparge under 80% N2 + 20% CO2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile methanol, 10.0mL of sterile vitamin solution, 10.0mL of sterile Lcysteine·HCl·H2O solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile screw-cappped bottles under 80% N2 + 20% CO2.

Use: For the cultivation and maintenance of Methanosarcina species.

Methanosarcina MP Medium Composition per 1015.0mL: NH4Cl ........................................................................................... 1.0g Yeast extract.................................................................................. 1.0g L-Cysteine·HCl.............................................................................. 0.5g K2HPO4 ......................................................................................... 0.4g MgCl2·6H2O.................................................................................. 0.2g Resazurin ................................................................................... 1.0mg Mineral solution .......................................................................50.0mL © 2010 by Taylor and Francis Group, LLC

NaCl............................................................................................ 12.0g KH2PO4......................................................................................... 6.0g (NH4)2SO4 .................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.6g CaCl2·2H2O ................................................................................ 0.16g

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 1.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................. 0.25g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Add components, except L-cysteine·HCl, Na2CO3 solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 80% N2 + 20% CO2. Add L-cysteine·HCl. Mix thoroughly. Adjust pH to 6.8–7.0. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile Na2CO3 solution and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Use: For the cultivation and maintenance of Methanosarcina species.

Methanosarcina semesiae Medium

Methanosarcina Nitrogen-Fixing Medium

1127

NaH2PO4 ..................................................................................... 1.38g K2HPO4 ....................................................................................... 0.34g MgCl2·2H2O.................................................................................. 0.1g Yeast extract.................................................................................. 0.1g CaCl2·2H2O................................................................................. 0.05g Resazurin .................................................................................. 0.001g Trace elements solution ...........................................................10.0mL Methanol ....................................................................................2.0mL Na2S·9H2O solution ...................................................................2.0mL NaHCO3 solution .......................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Solution E ................................................................................30.0mL Na2S·9H2O solution .................................................................15.0mL Solution D................................................................................10.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol solution ......................................................................5.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Seven vitamin solution ..............................................................1.0mL Dithionite solution .....................................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter:

Solution A: Composition per 950.0mL:

Composition per 1005.0mL:

Nitrilotriacetic acid ....................................................................... 4.5g FeCl2·7H2O ................................................................................... 0.4g H3BO3 ......................................................................................... 0.19g CoCl2·6H2O ................................................................................ 0.17g MnCl2·4H2O.................................................................................. 0.1g ZnCl2 ............................................................................................. 0.1g NiCl2·6H2O ................................................................................. 0.02g Na2MoO4·6H2O .......................................................................... 0.01g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Adjust pH to 7.0 with 10N NaOH. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Methanol: Sparge 2-propanol with 100% N2. Filter

sterilize.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 2.0g NaOH ............................................................................................ 0.1g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O and

NaOH to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.0g

KH2PO4......................................................................................... 1.4g NH4Cl ........................................................................................... 0.5g MgCl2·6H2O ................................................................................. 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 1.0mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize.

Solution B: Composition per liter: FeCl2·4H2O................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

lution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Solution C: Composition per liter:

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deion-

ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Prepare and dispense medium under

Preparation of Solution C: Add components to distilled/deionized

100% N2. Add components, except methanol, Na2S·9H2O solution, and NaHCO3 solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 2.0mL of sterile methanol, 2.0mL of sterile Na2S·9H2O solution, and 2.0mL of sterile NaHCO3 solution. Mix thoroughly. Adjust pH to 7.0 by adding sterile anaerobic 10N NaOH. Aseptically and anaerobically distribute into sterile tubes or bottles.

water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Use: For the cultivation of nitrogen-fixing Methanosarcina species.

Methanosarcina semesiae Medium Composition per 1214.0mL: Solution A ..............................................................................950.0mL NaHCO3 solution .....................................................................50.0mL © 2010 by Taylor and Francis Group, LLC

Solution D: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

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Methanosarcina Thermophilic Medium

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution E: Add NaHCO3 to distilled/deionized water and bring volume to 150.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize.

Solution F: Composition per 10.0mL: Na2-maleate................................................................................... 1.6g

Preparation of Solution F: Add Na2-maleate to distilled/deionized water and bring volume to 150.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Solution G: Composition per 10.0mL: Na2S·9H2O .................................................................................. 0.25g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. Methanol Solution: Composition per 100.0mL: Methanol ..................................................................................50.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

Na2S·9H2O Solution: Composition per 20.0mL:

Trace Elements Solution: Composition per liter:

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O ............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg © 2010 by Taylor and Francis Group, LLC

Na2S·9H2O.................................................................................... 0.6g

Dithionite Solution Composition per 10.0mL: Na-dithionite ............................................................................... 0.25g

Preparation of Dithionite Solution: Add Na-dithionite to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2. Combine component solutions A-G; do not add NaHCO3 solution, Na2S·9H2O solution, trace elements solution, vitamin solution, methanol solution, seven vitamin solution, and dithionite solution. Mix thoroughly. Flush medium with 80% N2 + 20% CO2 for 5 min. Distribute into serum bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. For every 10.0mL medium aseptically and anaerobically add from sterile anaerobic solution: 0.5mL NaHCO3 solution, 0.1mL trace elements solution, 0.1mL vitamin solution, 0.01mL seven vitamin solution, 0.01mL dithionite solution, 0.15mL Na2S·9H2O solution, and 0.05mL methanol solution. Final pH should be 7.0. Use: For the cultivation of Methanosarcina semesiae.

Methanosarcina Thermophilic Medium

Casitone ........................................................................................ 2.0g NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g K2HPO4 ..................................................................................... 0.348g CaCl2·2H2O................................................................................. 0.25g KH2PO4 ..................................................................................... 0.227g FeSO4·7H2O.............................................................................. 0.002g Resazurin .................................................................................. 0.001g Rumen fluid, clarified ..............................................................50.0mL Methanol solution ....................................................................10.0mL Vitamin solution.......................................................................10.0mL NaHCO3 solution .....................................................................10.0mL L-Cysteine solution ..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.5–6.8 at 25°C

Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filter sterilize. Methanol Solution: Composition per 100.0mL: Methanol ..................................................................................50.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 100.0mL: Na2S·9H2O .................................................................................... 3.0g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 8.5g

1129

Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL vitamin solution, 10.0mL NaHCO3 solution, 10.0mL methanol solution, 10.0mL L-cysteine-HCl·H2O solution, 10.0mL Na2S·9H2O solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure. Alternately, the medium without vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, and trace elements solution SL-10 can be distributed to tubes anaerobically prior to autoclaving. After autoclaving in tubes the appropriate volumes of the individual solutions can be injected through the stoppers so that the final concentrations of the medium are achieved.

Use: For the cultivation of Methanosarcina thermophila.

Methanosarcina Thermophilic Medium (DSMZ Medium 164) Composition per 1051.0mL: NaCl............................................................................................ 2.25g Yeast extract.................................................................................. 2.0g Casitone ........................................................................................ 2.0g NH4Cl ........................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g K2HPO4..................................................................................... 0.348g CaCl2·2H2O ................................................................................ 0.25g KH2PO4..................................................................................... 0.227g FeSO4·7H2O.............................................................................. 0.002g Resazurin .................................................................................. 0.001g Sludge fluid..............................................................................50.0mL Methanol solution ....................................................................10.0mL Vitamin solution.......................................................................10.0mL NaHCO3 solution .....................................................................10.0mL L-Cysteine solution ..................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 6.5–6.8 at 25°C

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Methanosphaera Medium 1

Preparation of Sludge Fluid: Add yeast extract to sludge from an anaerobic digester. Gas with nitrogen gas for a few minutes. Incubate at 37°C for 24 hr. Centrifuge the sludge at 13,000 x g. Autoclave for 15 min at 15 psi pressure–121°C. Place the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at room temperature in the dark.

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 100.0mL:

Preparation of Trace Elements Solution SL-10: Add 1.5g of FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/ deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. L-Cysteine

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, and trace elements solution SL-10, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL vitamin solution, 10.0mL NaHCO3 solution, 10.0mL methanol solution, 10.0mL L-cysteine-HCl·H2O solution, 10.0mL Na2S·9H2O solution, and 1.0mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles. After inoculation, flush and repressurize the gas head space of culture bottles with sterile 80% H2 + 20% CO2 to 1 bar overpressure. Alternately, the medium without vitamin solution, NaHCO3 solution, methanol solution, L-cysteine-HCl·H2O solution, Na2S·9H2O solution, and trace elements solution SL-10 can be distributed to tubes anaerobically prior to autoclaving. After autoclaving in tubes the appropriate volumes of the individual solutions can be injected through the stoppers so that the final concentrations of the medium are achieved.

Use: For the cultivation of Methanosarcina thermophila.

Methanosphaera Medium 1

Na2S·9H2O .................................................................................... 3.0g

Composition per liter:

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

KH2PO4......................................................................................... 2.8g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g NH4Cl ........................................................................................... 1.0g K2HPO4......................................................................................... 0.6g NaCl.............................................................................................. 0.6g Sodium acetate.............................................................................. 0.5g Sodium formate ............................................................................ 0.5g (NH4)2SO4 .................................................................................... 0.3g MgSO4·7H2O .............................................................................. 0.15g CaCl2·2H2O .............................................................................. 0.076g FeSO4·7H2O............................................................................... 3.0mg Na2SeO4 ..................................................................................... 1.9mg Resazurin ................................................................................... 1.0mg NiCl2·6H2O ................................................................................ 0.7mg Rumen fluid, clarified............................................................100.0mL Trace elements solution ...........................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol solution ......................................................................5.0mL Vitamin solution.........................................................................1.0mL pH 6.5 ± 0.2 at 25°C

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl.

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 8.5g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared freshly. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g H3BO3 .................................................................................... 300.0mg CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ....................................................................7.7mL © 2010 by Taylor and Francis Group, LLC

Methanosphaera Medium II

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.375g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Methanol Solution: Composition per 10.0mL: Methanol ..................................................................................10.0mL

Preparation of Methanol Solution: Sparge 10.0mL of methanol with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin Solution: Composition per 10.0mL: Calcium-D-pantothenate .......................................................... 20.0mg Nicotinamide............................................................................ 20.0mg Pyridoxine·HCl ........................................................................ 20.0mg Riboflavin ................................................................................ 20.0mg Thiamine·HCl .......................................................................... 20.0mg Biotin ....................................................................................... 10.0mg p-Aminobenzoic acid................................................................. 1.0mg Folic acid.................................................................................... 0.5mg Vitamin B12 ................................................................................ 0.2mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2. Preparation of Medium: Add components, except methanol solution, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 975.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% N2. Autoclave for 15 min © 2010 by Taylor and Francis Group, LLC

1131

at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile L-cysteine·HCl·H2O solution, 10.0mL of sterile Na2S·9H2O solution, and 5.0mL of sterile methanol solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile screw-cappped bottles under 100% N2.

Use: For the cultivation and maintenance of Methanosphaera stadtmanae.

Methanosphaera Medium II Composition per 1005.5mL: NaHCO3 ........................................................................................ 3.0g Sodium acetate·3H2O.................................................................... 3.0g Trypticase™.................................................................................. 1.0g Yeast extract.................................................................................. 1.0g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg NiCl2·6H2O................................................................................ 0.2mg Na2SeO3·5H2O (1mM)............................................................... 0.1mg Mineral solution 1....................................................................40.0mL Mineral solution 2....................................................................40.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Methanol solution ......................................................................5.0mL pH 7.0 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4......................................................................................... 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Mineral Solution 2: Composition per liter: NaCl............................................................................................ 12.0g KH2PO4......................................................................................... 6.0g (NH4)2SO4 .................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.6g CaCl2·2H2O ................................................................................ 0.16g

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH.

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Methanosphaerula Medium

Add remaining components. Adjust pH to 7.0. Add distilled/deionized water to 1.0L.

Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. L-Cysteine·HCl·H2O Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cyste-

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Methanol Solution: Composition per 10.0mL: Methanol ..................................................................................10.0mL

Preparation of Methanol Solution: Sparge 10.0mL of methanol with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. L-cyste-

ine·HCl·H2O solution, Na2S·9H2O solution, and methanol solution, to distilled/deionized water and bring volume to 975.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile Lcysteine·HCl·H2O solution, 10.0mL of sterile Na2S·9H2O solution, and 5.0mL of sterile methanol solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile screw-cappped bottles under 100% N2.

Use: For the cultivation and maintenance of Methanosphaera cuniculi.

Methanosphaerula Medium (DSMZ Medium 1094) Composition per liter: Solution A .....................................................................................1.0L Solution B ..................................................................................1.0mL Solution C ..............................................................................12.55mL Solution D ................................................................................20.0mL Solution E ................................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

Solution A: Composition per liter: NH4 Cl ..................................................................................... 26.8mg KH2PO4 ................................................................................... 13.6mg KCl ............................................................................................ 1.5mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Solution B: Composition per liter: Na2EDTA ............................................................................... 37.229g KAl(SO4)2·12H2O..................................................................... 3.446g CaCl2·2H2O .............................................................................. 2.336g MgSO4·7H2O ............................................................................ 1.556g FeCl2·4H2O ............................................................................... 1.344g CoCl2·6H2O ................................................................................ 0.24g ZnCl2 ......................................................................................... 0.075g MnSO4·2H2O ............................................................................ 0.026g NiCl2·6H2O ............................................................................... 0.024g NaMoO4·2H2O.......................................................................... 0.024g H3BO3 ....................................................................................... 0.019g CuSO4 ....................................................................................... 0.009g

Preparation of Solution B: Add components to distilled/deionized

Na2S·9H2O .................................................................................... 0.3g

Preparation of Medium: Add components, except

Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution H................................................................................10.0mL pH 6.9 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly.

Solution C: Composition per 12.55mL: TRIS-HCl, 1.0M, pH 8.0 ...........................................................7.2mL Sodium nitrilotriacetic acid, 0.5M .............................................4.8mL TiCl3, 15% in HCl....................................................................0.55mL

Preparation of Solution C: Combine solutions. Mix thoroughly. Filter sterilize. Store under N2 gas atmosphere. Solution D: Composition per 20.0mL: MES ......................................................................................... 3.905g NaOH ........................................................................................... 0.8g

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Adjust to pH 7.5 with NaOH. Filter sterilize. Store under N2 gas atmosphere.

Solution E: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Solution E: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Store under N2 gas atmosphere.

Methanospirillum hungatei JMA Medium

1133

Solution F: Composition per 10.0mL:

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/de-

2-Mercaptoethanesulfonic acid (coenzyme M) ....................... 0.082g

Mineral Solution 2: Composition per liter:

Preparation of Solution F: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with N2 gas. Autoclave for 15 min at 15 psi pressure–121°C. Store under N2 gas atmosphere.

ionized water and bring volume to 1.0L. Mix thoroughly.

Solution G: Composition per 10.0mL:

KH2PO4....................................................................................... 24.0g (NH4)2SO4 .................................................................................... 6.0g MgSO4·7H2O ................................................................................ 1.2g CaCl2·2H2O ................................................................................ 0.79g NaCl............................................................................................ 0.59g

Na-acetate .................................................................................. 0.68g

Preparation of Mineral Solution 2: Add components to distilled/

Preparation of Solution G: Add Na-acetate to distilled/deionized

deionized water and bring volume to 1.0L. Mix thoroughly.

water and bring volume to 10.0mL. Mix thoroughly. Sparge with N2 gas. Autoclave for 15 min at 15 psi pressure–121°C. Store under N2 gas atmosphere.

Wolfe’s Mineral Solution: Composition per liter:

Solution H: Composition per 10.0mL: Na2S·9H2O ............................................................................... 0.96mg

Preparation of Solution H: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with N2 gas. Filter sterilize. Store in sealed vial under N2 gas atmosphere. Preparation of Medium: Add 1.0mL of solution B to 1.0L of solution A. Gently heat and bring to boiling. Boil for 1 min. Cool to room temperature under 80% N2 + 20% CO2 gas mixture. Dispense under same gas atmosphere into culture vessels. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add solutions C to H in the sequence indicated and let medium equilibrate overnight. After inoculation pressurize vials to 1 bar overpressure with 80% H2 + 20% CO2 gas mixture. The final pH should be 5.7. Use: For the cultivation of Methanosphaerula spp.

Methanospirillum hungatei JMA Medium Composition per liter: Sodium acetate .............................................................................. 2.5g NH4Cl ........................................................................................... 1.9g Mineral solution 1....................................................................75.0mL Mineral solution 2....................................................................75.0mL Wolfe’s mineral solution ..........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL FeSO4·6H2O solution.................................................................6.0mL Na2CO3 solution.........................................................................4.8mL Resazurin ...................................................................................1.0mL NiCl2·6H2O solution ..................................................................0.1mL

FeSO4·6H2O Solution: Composition per 100.0mL:

MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoCl2·6H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Vitamin Solution: Composition per liter: Calcium DL-pantothenate.............................................................. 5.0g Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.4mg

FeSO4·6H2O.................................................................................. 0.2g

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Preparation of FeSO4·6H2O Solution: Add FeSO4·6H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Na2CO3 Solution: Composition per liter:

NiCl2·6H2O Solution: Composition per liter:

Na2CO3 ....................................................................................... 0.84g

NiCl2·6H2O ................................................................................... 1.2g

Preparation of NiCl2·6H2O Solution: Add NiCl2·6H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure– 121°C.

Mineral Solution 1: Composition per liter:

Na2S·9H2O Solution: Composition per 10.0mL:

K2HPO4 ....................................................................................... 39.0g

Na2S·9H2O.................................................................................... 0.2g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

1134

Methanospirillum hungatei Medium

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Before use, neutralize to pH 7.0 with sterile HCl. L-Cysteine·HCl·H2O

Solution: Composition per 10.0mL:

L-Cysteine·HCl·H2O ................................................................... 0.22g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cyste-

ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Trace Metal Solution: Composition per liter: K2HPO4·3H2O .............................................................................. 9.0g K2HPO4......................................................................................... 6.0g NH4Cl ........................................................................................... 5.0g MgCl2·6H2O ................................................................................. 1.0g CaCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Metal Solution: Add components to dis-

Preparation of Medium: Prepare and dispense medium under 80%

tilled/deionized water and bring volume to 1.0L. Mix thoroughly.

N2 + 20% CO2. Add components, except Na2CO3 solution, Lcysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 975.2mL. Mix thoroughly. Gently heat and bring to boiling. Cool while sparging with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 4.8mL of sterile Na2CO3 soltuion, 10.0mL of sterile Lcysteine·HCl solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Preparation of Medium: Prepare and distribute medium anaerobi-

Use: For the cultivation of Methanospirillum hungatei.

Methanospirillum hungatei Medium Composition per 100.0mL: Na2CO3 ......................................................................................... 0.4g Sodium formate............................................................................. 0.2g Pancreatic digest of casein ............................................................ 0.2g Yeast extract.................................................................................. 0.2g NaCl ............................................................................................ 0.05g L-Cysteine·HCl·H2O.................................................................... 0.03g K2HPO4 ....................................................................................... 0.02g KH2PO4 ....................................................................................... 0.02g (NH4)2SO4 ................................................................................... 0.02g MgSO4·7H2O ............................................................................. 9.0mg CaCl2·2H2O................................................................................ 6.0mg Resazurin ................................................................................... 0.1mg Na2S·9H2O solution .................................................................10.0mL Vitamin solution.........................................................................1.0mL Trace metal solution...................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Na2S·9H2O Solution: Composition per 10.0.mL: Na2S·9H2O .................................................................................. 0.03g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Vitamin Solution: Composition per 1000.0mL: Pyridoxine·HCl .......................................................................... 1.0mg p-Aminobenzoic acid ................................................................. 0.5mg Calcium-D-pantothenate............................................................. 0.5mg Nicotinic acid ............................................................................. 0.5mg Riboflavin .................................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.5mg Thioctic acid .............................................................................. 0.5mg Biotin ......................................................................................... 0.2mg Folic acid.................................................................................... 0.2mg Vitamin B12 .............................................................................. 0.01mg © 2010 by Taylor and Francis Group, LLC

cally under 80% H2 + 20% CO2. Add components, except Na2S·9H2O solution, to distilled/deionized water and bring volume to 90.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling until resazurin turns colorless, indicating reduction. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or flasks.

Use: For the cultivation of Methanospirillum hungatei.

Methanospirillum hungatei SAM Medium Composition per liter: Na2CO3 ......................................................................................... 2.63 Sodium acetate·3H2O.................................................................... 2.5g (NH4)2SO4 .................................................................................. 0.45g K2HPO4......................................................................................... 0.3g KH2PO4....................................................................................... 0.18g MgSO4·9H2O .............................................................................. 0.12g CaCl2·2H2O ................................................................................ 0.06g NaCl............................................................................................ 0.05g Na2CO3 solution.......................................................................20.0mL Trace minerals solution............................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine/Na2S solution.........................................................10.0mL FeSO4·7H2O solution.................................................................5.0mL Resazurin solution .....................................................................0.2mL pH 7.1 ± 0.2 at 25°C

Na2CO3 Solution: Composition per liter: Na2CO3 ..................................................................................... 100.0g

Preparation of Na2CO3 Solution: Bring 1.0L of distilled/deion-

ized water to boiling. Cool to room temperature while sparging with 80% H2 + 20% CO2. Add Na2CO3. Distribute into serum bottles fitted with butyl rubber stoppers and aluminum seals. Do not grease stoppers. Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber.

Trace Minerals Solution: Composition per liter: Nitrolotriacetic acid ...................................................................... 1.5g MnSO4·2H2O ................................................................................ 0.5g Na2MoO4·2H2O .......................................................................... 0.24g Na2WO4·2H2O .......................................................................... 0.165g Na2SeO3 ...................................................................................... 0.15g CoCl2·6H2O .................................................................................. 0.1g NiCl2·6H2O ................................................................................... 0.1g ZnSO4·7H2O ................................................................................. 0.1g AIK(SO4)2·12H2O ...................................................................... 0.01g

Methanospirillum SK Medium

CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g

Preparation of Trace Minerals Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Mix thoroughly. Add distilled/deionized water to 1.0L. Adjust pH to 7.0. Vitamin Solution: Composition per liter: Vitamin B12 ................................................................................... 0.1g Pyridoxine·HCl ........................................................................ 10.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.4mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. L-Cysteine/Na2S Solution: Composition per liter: L-Cysteine·HCl............................................................................ 1.25g Na2S·7H2O .................................................................................. 1.25g

Preparation of L-Cysteine/Na2S Solution: Gently heat and bring 75.0mL of distilled/deionized water to boiling. Add Lcysteine·HCl. Adjust pH to 10.0 with 3N NaOH. Add Na2S·9H2O. Mix thoroughly. Gently heat and bring to boiling. Cool to room temperature while sparging with 100% N2. Bring volume to 100.0mL with distilled/ deionized water. Gently heat and bring to boiling. Cool to 60°C while sparging with 100% N2. Anaerobically distribute into serum bottles. Autoclave for 15 min at 15 psi pressure–121°C. FeSO4·6H2O Solution: Composition per 100.0mL:

1135

NaHCO3 ........................................................................................ 5.0g MgCl2·6H2O ................................................................................. 4.0g MgSO4·7H2O ............................................................................. 3.45g Trypticase™.................................................................................. 2.0g Yeast extract.................................................................................. 2.0g Sodium acetate.............................................................................. 1.0g L-Cysteine·HCl ............................................................................. 0.5g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4....................................................................................... 0.14g Fe(NH4)2(SO4)2·7H2O ............................................................... 2.0mg Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0–7.4 at 25°C

Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of FeSO4·6H2O Solution: Add FeSO4·6H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Resazurin Solution: Composition per 10.0mL:

Vitamin Solution: Composition per liter:

FeSO4·6H2O.................................................................................. 0.2g

Resazurin ................................................................................. 10.0mg

Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly.

Preparation of Medium: Prepare and dispense medium under 80% H2 + 20% CO2. Add components, except Na2CO3, Na2CO3 solution, and L-cysteine/Na2S solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Continue boiling for 3 min. Cool to 60°C while sparging with 80% H2 + 20% CO2. Add Na2CO3 and L-cysteine/Na2S solution. Mix thoroughly. Anaerobically distribute 10.0mL volumes into anaerobic tubes. Autoclave for 20 min at 15 psi pressure–121°C. Aseptically and anaerobically add 0.2mL of sterile Na2CO3 solution to each tube. Mix thoroughly. Adjust pH to 7.1.

Use: For the cultivation of Methanospirillum hungatei.

Methanospirillum SK Medium

Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Na2S·9H2O Solution: Composition per 10.0mL:

Composition per liter:

Na2S·9H2O.................................................................................... 0.4g

NaCl ............................................................................................ 18.0g Isopropanol ................................................................................... 7.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

1136

Methanothermobacter/Methanobacterium Transduction Agar

Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

NaS Solution: Composition per 10.0mL:

Preparation of Medium: Add components, except Na2S·9H2O so-

Na2S·9H2O.................................................................................... 1.0g

lution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Preparation of NaS Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Adjust pH to 10.0. Sparge with N2. Store anaerobically.

Use: For the cultivation and maintenance of Methanospirillum hun-

Sodium citrate, 0.2M................................................................50.0mL Titanium(III) chloride, 15%.......................................................5.0mL

gatei.

Methanothermobacter/Methanobacterium Transduction Agar (DSMZ Medium 863) Composition per 1016.0mL: Agar ............................................................................................ 15.0g Basal salts solution.................................................................100.0mL Sodium carbonate solution.......................................................24.0mL Titanium (III) citrate solution ..................................................16.0mL Trace elements solution ...........................................................10.0mL Cysteine solution........................................................................5.0mL NaS solution...............................................................................5.0mL pH 7.0 ± 0.2 at 25°C

Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ....................................................................................... 10.6g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Store anaerobically.

Preparation of Basal Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ..................................................................... 9.55g MgCl2·6H2O................................................................................ 4.06g FeCl2·4H2O ................................................................................. 0.98g NaWO4·6H2O............................................................................ 0.264g NiCl2·6H2O ............................................................................... 0.118g Na2Se2O3 ....................................................................................... 0.1g Na2MoO4·4H2O ........................................................................ 0.024g CoCl2·6H2O .............................................................................. 0.023g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 7.0 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly.

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 1.0g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Adjust pH to 10.0. Sparge with 100% N2. Store anaerobically. © 2010 by Taylor and Francis Group, LLC

Titanium (III) Citrate Solution:

Preparation of Titanium (III) Citrate Solution: Mix solutions. Neutralize with sodium carbonate solution. Sparge with N2. Store anaerobically.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components, except titanium citrate solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C. Add 16.0mL titanium citrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute to sterile tubes. Store anaerobically.

Use: For the transduction of Methanobacter spp. and Methanobacterium spp.

Methanothermobacter/Methanobacterium Transduction Medium (DSMZ Medium 863) Composition per liter: Basal salts solution ................................................................100.0mL Sodium carbonate solution ......................................................24.0mL Trace elements solution ...........................................................10.0mL Cysteine solution .......................................................................5.0mL NaS solution...............................................................................5.0mL pH 7.0 ± 0.2 at 25°C

Sodium Carbonate Solution: Composition per 100.0mL: Na2CO3 ....................................................................................... 10.6g

Preparation of Sodium Carbonate Solution: Add Na2CO3 to

distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Store anaerobically.

Basal Salts Solution: KH2PO4....................................................................................... 68.0g NH4Cl ......................................................................................... 21.2g Resazurin ................................................................................. 10.0mg

Preparation of Basal Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Trace Elements Solution: Nitrilotriacetic acid ..................................................................... 9.55g MgCl2·6H2O ............................................................................... 4.06g FeCl2·4H2O ................................................................................. 0.98g NaWO4·6H2O ........................................................................... 0.264g NiCl2·6H2O ............................................................................... 0.118g Na2Se2O3 ...................................................................................... 0.1g Na2MoO4·4H2O ........................................................................ 0.024g CoCl2·6H2O .............................................................................. 0.023g

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH

Methanothermus fervidus Medium

to 7.0 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly.

Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 1.0g

Preparation of NaS Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Adjust pH to 10.0. Sparge with N2. Store anaerobically. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Anaerobically distribute 10.0mL aliquots into serum vials under 80% N2 + 20% CO2 gas atmosphere. Seal vials. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Pressurize to 2 bar with 80% N2 + 20% CO2 gas atmosphere.

Use: For the transduction of Methanobacter spp. and Methanobacterium spp.

Methanothermus fervidus Medium Composition per liter: Na2SO4 .......................................................................................... 3.4g NaHCO3 ........................................................................................ 2.0g Tryticase™.................................................................................... 2.0g Yeast extract.................................................................................. 2.0g L-Cysteine·HCl.............................................................................. 0.5g Na2S·9H2O .................................................................................... 0.5g FeSO4·7H2O............................................................................... 2.0mg Ni(NH4)2(SO4)2.......................................................................... 2.0mg Resazurin ................................................................................... 1.0mg Mineral solution 1....................................................................37.5mL Mineral solution 2....................................................................37.5mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL pH 6.5 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4 ......................................................................................... 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/de-

ionized water and bring volume to 1.0L. Mix thoroughly.

Mineral Solution 2: Composition per liter: NaCl ............................................................................................ 12.0g K2HPO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.4g CaCl2·2H2O................................................................................... 1.6g

1137

NaCl.............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL of distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Preparation of Medium: Add components, except NaHCO3 and

Na2S·9H2O, to distilled/deionized water and bring volume to 1.0L. Adjust pH to 6.5 with 10N H2SO4. Add NaHCO3 and sparge with 80% N2 + 20% CO2 for 15 min. Add Na2S·9H2O. Mix thoroughly. Anaerobically distribute 20.0mL of medium into 100mL alkali-rich soda lime glass bottles. Pressurize to 2 bar with 80% H2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Methanothermus fervidus.

Methanothermus fervidus Medium Composition per liter: Na2SO4 .......................................................................................... 3.4g NaHCO3 ........................................................................................ 2.0g L-Cysteine·HCl ............................................................................. 0.5g Na2S·9H2O.................................................................................... 0.5g FeSO4·7H2O............................................................................... 2.0mg Ni(NH4)2(SO4)2 ......................................................................... 2.0mg Resazurin ................................................................................... 1.0mg Mineral solution 1....................................................................37.5mL Mineral solution 2....................................................................37.5mL Trace elements solution ...........................................................10.0mL pH 6.5 ± 0.2 at 25°C

Mineral Solution 1: Composition per liter: K2HPO4......................................................................................... 6.0g

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Methanothrix Medium

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/de-

Trace Elements Solution: Composition per liter:

Mineral Solution 2: Composition per liter:

Nitrilotriacetic acid ..................................................................... 12.8g FeCl3·6H2O ................................................................................. 1.35g NaCl.............................................................................................. 1.0g NiCl2·6H2O ................................................................................. 0.12g CaCl2·2H2O ................................................................................ 0.10g MnCl2·4H2O ............................................................................... 0.10g ZnCl2 ........................................................................................... 0.10g Na2SeO3·5H2O.......................................................................... 0.026g CuCl2·2H2O .............................................................................. 0.025g CoCl2·6H2O .............................................................................. 0.024g Na2MoO4·2H2O ........................................................................ 0.024g H3BO3 ......................................................................................... 0.01g

ionized water and bring volume to 1.0L. Mix thoroughly.

NaCl ............................................................................................ 12.0g K2HPO4 ......................................................................................... 6.0g (NH4)2SO4 ..................................................................................... 6.0g MgSO4·7H2O ................................................................................ 2.4g CaCl2·2H2O................................................................................... 1.6g

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution: Composition per liter: MgSO4·7H2O ............................................................................... 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·2H2O ............................................................................... 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O................................................................................... 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O ......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Use: For the cultivation and maintenance of Methanothermus sociabilis.

Methanothrix Medium Composition per liter: Sodium acetate .............................................................................. 6.8g KHCO3 .......................................................................................... 4.0g NH4Cl ........................................................................................... 1.0g NaCl .............................................................................................. 0.6g KH2PO4 ......................................................................................... 0.3g MgCl2·6H2O.................................................................................. 0.1g CaCl2·2H2O................................................................................. 0.08g Resazurin ................................................................................... 1.0mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl·H2O solution ..................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL of distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 80% N2 + 20% CO2. L-Cysteine·HCl·H2O Solution: Composition per 10.0mL: L-Cysteine·HCl·H2O ..................................................................... 0.3g

Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C. Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O.................................................................................... 0.3g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except vitamin solution, L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of sterile vitamin solution, 10.0mL of sterile Lcysteine·HCl·H2O solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Check that final pH is 7.0. Use 20% inoculum.

Use: For the cultivation and maintenance of Methanosaeta concilii.

Methylene Blue Milk Medium

Methermicoccus Medium (DSMZ Medium 1084) Composition per liter: NaCl ........................................................................................... 24.0g MgCl2·6H2O................................................................................ 10.2g Yeast extract ................................................................................. 2.0g KCl ............................................................................................. 0.34g NH4Cl ........................................................................................ 0.25g K2HPO4 ........................................................................................ 0.2g Resazurin .................................................................................. 0.5mg Cysteine solution......................................................................10.0mL Sulfide solution ........................................................................10.0mL Bicarbonate solution ................................................................10.0mL Coenzyme M solution ..............................................................10.0mL Methanol ...................................................................................8.0mL Sludge fluid................................................................................5.0mL pH 9.5 ± 0.2 at 25°C

Methanol: Composition per 10.0mL:

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screw-capped vessels under nitrogen gas. The sludge fluid can be stored at room temperature in the dark.

Preparation of Medium: Add components, except bicarbonate, sludge fluid, methanol, coenzyme M, cysteine, and sulfide solutions, to distilled/deionized water and bring volume to 947.0mL. Gently heat and bring to boiling. Boil medium for 1 min. Cool to room temperature under 80% N2 + 20% CO2 gas mixture. Dispense under same gas atmosphere into culture vessels. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature under 80% N2 + 20% CO2 gas mixture. Aseptically add coenzyme M, methanol, sludge fluid, cysteine, and sulfide from sterile anoxic stock solutions. Adjust pH to 6.0–6.5. Use: For the cultivation of Methermicoccus spp. Methyl Red Voges-Proskauer Broth See: MRVP Broth Methyl Red Voges-Proskauer Medium See: MRVP Medium

Methylamine Salts Medium

Methanol .................................................................................10.0mL

Composition per liter:

Preparation of Methanol: Sparge 10.0mL of methanol with 100%

Agar ............................................................................................ 15.0g Methylamine·HCl ....................................................................... 6.75g K2HPO4....................................................................................... 2.12g KH2PO4......................................................................................... 1.0g Solution A..................................................................................5.0mL Solution B ..................................................................................1.0mL pH 7.0 ± 0.2 at 25°C

N2. Filter sterilize.

Coenzyme M Solution: Composition per 10.0mL: Mercaptoethanesulfonic acid (coenzyme M) .............................. 2.5g

Preparation of Coenzyme M: Add coenzyme M to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

B icarbonate Solution: Composition per 10.0mL:

Solution A: Composition per 100.0mL:

NaHCO3 ........................................................................................ 2.5g

MgSO4·7H2O ................................................................................ 2.0g CaCl2·2H2O .................................................................................. 0.2g FeSO4·7H2O.................................................................................. 0.2g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

Preparation of Solution A: Add components to distilled/deionized

deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO2 + 80% H2. Filter sterilize.

Sulfide Solution: Composition per 10.0mL:

water and bring volume to 100.0mL. Mix thoroughly.

Solution B: Composition per 100.0mL:

Na2S·9H2O .................................................................................... 0.5g

MnSO4·7H2O .............................................................................. 0.05g Na2MoO4·2H2O .......................................................................... 0.05g

Preparation of Sulfide Solution: Add Na2S·9H2O to distilled/de-

Preparation of Solution B: Add components to distilled/deionized

ionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to room temperature.

water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized

Cysteine Solution: Composition per 10.0mL:

L-Cysteine-HCl·2H2O ................................................................... 0.3g

Use: For the cultivation and maintenance of Methylobacterium

Preparation of Cysteine Solution: Add L-cysteine-HCl·2H2O to

extorquens and Pseudomonas species.

distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Methylene Blue Milk Medium (MBM Medium) Composition per liter: Skim milk, dehydrated.............................................................. 100.0g Methylene Blue........................................................................... 10.0g pH 6.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 20 min at 10 psi pressure–115°C.

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Methylobacterium Agar

Use: For the cultivation and differentiation of group D streptococci (enterococci) from other Streptococcus species.

Methylobacterium Agar Composition per liter: Agar ............................................................................................ 12.0g KNO3 ............................................................................................ 1.0g Na2HPO4 ..................................................................................... 0.23g MgSO4·7H2O ............................................................................... 0.2g NaH2PO4 ..................................................................................... 0.07g CaCl2·2H2O................................................................................. 0.02g FeSO4·7H2O.............................................................................. 1.0mg ZnSO4·7H2O ............................................................................ 70.0μg H3BO3 .......................................................................................10.0μg MnSO4·5H2O ............................................................................10.0μg MoO3.........................................................................................10.0μg CuSO4·5H2O .............................................................................. 5.0μg Methanol, filter sterilized...........................................................5.0mL pH 6.8 ± 0.2 at 25°C

MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.07g CaCl2·2H2O ................................................................................ 0.02g FeSO4·7H2O............................................................................... 1.0mg ZnSO4·7H2O ............................................................................. 70.0µg MoO3 ........................................................................................ 10.0µg H3BO3 ....................................................................................... 10.0µg MnSO4·5H2O ............................................................................ 10.0µg CuSO4·5H2O ............................................................................... 5.0µg Methanol ....................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Methylobacterium organophilum.

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 995.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 5.0mL of filtersterilized methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylobacterium organophilum.

Methylobacterium Medium (DSMZ Medium 125) Composition per liter: Agar ............................................................................................ 12.0g KNO3 ............................................................................................ 1.0g Na2HPO4 ..................................................................................... 0.23g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.07g CaCl2·2H2O................................................................................. 0.02g FeSO4·7H2O............................................................................... 1.0mg ZnSO4·7H2O .............................................................................70.0µg MoO3.........................................................................................10.0µg H3BO3 .......................................................................................10.0µg MnSO4·5H2O ............................................................................10.0µg CuSO4·5H2O ...............................................................................5.0µg Methanol ..................................................................................15.0mL pH 4.0–5.4 at 25°C

Use: For the cultivation and maintenance of Acidomonas methanolica and Methanomonas methylovora.

Methylobacterium Medium (DSMZ Medium 125) Composition per liter: KNO3 ............................................................................................ 1.0g Na2HPO4 ..................................................................................... 0.23g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.07g CaCl2·2H2O ................................................................................ 0.02g FeSO4·7H2O............................................................................... 1.0mg ZnSO4·7H2O ............................................................................. 70.0µg MoO3 ........................................................................................ 10.0µg H3BO3 ....................................................................................... 10.0µg MnSO4·5H2O ............................................................................ 10.0µg CuSO4·5H2O ............................................................................... 5.0µg Methane/air mixture (4:1)....................................................... variable pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Methylobacterium organophilum.

Methylobacterium Medium Composition per liter: Agar ............................................................................................ 15.0g K2HPO4......................................................................................... 1.2g KH2PO4....................................................................................... 0.62g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g NaCl.............................................................................................. 0.1g CaCl2·2H2O ............................................................................. 34.0mg FeCl3·H2O.................................................................................. 1.0mg Trace elements solution .............................................................1.0mL Methanol, filter sterilized.........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: ZnSO4·7H2O ............................................................................ 70.0mg H3BO3 ...................................................................................... 10.0mg

Methylocella silverstris Medium

Na2MoO4·2H2O ....................................................................... 10.0mg MnSO4·H2O ............................................................................... 7.0mg CoCl2·H2O ................................................................................. 5.0mg CuSO4·5H2O .............................................................................. 5.0mg

should be checked before and after autoclaving. Incubate under atmosphere of 10–30% methane.

Use: For the cultivation of Methylocapsa acidiphila.

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylobacterium species.

Methylobacterium thiocyanatum Medium (DSMZ Medium 805) Composition per liter: Na2HPO4·2H2O............................................................................. 7.9g Glucose ......................................................................................... 4.5g K2HPO4 ......................................................................................... 1.5g KSCN.......................................................................................... 0.25g MgSO4·7H2O ................................................................................ 0.1g Iron sulfate solution ...................................................................1.0mL pH 7.1 ± 0.1 at 25°C

Iron Sulfate Solution: Composition per 10.0mL: FeSO4·7H2O.................................................................................. 0.2g

Preparation of Iron Sulfate Solution: Add FeSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 10 min at 10 psi pressure–115°C.

Use: For the cultivation of Methylobacterium thiocyanatum.

Methylocapsa acidophila Medium (DSMZ Medium 922) Composition per liter: KH2PO4 .................................................................................. 100.0mg MgSO4·7H2O ........................................................................... 50.0mg CaCl2·2H2O.............................................................................. 10.0mg Trace elements ...........................................................................1.0mL pH 4.5-5.8 at 25°C

Trace Elements: Composition per liter: EDTA ............................................................................................ 5.0g FeSO4·7H2O.................................................................................. 2.0g CoCl2·6H2O .................................................................................. 0.2g CuCl2·5H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g Na2MoO4..................................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g

Preparation of Trace Elements: Add components to distilled/de-

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Methylocapsa acidophila Medium (DSMZ Medium 922) Composition per liter: KNO3 ..................................................................................... 100.0mg KH2PO4.................................................................................. 100.0mg MgSO4·7H2O ........................................................................... 50.0mg CaCl2·2H2O ............................................................................. 10.0mg Trace elements ...........................................................................1.0mL pH 4.5–5.8 at 25°C

Trace Elements: Composition per liter: EDTA ............................................................................................ 5.0g FeSO4·7H2O.................................................................................. 2.0g CoCl2·6H2O .................................................................................. 0.2g CuCl2·5H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g Na2MoO4 .................................................................................... 0.03g NiCl2·6H2O................................................................................. 0.02g

Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. The final pH should be 4.5–5.8. The medium is fairly weakly buffered so the pH should be checked before and after autoclaving. Incubate under atmosphere of 10–30% methane.

Use: For the cultivation and enhanced growth of Methylocapsa acidiphila.

Methylocella silverstris Medium (DSMZ Medium 1181) Composition per liter: Na2HPO4·12H2O....................................................................... 0.717g KH2PO4·2H2O .......................................................................... 0.272g MgSO4·7H2O ................................................................................ 0.2g NaNO3 ......................................................................................... 0.2g CaCl2·2H2O ............................................................................... 6.0mg Fe(III)NH4-EDTA ..................................................................... 4.0mg Phosphate buffer solution ..........................................................5.0mL Trace elements solution .............................................................0.5mL pH 5.8 ± 0.2 at 25°C

Phosphate Buffer Solution: Composition per liter: NaH2PO4·2H2O......................................................................... 28.71g Na2HPO4·12H2O......................................................................... 5.73g

Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Filter sterilize

Preparation of Medium: Add components to distilled/deionized

Trace Elements Solution: Composition per liter: Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. The final pH should be 4.5–5.8. The medium is fairly weakly buffered so the pH

H3BO3 ...................................................................................... 30.0mg CaCl2·2H2O ............................................................................. 20.0mg ZnSO4·2H2O ............................................................................ 10.0mg

ionized water and bring volume to 1.0L. Mix thoroughly.

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Methylococcus Medium

MnCl2·4H2O............................................................................... 3.0mg Na2MoO4·2H2O ......................................................................... 3.0mg CuCl2·6H2O ............................................................................... 1.0mg

Methanol ...................................................................................2.0mL Trace elements solution SL-6 ...................................................1.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Trace Elements Solution: Add components to

Trace Elements Solution SL-6: Composition per liter:

distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except phosphate buffer solution, to distilled/deionized water and bring volume to 995.0mL. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 5.0mL sterile phosphate buffer solution. Adjust pH to 5.8.

Use: For the cultivation of Methylocella silverstris.

Methylococcus Medium Composition per liter: Agar .............................................................................................. 8.0g NaNO3 (20% solution).............................................................10.0mL L-F salts solution......................................................................10.0mL Sodium-potassium phosphate buffer ...................................................................................6.5mL pH 7.1 ± 0.2 at 25°C

Sodium-Potassium Phosphate Buffer: Composition per liter: KH2PO4 ..................................................................................... 136.0g NaOH .......................................................................................... 28.8g

Preparation of Sodium-Potassium Phosphate Buffer: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1.

L-F Salts Solution: Composition per liter: MgSO4·7H2O (10% solution) ................................................200.0mL CaCl2·2H2O (10% solution).....................................................20.0mL FeSO4 (10% solution) ..............................................................10.0mL ZnSO4·7H2O (1% solution) .......................................................4.9mL H3BO3 (1% solution) .................................................................0.6mL MnSO4·H2O (1% solution) ......................................................0.27mL CuSO4·5H2O (1% solution) .......................................................0.2mL

Preparation of L-F Salts Solution: Filter sterilize FeSO4 solution

immediately prior to use. Add all components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized

MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Magnesium Sulfate Solution: Composition per 10.0mL: MgSO4·7H2O ................................................................................ 2.0g

Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O

to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Vitamin Solution: Composition per 10.0mL: Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Methanol: Composition per 10.0mL: Methanol .................................................................................10.0mL

Preparation of Methanol: Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ...................................................................................... 11.0g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Thiosulfate Solution: Composition per 10.0mL:

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.1. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

NaS2O3·5H2O ............................................................................. 18.0g

Use: For the cultivation and maintenance of Methylococcus species.

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

Methylohalomonas Medium (DSMZ Medium 1171) Composition per liter: NaCl ......................................................................................... 120.0g K2HPO4 ......................................................................................... 2.0g NH4Cl .......................................................................................... 0.5g Magnesium sulfate solution .....................................................10.0mL Thiosulfate solution .................................................................10.0mL Bicarbonate solution ................................................................10.0mL Vitamin solution.......................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Add components, except methanol, vitamin, bicarbonate, thiosulfate, and magnesium sulfate solutions, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Distribute into closed vessels with a medium to headspace ratio of 1:5 to 1:10. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the methanol, vitamin, bicarbonate, thiosulfate, and magnesium sulfate solutions. Use: For the cultivation of Methylohalomonas spp.

Methylomicrobium Medium

Methylohalomonas Medium with Acetate (DSMZ Medium 1171) Composition per liter: NaCl ......................................................................................... 120.0g K2HPO4 ......................................................................................... 2.0g NH4Cl .......................................................................................... 0.5g Magnesium sulfate solution .....................................................10.0mL Thiosulfate solution .................................................................10.0mL Bicarbonate solution ................................................................10.0mL Acetate solution .......................................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution SL-6 ...................................................1.0mL pH 7.5 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Magnesium Sulfate Solution: Composition per 10.0mL: MgSO4·7H2O ................................................................................ 2.0g

Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O

to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

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Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Add components, except acetate, vitamin, bicarbonate, thiosulfate, and magnesium sulfate solutions, to distilled/ deionized water and bring volume to 950.0mL. Mix thoroughly. Distribute into closed vessels with a medium to headspace ratio of 1:5 to 1:10. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the acetate, vitamin, bicarbonate, thiosulfate, and magnesium sulfate solutions. Use: For the cultivation with faster growth rates of Methylohalomonas spp.

Methylomicrobium Medium (DSMZ Medium 1180) Composition per liter: NaCl ........................................................................................... 30.0g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 0.02g KNO3 ............................................................................................ 1.0g Bicarbonate solution ................................................................50.0mL Phosphate buffer solution ........................................................20.0mL Carbonate solution .....................................................................5.0mL Trace elements solution .............................................................1.0mL pH 8.7 ± 0.2 at 25°C

Phosphate Buffer Solution: Composition per liter: Na2HPO4·12H2O......................................................................... 30.0g KH2PO4....................................................................................... 14.0g

Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Filter sterilize Bicarbonate Solution: Composition per 100.0mL:

Vitamin Solution: Composition per 10.0mL:

NaHCO3 ........................................................................................ 7.2g

Vitamin B12 ................................................................................ 0.1mg

deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Carbonate Solution: Composition per 10.0mL:

Aceate Solution: Composition per 10.0mL:

Na2CO3 ......................................................................................... 1.1g

Sodium acetate .............................................................................. 0.2g

Preparation of Acetate Solution: Add sodium acetate to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. B icarbonate Solution: Composition per 10.0mL: NaHCO3 ...................................................................................... 11.0g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O.............................................................................. 18.0g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.

Preparation of Carbonate Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Trace Elements Solution: Composition per liter: EDTA ............................................................................................ 5.0g FeSO4·7H2O................................................................................. 2.0g CoCl2·6H2O .................................................................................. 0.2g CuCl2·2H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................ 0.1g MnCl2·4H2O ............................................................................... 0.03g H3BO3 ......................................................................................... 0.03g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O................................................................................. 0.02g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

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Methylonatrum Medium

Preparation of Medium: Add components, except carbonate, bicarbonate, and phosphate buffer solutions, to distilled/deionized water and bring volume to 925.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the carbonate, bicarbonate, and phosphate buffer solutions. Aseptically distribute into culture vessels. Use: For the cultivation with faster growth rates of Methylomicrobium spp.

Methylonatrum Medium (DSMZ Medium 1170) Composition per liter: NaCl ............................................................................................ 18.0g K2HPO4 ......................................................................................... 1.0g Magnesium sulfate solution .....................................................10.0mL Thiosulfate solution .................................................................10.0mL Carbonate solution ...................................................................10.0mL Vitamin solution.......................................................................10.0mL Methanol ....................................................................................2.0mL Trace elements solution SL-6 ...................................................1.0mL pH 10.0 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Magnesium Sulfate Solution: Composition per 10.0mL: MgSO4·7H2O ................................................................................ 2.0g

Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Vitamin Solution: Composition per 10.0mL:

Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O ............................................................................. 18.0g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Add components, except methanol, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Distribute into closed vessels with a medium to headspace ratio of 1:5 to 1:10. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the methanol, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions. Use: For the cultivation with faster growth rates of Methylonatrum spp.

Methylonatrum Medium with Acetate (DSMZ Medium 1170) Composition per liter: NaCl ........................................................................................... 18.0g K2HPO4......................................................................................... 1.0g Magnesium sulfate solution.....................................................10.0mL Thiosulfate solution .................................................................10.0mL Carbonate solution ...................................................................10.0mL Acetate solution .......................................................................10.0mL Vitamin solution.......................................................................10.0mL Trace elements solution SL-6 ...................................................1.0mL pH 10.0 ± 0.2 at 25°C

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin B12 ................................................................................ 0.1mg

Magnesium Sulfate Solution: Composition per 10.0mL:

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

MgSO4·7H2O ................................................................................ 2.0g

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Methanol: Composition per 10.0mL: Methanol .................................................................................10.0mL

Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O

to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Methanol: Autoclave for 15 min at 15 psi press-

Vitamin Solution: Composition per 10.0mL:

ure–121°C. Cool to room temperature.

Vitamin B12 ................................................................................ 0.1mg

Carbonate Solution: Composition per 10.0mL:

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

NaHCO3 ...................................................................................... 11.0g

Preparation of Carbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Carbonate Solution: Composition per 10.0mL: Na2CO3 ....................................................................................... 11.0g

Methylophaga alcalica Agar Preparation of Carbonate Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O.............................................................................. 18.0g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Acetate Solution: Composition per 10.0mL: Sodium acetate .............................................................................. 0.2g

Preparation of Medium: Add components, except acetate, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Distribute into closed vessels with a medium to headspace ratio of 1:5 to 1:10. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the acetate, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions.

Use: For the cultivation with faster growth rates of Methylonatrum spp.

Methylophaga Agar Composition per 103.0mL: Agar solution............................................................................50.0mL Mineral base, 2X......................................................................50.0mL Solution T ..................................................................................2.0mL Vitamin B12 solution ..................................................................1.0mL Methanol ....................................................................................0.3mL pH 7.3 ± 0.2 at 25°C

Agar Solution: Composition per 500.0mL: Agar ............................................................................................ 15.0g

Preparation of Agar Solution: Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.

Mineral Base, 2X: Composition per 500.0mL: NaCl ............................................................................................ 24.0g MgCl2·6H2O.................................................................................. 3.0g MgSO4·7H2O ................................................................................ 2.0g CaCl2·2H2O................................................................................... 1.0g KCl................................................................................................ 0.5g Bis-Tris buffer (bis[2-hydroxyethyl]aminotris[hydroxymethyl]-methane)............................................ 0.5g Wolfe’s mineral solution ..........................................................10.0mL

Preparation of Mineral Base, 2X: Add components to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Wolfe’s Mineral Solution: Composition per liter: © 2010 by Taylor and Francis Group, LLC

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Solution T: Composition per 100.0mL: NH4Cl ......................................................................................... 10.0g Bis-Tris buffer (bis[2-hydroxyethyl]aminotris[hydroxymethyl]-methane) ......................................... 10.0g KH2PO4......................................................................................... 0.7g Ferric ammonium citrate............................................................... 0.3g

Preparation of Solution T: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin B12 Solution: Composition per 10.0mL: Vitamin B12 ................................................................................. 1.0μg

Preparation of Vitamin B12 Solution: Add vitamin B12 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically mix 50.0mL of the sterile agar solution with 50.0mL of the sterile mineral base, 2X. Aseptically combine sterile solution T and sterile vitamin B12 solution with the sterile mineral base. Filter sterilize methanol and add to basal medium. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylophaga marina.

Methylophaga alcalica Agar (DSMZ Medium 976) Composition per liter: NaCl............................................................................................ 30.0g Agar ............................................................................................ 20.0g KH2PO4......................................................................................... 1.0g KNO3 ............................................................................................ 1.0g MgSO4·7H2O .............................................................................. 0.22g Na2CO3 solution......................................................................50.0mL Methanol solution ....................................................................50.0mL Trace elements solution .............................................................1.0mL pH 9.5 ± 0.2 at 25°C

Methanol Solution: Composition per 50.0mL: Methanol ..................................................................................10.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

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Methylophaga alcalica Medium

Na2CO3 Solution: Composition per 50.0mL: Na2CO3 ......................................................................................... 5.0g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: Ferric citrate ............................................................................. 30.0mg CaCl2·2H2O.............................................................................. 30.0mg MgCl2·4H2O............................................................................... 5.0mg ZnSO4·7H2O .............................................................................. 5.0mg CuSO4·5H2O .............................................................................. 0.5mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except methanol solution and Na2CO3 solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 55°C. Aseptically add 50.0mL warm sterile Na2CO3 solution and 50.0mL warm sterile methanol solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylophaga alcalica.

Methylophaga alcalica Medium (DSMZ Medium 976) Composition per liter: NaCl ............................................................................................ 30.0g KH2PO4 ......................................................................................... 1.0g KNO3 ............................................................................................ 1.0g MgSO4·7H2O .............................................................................. 0.22g Na2CO3 solution......................................................................50.0mL Methanol solution ....................................................................50.0mL Trace elements solution .............................................................1.0mL pH 9.5 ± 0.2 at 25°C

Methanol Solution: Composition per 50.0mL: Methanol ..................................................................................10.0mL

Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Na2CO3 Solution: Composition per 50.0mL: Na2CO3 ......................................................................................... 5.0g

Preparation of Medium: Add components, except methanol solution and Na2CO3 solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 50.0mL sterile Na2CO3 solution and 50.0mL sterile methanol solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Methylophaga alcalica.

Methylophaga Broth Composition per 103.0mL: Mineral base...........................................................................100.0mL Solution T ..................................................................................2.0mL Vitamin B12 solution ..................................................................1.0mL Methanol ....................................................................................0.3mL pH 7.3 ± 0.2 at 25°C

Mineral Base: Composition per liter: NaCl............................................................................................ 24.0g MgCl2·6H2O ................................................................................. 3.0g MgSO4·7H2O ................................................................................ 2.0g CaCl2·2H2O .................................................................................. 1.0g KCl................................................................................................ 0.5g Bis-Tris buffer (bis[2-hydroxyethyl]aminotris[hydroxymethyl]-methane) ........................................... 0.5g Wolfe’s mineral solution..........................................................10.0mL

Preparation of Mineral Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C.

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Solution T: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C.

Methylosarcina quisquillarum/ Methylosarcina fibrata Medium

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Vitamin B12 Solution: Composition per 10.0mL:

water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 80% N2 + 20% CO2.

Vitamin B12 .................................................................................1.0μg

Vitamin Solution: Composition per liter:

Preparation of Vitamin B12 Solution: Add vitamin B12 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine sterile solution T and sterile vitamin B12 solution with the sterile mineral base. Filter sterilize methanol and add to basal medium. Aseptically distribute into sterile tubes or sterile flasks.

Use: For the cultivation and maintenance of Methylophaga marina.

Methylophaga Medium Composition per liter: NaCl ........................................................................................... 25.0g Agar ............................................................................................ 20.0g Peptone........................................................................................ 10.0g Beef extract ................................................................................... 7.0g K2HPO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 1.0g Methanol, filter sterilized.........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Methylophaga marina and Methylophaga thalassica.

Methylophaga sulfidovorans Medium (DSMZ Medium 951) Composition per liter: NaCl ............................................................................................ 15.0g Na2CO3 ......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 1.0g (NH4)2SO4 ..................................................................................... 0.5g CaCl2·6H2O................................................................................. 0.33g KCl................................................................................................ 0.2g KH2PO4 ....................................................................................... 0.02g DMS (Dimethylsulphide) ........................................................ 62.0mg FeSO4·7H2O............................................................................... 1.0mg Trace elements solution SL-10 ..................................................1.0mL Vitamin solution.........................................................................1.0mL pH 7.5 ± 0.3 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Pyridoxine-HCl...................................................................... 500.0mg Nicotinic acid......................................................................... 200.0mg Thiamine ................................................................................ 100.0mg p-Aminobenzoic acid............................................................. 100.0mg Pantothenate............................................................................. 50.0mg Biotin ....................................................................................... 20.0mg Riboflavin ................................................................................ 10.0mg Vitamin B12 .............................................................................. 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Methylophaga sulfidovorans.

Methylophaga thalassica Agar (LMG Medium 73) Composition per liter: NaCl............................................................................................ 25.0g Agar ............................................................................................ 20.0g Peptone ....................................................................................... 10.0g Lab Lemco beef extract ................................................................ 7.0g K2HPO4......................................................................................... 1.0g (NH4)2SO4 .................................................................................... 1.0g pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Methylophaga thalassica.

Methylosarcina quisquillarum/ Methylosarcina fibrata Medium (DSMZ Medium 921) Composition per 1012.1mL: Solution 1...............................................................................100.0mL Phosphate buffer ......................................................................10.0mL Solution 3...................................................................................1.0mL Trace elements ...........................................................................1.0mL Solution 2...................................................................................0.1mL pH 7.0 ± 0.2 at 25°C

Solution 1 (10X NMS Salts): Composition per liter: KNO3 .......................................................................................... 10.0g MgSO4·6H2O .............................................................................. 10.0g CaCl2·2H2O .................................................................................. 2.0g

Preparation of Solution 1 (10X NMS Salts): Add components to 700.0mL distilled/deionized water. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly.

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Methylotrophic Arthrobacter Hyphomicrobium Medium

Solution 2 (Fe EDTA): Composition per liter: Fe EDTA ....................................................................................... 3.8g

CoCl2·6H2O .................................................................................. 0.5g NH4(MoO4)................................................................................... 0.5g CuSO4·5H2O ................................................................................. 0.2g

Preparation of Solution 2 (Fe EDTA): Add Fe EDTA to distilled/

Preparation of Trace Elements Solution: Add Na2-EDTA to

deionized water and bring volume to 1.0L. Mix thoroughly.

Solution 3 (Sodium Molybdate): Composition per liter: Na2MoO4·4H2O .......................................................................... 0.26g

Preparation of Solution 3 (Sodium Molybdate): Add Na2MoO4·4H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements: Composition per 100.0mL: CuSO4·5H2O .......................................................................... 100.0mg FeSO4·7H2O............................................................................. 50.0mg ZnSO4·7H2O ............................................................................ 40.0mg EDTA disodium salt................................................................. 25.0mg CoCl2·6H2O ............................................................................... 5.0mg MnCl2·4H2O............................................................................... 2.0mg H3BO3 ........................................................................................ 1.5mg NiCl2·6H2O ................................................................................ 1.0mg

Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Phosphate Buffer: Composition per liter: Na2HPO4·2H2O........................................................................... 71.6g KH2PO4 ....................................................................................... 26.0g

Preparation of Phosphate Buffer: Add components to 800.0mL distilled/deionized water. Mix thoroughly. Adjust pH to 6.8. Bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 55°C.

Preparation of Medium: Add 100.0mL solution 1 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Add 1.0mL of solution 3, 1.0mL of the trace elements, and 0.1mL of solution 2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 55°C. Aseptically add 10.0mL phosphate buffer. Mix thoroughly. Aseptically distribute to sterile tubes or bottles.

400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Hyphomicrobium sulfonivorans.

Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na2HPO4·2H2O............................................................................. 7.9g KH2PO4......................................................................................... 1.5g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Trace elements solution ...........................................................10.0mL Methanol ..................................................................................10.0mL pH 7.2–7.5 at 25°C

Trace Elements Solution: Composition per liter: EDTA disodium salt.................................................................... 50.0g NaOH............................................................................................ 9.0g CaCl2·2H2O ................................................................................ 7.34g FeSO4·7H2O.................................................................................. 5.0g MnCl2·4H2O ................................................................................. 2.5g ZnSO4·7H2O ................................................................................. 1.0g CoCl2·6H2O .................................................................................. 0.5g NH4(MoO4)................................................................................... 0.5g CuSO4·5H2O ................................................................................. 0.2g

Preparation of Trace Elements Solution: Add Na2-EDTA to

Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939)

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Methylosarcina fibrata and Methylosarcina quisquiliarum.

Na2HPO4·2H2O............................................................................. 7.9g Dimethylsulfone............................................................................ 1.9g KH2PO4 ......................................................................................... 1.5g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Trace elements solution ...........................................................10.0mL pH 7.2-7.5 at 25°C

Trace Elements Solution: Composition per liter: EDTA disodium salt.................................................................... 50.0g NaOH ............................................................................................ 9.0g CaCl2·2H2O................................................................................. 7.34g FeSO4·7H2O.................................................................................. 5.0g MnCl2·4H2O.................................................................................. 2.5g ZnSO4·7H2O ................................................................................. 1.0g © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Hyphomicrobium sulfonivorans.

Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na2HPO4·2H2O............................................................................. 7.9g KH2PO4......................................................................................... 1.5g Methylamine ................................................................................. 1.0g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Trace elements solution ...........................................................10.0mL pH 7.2–7.5 at 25°C

Methylpyridine Medium

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Trace Elements Solution: Composition per liter:

Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939)

EDTA disodium salt.................................................................... 50.0g NaOH ............................................................................................ 9.0g CaCl2·2H2O................................................................................. 7.34g FeSO4·7H2O.................................................................................. 5.0g MnCl2·4H2O.................................................................................. 2.5g ZnSO4·7H2O ................................................................................. 1.0g CoCl2·6H2O .................................................................................. 0.5g NH4(MoO4)................................................................................... 0.5g CuSO4·5H2O ................................................................................. 0.2g

Composition per liter:

Na2HPO4·2H2O............................................................................. 7.9g KH2PO4......................................................................................... 1.5g Fructose......................................................................................... 1.0g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Trace elements solution ...........................................................10.0mL pH 7.2–7.5 at 25°C

Trace Elements Solution: Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

EDTA disodium salt.................................................................... 50.0g NaOH............................................................................................ 9.0g CaCl2·2H2O ................................................................................ 7.34g FeSO4·7H2O.................................................................................. 5.0g MnCl2·4H2O ................................................................................. 2.5g ZnSO4·7H2O ................................................................................. 1.0g CoCl2·6H2O .................................................................................. 0.5g NH4(MoO4)................................................................................... 0.5g CuSO4·5H2O................................................................................. 0.2g

Use: For the cultivation of Hyphomicrobium sulfonivorans.

Preparation of Trace Elements Solution: Add Na2-EDTA to

Preparation of Medium: Add components to distilled/deionized

Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter:

Na2HPO4·2H2O............................................................................. 7.9g KH2PO4 ......................................................................................... 1.5g Glucosel ........................................................................................ 1.0g NH4Cl ........................................................................................... 0.8g MgSO4·7H2O ................................................................................ 0.1g Trace elements solution ...........................................................10.0mL pH 7.2–7.5 at 25°C

Preparation of Medium: Add components to distilled/deionized

Trace Elements Solution: Composition per liter:

K2HPO4....................................................................................... 0.61g KH2PO4....................................................................................... 0.39g KCl.............................................................................................. 0.25g Yeast extract.................................................................................. 0.1g Wolfe’s mineral solution..........................................................10.0mL 2-Methylpyridine .......................................................................1.0mL

Preparation of Trace Elements Solution: Add Na2-EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Hyphomicrobium sulfonivorans.

Methylpyridine Medium Composition per 1002.0mL:

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoCl2·6H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with

1150

M-FC HiVeg Agar Base with Rosalic Acid

KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Rosolic Acid Solution: Composition per 100.0mL:

Preparation of Medium: Add components, except 2-methylpyridine, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. In a fume hood, aseptically add 1.0mL of 2-methylpyridine. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use polyurethane foam closures to eliminate odors caused by volatilization of 2-methylpyridine.

Rosolic acid .................................................................................. 1.0g

Use: For the cultivation of Arthrobacter species.

Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly.

Preparation of Medium: Add 10.0mL rosolic acid solution to 950.0mL of distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling with frequent mixing. Do not autoclave. Pour into sterile Petri dishes or leave in tubes.

M-FC Agar See: FC Agar

Use: For the detection and enumeration of fecal coliforms using membrane filter technique.

M-FC Broth See: FC Broth

Composition per liter:

M-FC HiVeg Agar Base with Rosalic Acid Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 12.5g Plant hydrolysate No. 1............................................................... 10.0g Plant peptone No. 3....................................................................... 5.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Synthetic detergent No. I .............................................................. 1.5g Aniline Blue .................................................................................. 0.1g Rosolic acid solution................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium, without rosolic acid, is available as a premixed

M-FC HiVeg Broth Base with Rosalic Acid Lactose........................................................................................ 12.5g Plant hydrolysate No. 1............................................................... 10.0g Plant peptone No. 3....................................................................... 5.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Synthetic detergent No. I .............................................................. 1.5g Aniline blue .................................................................................. 0.1g Rosolic acid solution................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium, without rosolic acid, is available as a premixed powder from HiMedia.

Rosolic Acid Solution: Composition per 100.0mL: Rosolic acid .................................................................................. 1.0g

powder from HiMedia.

Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N

Rosolic Acid Solution: Composition per 100.0mL:

NaOH and bring volume to 100.0L. Mix thoroughly.

Rosolic acid................................................................................... 1.0g

Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly.

Use: For the detection and enumeration of fecal coliforms using the membrane filter technique.

M-FC HiVeg Agar Base, Modified with Rosalic Acid Composition per liter: Agar ............................................................................................ 15.0g Plant hydrolysate No. 1............................................................... 10.0g Inositol ........................................................................................ 10.0g Plant peptone No. 3....................................................................... 5.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Synthetic detergent No. I .............................................................. 1.5g Aniline blue................................................................................... 0.1g Rosolic acid solution................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add 10.0mL of rosolic acid solution to 950.0mL of distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling with frequent mixing. Do not autoclave. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of fecal coliform bacteria from waters and the enumeration of coliform bacteria using the membrane filtration method.

M-FC Agar Composition per liter: Agar ............................................................................................ 15.0g Tryptose ...................................................................................... 10.0g Inositol ........................................................................................ 10.0g Proteose peptone........................................................................... 5.0g NaCl.............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Bile salts mixture .......................................................................... 1.5g Aniline Blue.................................................................................. 0.1g Selective supplement solution .................................................10.0mL Rosolic acid solution................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

Source: This medium, without rosolic acid, is available as a premixed

Rosolic Acid Solution: Composition per 10.0mL:

powder from HiMedia.

Rosolic acid .................................................................................. 0.1g

MG Medium Preparation of Rosolic Acid Solution: Add rosoloic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Selective Supplement Solution: Composition per 10.0mL: Carbenicillin................................................................................ 0.05g

Preparation of Selective Supplement Solution: Add carbenicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

M-FC Agar Base Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 12.5g Tryptose ...................................................................................... 10.0g Proteose peptone ........................................................................... 5.0g NaCl .............................................................................................. 5.0g Yeast extract.................................................................................. 3.0g Bile salts mixture .......................................................................... 1.5g Aniline Blue .................................................................................. 0.1g Rosolic acid solution................................................................10.0mL pH 7.4 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Rosolic Acid Solution: Composition per 10.0mL: Rosolic acid................................................................................... 0.1g

Preparation of Rosolic Acid Solution: Add rosolic acidto distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat to dissolve components. Do not autoclave. Cool to 50°C. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the detection and enumeration of fecal coliforms using the membrane filter technique at 44.5°C.

m-Fecal Coliform Agar See: FC Agar m-Fecal Coliform Agar, Modified See: Fecal Coliform Agar, Modified m-Fecal Coliform Broth See: FC Broth

MG Medium Composition per liter: NaCl .......................................................................................... 100.0g MgSO4·7H2O .............................................................................. 3.45g MgCl2·6H2O................................................................................ 2.75g Sodium acetate .............................................................................. 1.0g © 2010 by Taylor and Francis Group, LLC

1151

KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O ................................................................................ 0.14g K2HPO4·3H2O ............................................................................ 0.14g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................80.0mL Trimethylamine·HCl solution ..................................................20.0mL Na2CO3 solution ......................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution ..........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.9 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Trimethylamine·HCl Solution: Composition per 20.0mL: Trimethylamine·HCl ..................................................................... 5.0g

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 0.5g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg

1152

MG Medium

Nicotinic acid ............................................................................. 5.0mg p-Aminobenzoic acid ................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N2. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl.............................................................................. 0.5g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except NaHCO3 solu-

tion, trimethylamine·HCl solution, Na2CO3 solution, vitamin solution, L-cysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 860.0mL. Mix thoroughly. Sparge with 100% N2 for 20 min. Then sparge with 80% N2 + 20% CO2 for 10 min. Anaerobically distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 80.0mL of sterile NaHCO3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na2CO3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly.

Use: For the cultivation and maintenance of Methanohalobium species, Methanohalophilus halophilus, and Methanohalophilus species.

MG Medium Composition per liter: NaCl .......................................................................................... 150.0g MgSO4·7H2O .............................................................................. 3.45g MgCl2·6H2O................................................................................ 2.75g Sodium acetate .............................................................................. 1.0g KCl............................................................................................ 0.335g NH4Cl ......................................................................................... 0.25g CaCl2·2H2O................................................................................. 0.14g K2HPO4·3H2O............................................................................. 0.14g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................80.0mL Trimethylamine·HCl solution ..................................................20.0mL Na2CO3 solution ......................................................................10.0mL Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL L-Cysteine·HCl solution...........................................................10.0mL Na2S·9H2O solution .................................................................10.0mL pH 6.9 ± 0.2 at 25°C

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Trimethylamine·HCl Solution: Composition per 20.0mL: Trimethylamine·HCl ..................................................................... 5.0g

Na2CO3 Solution: Composition per 10.0mL: Na2CO3 ......................................................................................... 0.5g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/de-

ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL of distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................................................................ 10.0mg Calcium DL-pantothenate........................................................... 5.0mg Lipoic acid ................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Thiamine·HCl ............................................................................ 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N2. L-Cysteine·HCl

Solution: Composition per 10.0mL:

L-Cysteine·HCl ............................................................................. 0.5g

Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

MH IH Agar

Na2S·9H2O Solution: Composition per 10.0mL: Na2S·9H2O .................................................................................... 0.5g

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except NaHCO3 solution, trimethylamine·HCl solution, Na2CO3 solution, vitamin solution, Lcysteine·HCl solution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 860.0mL. Mix thoroughly. Sparge with 100% N2 for 20 min. Then sparge with 80% N2 + 20% CO2 for 10 min. Anaerobically distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 80.0mL of sterile NaHCO3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na2CO3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile Lcysteine·HCl solution, and 10.0mL of sterile Na2S·9H2O solution. Mix thoroughly. Use: For the cultivation and maintenance of Methanohalophilus species.

MGA Agar Composition per liter:

1153

MgSO4·7H2O .............................................................................. 14.4g MgCl2.......................................................................................... 10.5g Yeast extract................................................................................ 10.0g Proteose peptone no.3................................................................... 5.0g KCl................................................................................................ 3.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.54g NaBr......................................................................................... 39.0mg NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ...................................................................................... 0.09g

Preparation of NaHCO3 Solution: Add NaHCO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except NaHCO3 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL sterile NaHCO3 solution. Pour into sterile Petri dishes or aseptically distribute into sterile tubes. Use: For cultivation and maintenance of Bacillus halophilus.

Agar ............................................................................................ 20.0g Glucose ......................................................................................... 2.0g L-Asparagine ................................................................................. 1.0g K2HPO4 ......................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.5g Trace salts solution ....................................................................1.0mL pH 7.4 ± 0.2 at 25°C

Solution A..............................................................................490.0mL Solution B ..............................................................................490.0mL Supplement solution ................................................................20.0mL pH 6.9 ± 0.2 at 25°C

Trace Salts Solution: Composition per liter:

Solution A: Composition per 490.0mL:

MH IH Agar Composition per liter:

FeSO4·7H2O................................................................................ 10.0g CuSO4·5H2O ................................................................................. 1.0g MnSO4·7H2O ................................................................................ 1.0g ZnSO4·7H2O ................................................................................. 1.0g

Beef infusion............................................................................. 300.0g Acid hydrolysate of casein.......................................................... 17.5g Agar ............................................................................................ 17.0g Starch ............................................................................................ 1.5g

Preparation of Trace Salts Solution: Add components to dis-

Preparation of Solution A: Add components to distilled/deionized

tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 8.0.

water and bring volume to 490.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Solution B: Composition per 490.0mL:

Use: For the isolation and cultivation of Actinomadura species, Actinopolyspora species, Excellospora species, and Microspora species.

Preparation of Solution B: Add hemoglobin to distilled/deionized

m-Green Yeast and Mold Broth See: Green Yeast and Mold Broth MGTY Agar See: Marine Glucose Trypticase™ Yeast Extract Agar MGTY Broth See: Marine Glucose Trypticase™ Yeast Extract Broth

MH Agar 15% (LMG Medium 258) Composition per liter: NaCl .......................................................................................... 121.5g Agar ............................................................................................ 20.0g © 2010 by Taylor and Francis Group, LLC

Hemoglobin ................................................................................ 10.0g water and bring volume to 490.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.

Supplement Solution: Composition per liter: Glucose ..................................................................................... 100.0g L-Cysteine·HCl............................................................................ 25.9g L-Glutamine ................................................................................ 10.0g L-Cystine ....................................................................................... 1.1g Adenine......................................................................................... 1.0g Nicotinamide adenine dinucleotide ............................................ 0.25g Vitamin B12 ................................................................................... 0.1g Thiamine pyrophosphate .............................................................. 0.1g Guanine·HCl ............................................................................... 0.03g Fe(NO3)3·6H2O........................................................................... 0.02g

1154

MH Medium

p-Aminobenzoic acid ................................................................ 0.013g Thiamine·HCl ............................................................................ 3.0mg

Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems.

Preparation of Supplement Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine cooled, sterile solu-

NaCl.............................................................................................. 0.5g MgSO4·7H2O ............................................................................ 0.025g FeSO4·7H2O............................................................................... 2.0mg Glucose solution ......................................................................20.0mL Methanol, filter sterilized.........................................................20.0mL pH 7.0–7.5 at 25°C

Glucose Solution: Composition per 20.0mL: Glucose ......................................................................................... 1.5g

tion A and cooled, sterile solution B. Mix thoroughly. Adjust pH to 6.9 with sterile 1N HCl or sterile 1N KOH. Aseptically add 20.0mL of sterile supplement solution. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Glucose Solution: Add glucose to distilled/deion-

Use: For the cultivation and differentiation of Legionella species.

tion and methanol, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 20.0mL of sterile glucose solution and 20.0mL of filter-sterilized methanol. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

MH Medium Composition per liter: NaCl ............................................................................................ 60.7g Agar ............................................................................................ 20.0g MgCl2·6H2O................................................................................ 15.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O ................................................................................ 7.4g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 1.5g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.27g NaHCO3 ...................................................................................... 0.45g NaBr............................................................................................ 0.19g

Use: For the cultivation and maintenance of Deleya salina and Volcaniella eurihalina.

MH Medium Composition per liter: NaCl ............................................................................................ 60.7g MgCl2·6H2O................................................................................ 15.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O ................................................................................ 7.4g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 1.5g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.27g NaHCO3 .................................................................................... 0.045g NaBr.......................................................................................... 0.019g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation of Halomonas eurihalina.

MH Medium, 2% Composition per liter: KH2PO4 ......................................................................................... 2.0g (NH4)2SO4 ..................................................................................... 2.0g © 2010 by Taylor and Francis Group, LLC

ized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except glucose solu-

Use: For the cultivation of Methylobacillus fructoseoxidans and Methylophilus glucoseoxidans.

MH Medium, 10% (LMG Medium 270) Composition per liter: NaCl............................................................................................ 81.0g Agar ............................................................................................ 15.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O ................................................................................ 9.6g MgCl2·6H2O ................................................................................. 7.0g Proteose peptone No.3 .................................................................. 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.54g NaBr......................................................................................... 26.0mg NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ...................................................................................... 0.06g

Preparation of NaHCO3 Solution: Add NaHCO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except NaHCO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptically add 10.0mL sterile NaHCO3 solution. Pour into sterile Petri dishes or aseptically distribute into sterile tubes.

Use: For cultivation and maintenance of Chromohalobacter israelensis and Chromohalobacter canadensis.

MH Medium, 10% Composition per liter: NaCl............................................................................................ 81.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O ................................................................................ 9.6g MgCl2·6H2O ................................................................................. 7.0g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g

Micro Assay Culture Agar

CaCl2 ........................................................................................... 0.36g NaBr.......................................................................................... 0.026g NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL:

1155

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Bacillus halophilus.

NaHCO3 ...................................................................................... 0.06g

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except NaHCO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile NaHCO3 solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Zygomonas mobilis, Salinicoccus hispanicus, Salinicoccus roseus, and Pseudomonas beijerinckii.

MH Medium 15% (LMG Medium 258) Composition per liter: NaCl .......................................................................................... 121.5g MgSO4·7H2O .............................................................................. 14.4g MgCl2 .......................................................................................... 10.5g Yeast extract................................................................................ 10.0g Proteose peptone No.3 .................................................................. 5.0g KCl................................................................................................ 3.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.54g NaBr......................................................................................... 39.0mg NaHCO3 solution .....................................................................10.0mL pH 7.5 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 10.0mL: NaHCO3 ...................................................................................... 0.09g

Preparation of NaHCO3 Solution: Add NaHCO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except NaHCO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL sterile NaHCO3 solution. Aseptically distribute into sterile tubes or flasks.

Use: For cultivation and maintenance of Bacillus halophilus.

MIB with Maltose Composition per liter: Yeast extract................................................................................ 20.0g Maltose ....................................................................................... 10.0g Glucose ....................................................................................... 10.0g Proteose peptone No. 3 ................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 0.1g pH 6.7 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Lactobacillus sanfrancisco.

Micro Assay Culture Agar Composition per liter: Yeast extract................................................................................ 20.0g Agar ............................................................................................ 10.0g Glucose ....................................................................................... 10.0g Proteose peptone No. 3 ................................................................. 5.0g KH2PO4......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 0.1g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Just prior to solidification of the agar, disperse precipitate by gently twirling tube.

Use: For carrying stock cultures of lactobacilli and other test microorganisms used in microbiological assays. For the general cultivation of lactobacilli.

Micro Assay Culture Agar Composition per liter:

MH Salts Composition per liter: NaCl .......................................................................................... 120.5g MgCl2·6H2O................................................................................ 22.4g Agar ............................................................................................ 20.0g MgSO4 ........................................................................................ 14.4g Yeast extract................................................................................ 10.0g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 3.0g Glucose ......................................................................................... 1.0g CaCl2 ........................................................................................... 0.54g NaHCO3 ...................................................................................... 0.09g NaBr.......................................................................................... 0.039g pH 7.5 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Agar ............................................................................................ 10.0g Glucose ....................................................................................... 10.0g Proteose peptone No. 3 ................................................................. 5.0g Yeast extract.................................................................................. 5.0g KH2PO4......................................................................................... 2.0g Polysorbate 80 .............................................................................. 0.1g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

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Micro Assay Culture Broth

Use: For the cultivation of lactobacilli and other microorganisms used in microbiological assays.

Micro Assay Culture Broth Composition per liter: Glucose ....................................................................................... 10.0g Proteose Peptone No. 3 ................................................................. 5.0g Yeast Extract ................................................................................. 5.0g KH2PO4 ......................................................................................... 2.0g Polysorbate 80............................................................................... 0.1g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Plant peptone No. 3....................................................................... 5.0g KH2PO4......................................................................................... 2.0g Polysorbate 80 .............................................................................. 0.1g pH 6.7 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of lactobacilli and other microorganisms used in microbiological assays.

Micro Inoculum Broth Composition per liter: Yeast extract................................................................................ 20.0g Glucose ....................................................................................... 10.0g Proteose peptone No. 3 ................................................................. 5.0g KH2PO4 ......................................................................................... 2.0g Sorbitan monooleate complex ...................................................... 0.1g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of lactobacilli used in microbiological assays. It is of particular value in the preparation of the inoculum for these tests.

Micro Vitamin Test Culture HiVeg Agar Composition per liter: Yeast extract................................................................................ 20.0g Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Plant peptone................................................................................. 5.0g KH2PO4 ......................................................................................... 2.0g Polysorbate 80............................................................................... 0.1g pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of lactobacilli and other microorganisms used in microbiological assays.

Micro Vitamin Test Inoculum HiVeg Broth

Microbacterium Medium Composition per liter: Glucose ....................................................................................... 10.0g KH2PO4......................................................................................... 5.0g K2HPO4......................................................................................... 5.0g Potassium aspartate....................................................................... 5.0g (NH4)2SO4 .................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g Calcium pantothenate ................................................................... 0.2g β-Mercaptopurine ......................................................................... 0.1g FeSO4·7H2O................................................................................ 0.01g Thiamine·HCl ............................................................................. 0.01g Biotin ......................................................................................... 0.1mg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Microbacterium species.

Microbial Content Test Agar Composition per liter: Agar ............................................................................................ 15.0g Pancreatic digest of casein.......................................................... 15.0g NaCl.............................................................................................. 5.0g Tween™ 80................................................................................... 5.0g Enzymatic hydrolysate of soybean meal ...................................... 5.0g Lecithin ......................................................................................... 0.7g pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Boil for 1–2 min. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For use in the microbial content test of water-soluble cosmetic products. Also used for determining the efficiency of sanitization of containers, equipment, and environmental surfaces.

Microbial Content Test HiVeg Agar (Tryptone Soy HiVeg Agar with Lecithin and Tween 80)

Composition per liter:

Yeast extract................................................................................ 20.0g Glucose ....................................................................................... 10.0g

Micrococcus Medium, FDA

Papaic digest of soybean meal ...................................................... 5.0g NaCl .............................................................................................. 5.0g Lecithin ......................................................................................... 0.7g Polysorbate 80............................................................................5.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium, without polysorbate 80, is available as a premixed powder from HiMedia.

Use: For use in the microbial content test of water-soluble cosmetic products. Also used for determining the efficiency of sanitization of containers, equipment, and environmental surfaces.

Microcella alkaliphila Medium (DSMZ Medium 1063) Composition per liter: Pancreatic digest of casein .......................................................... 17.0g NaCl .............................................................................................. 5.0g Papaic digest of soybean meal ...................................................... 3.0g Yeast extract.................................................................................. 3.0g K2HPO4 ......................................................................................... 2.5g Glucose ......................................................................................... 2.5g Na-sesquicarbonate solution ..................................................100.0mL Mineral salt solution ................................................................50.0mL Vitamin solution.........................................................................5.0mL Trace elements solution SL-10 ..................................................1.0mL pH 9.5 ± 0.2 at 25°C

Sodium Sesquicarbonate Solution: Composition per 100.0mL: Na2CO3, anhydrous..................................................................... 10.6g NaHCO3 ...................................................................................... 8.42g

Preparation of Sodium Sesquicarbonate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Mineral Solution: Composition per liter: KH2PO4 ....................................................................................... 10.0g MgCl2·6H2O.................................................................................. 6.6g NaCl .............................................................................................. 8.0g NH4Cl ........................................................................................... 8.0g CaCl2·2H2O................................................................................... 1.0g

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Trace Elements Solution SL-10: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl.............................................................................................. 1.0g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Medium: Add components, except sodium sesquicarbonate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Aseptically add sterile 1M sodium sesquicarbonate solution to achieve a final pH of 9.5. Aseptically dispense into tubes, flasks, or bottles.

Use: For the cultivation of Microcella alkaliphila.

Micrococcus Medium Composition per liter: Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Beef extract................................................................................... 1.5g Glucose ......................................................................................... 1.0g pH 7.4 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Staphylococcus aureus and Micrococcus species.

Micrococcus Medium, FDA

Preparation of Mineral Solution: Add components to distilled/

Composition per liter:

deionized water and bring volume to 1.0L. Mix thoroughly.

Agar ............................................................................................ 15.0g Proteose peptone......................................................................... 10.0g Beef extract................................................................................... 5.0g NaCl.............................................................................................. 5.0g pH 7.2 ± 0.2 at 25°C

Vitamin Solution: Composition per liter: Pyridoxine·HCl .......................................................................... 6.2mg Nicotinic acid ............................................................................. 2.5mg Thiamine·HCl .......................................................................... 1.25mg p-Aminobenzoic acid............................................................... 1.25mg Pantothenic acid ....................................................................... 0.62mg Biotin ....................................................................................... 0.25mg

Preparation of Vitamin Solution: Add components to distilled/

Use: For the cultivation and maintenance of Staphylococcus aureus

deionized water and bring volume to 1.0L. Mix thoroughly.

and Micrococcus species.

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Micrococcus/Sarcina Medium

Use: For the cultivation of Microcyclus major.

Composition per liter: Agar ............................................................................................ 16.0g Pancreatic digest of casein ............................................................ 5.0g Sodium succinate·6H2O ................................................................ 2.0g Starch ............................................................................................ 2.0g Yeast autolysate............................................................................. 1.0g Sodium citrate·2H2O..................................................................... 0.5g Sodium acetate·3H2O.................................................................... 0.3g K2HPO4 ......................................................................................... 0.2g pH 7.0 ± 0.2 at 25°C

Microcyclus marinus Medium Composition per liter: NaCl............................................................................................ 23.5g MgCl2............................................................................................ 5.0g Na2SO4 .......................................................................................... 4.0g CaCl2·2H2O .................................................................................. 1.5g KCl................................................................................................ 0.7g NaHCO3 ........................................................................................ 0.2g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Microcyclus marinus.

Use: For the cultivation and maintenance of Micrococcus luteus and Sarcina species.

Microcyclus Medium Composition per liter:

Microcyclus eburneus Medium Composition per liter: K2HPO4 ......................................................................................... 7.0g (NH4)SO4 ...................................................................................... 3.0g KH2PO4 ......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g Yeast extract.................................................................................. 0.2g Thiamine·HCl ............................................................................ 0.2mg Biotin ....................................................................................... 0.02mg FeSO4·7H2O................................................................................2.0μg MnSO4·4H2O ..............................................................................2.0μg

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Microcyclus eburneus.

Microcyclus major Medium Composition per liter: Glucose ......................................................................................... 1.0g Peptone.......................................................................................... 1.0g KNO3 ............................................................................................ 0.1g K2HPO4 ....................................................................................... 0.07g MgSO4·7H2O .............................................................................. 0.03g Trace elements solution .............................................................1.0mL

Trace Elements Solution: Composition per liter: Disodium EDTA ......................................................................... 10.0g FeSO4·7H20................................................................................... 9.3g NaBO3·4H2O................................................................................. 2.6g MnCl2·4H2O.................................................................................. 1.8g CaCl2 ............................................................................................. 1.2g (NH4)6Mo7O24·4H2O .................................................................... 1.0g ZnSO4·7H2O ................................................................................. 0.2g CuSO4·5H2O ............................................................................... 0.08g Co(NO3)2·H2O ............................................................................ 0.02g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Use: For the cultivation and maintenance of Flectobacillus major and Microcyclus species.

Microcyclus/Spirosoma Medium Composition per liter: Agar ............................................................................................ 15.0g Glucose ......................................................................................... 1.0g Peptone ......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g pH 6.8–7.0 at 25°C

Use: For the cultivation and maintenance of Spirosoma linguale and Microcyclus species.

Microlunatus Medium (DSMZ Medium 776) Composition per liter: Glucose ......................................................................................... 0.5g Peptone ......................................................................................... 0.5g Yeast extract.................................................................................. 0.5g Na-glutamate................................................................................. 0.5g KH2PO4......................................................................................... 0.5g (NH4)2SO4 .................................................................................... 0.1g MgSO4·7H2O ................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0.

Middlebrook ADC Enrichment

Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Microlunatus phosphovorus and Kineosphaera limosa.

Micromonospora megalomicea Agar Composition per liter: Soluble starch.............................................................................. 20.0g Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g Pancreatic digest of casein ............................................................ 5.0g Yeast extract.................................................................................. 5.0g CaCO3 ........................................................................................... 1.0g

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Micromonospora rhodorangea and Micromonospora rosaria.

Micromonospora Starch Agar Composition per liter: Starch, soluble............................................................................. 20.0g Agar ............................................................................................ 15.0g Glucose ....................................................................................... 10.0g N-Z amine type A ......................................................................... 5.0g Yeast extract.................................................................................. 5.0g CaCO3 ........................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

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Middlebrook 13A Medium Composition per 112.5mL: Casein hydrolysate........................................................................ 0.1g Tween™ 80................................................................................. 0.02g Sodium polyanetholesulfonate.................................................. 0.025g Middlebrook 7H9 broth .........................................................100.0mL Middlebrook 13A enrichment..................................................12.5mL Catalase.................................................................................. 36,000U 14C-substrate ............................................................125μCi (185kBq) pH 6.6 ± 0.2 at 25°C

Middlebrook 7H9 Broth: Composition per liter: Na2HPO4 ....................................................................................... 2.5g KH2PO4......................................................................................... 1.0g Monosodium glutamate ................................................................ 0.5g (NH4)2SO4 .................................................................................... 0.5g Sodium citrate............................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g CuSO4·5H2O.............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Glycerol .....................................................................................2.0mL

Preparation of Middlebrook 7H9 Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Middlebrook 13A Enrichment: Composition per 20.0mL:

Preparation of Medium: Add components to distilled/deionized

Bovine serum albumin.................................................................. 3.0g

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Preparation of Middlebrook 13A Enrichment: Add bovine serum albumin to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Use: For the cultivation and maintenance of Ampullariella campanu-

add remaining components, except Middlebrook 13A enrichment. Mix thoroughly. Filter sterilize. Aseptically distribute into bottles in 4.0mL volumes. Prior to inoculation, aseptically add 0.5mL of Middlebrook 13A enrichment to each bottle. Mix thoroughly.

lata, Micromonospora aurantiaca, Micromonospora brunnea, Micromonospora chalcea, Micromonospora halophytica, Micromonospora inositola, Micromonospora melanosporea, Micromonospora purpurea, Micromonospora purpureochromogenes, Micromonospora rhodorangea, and Micromonospora glauca.

Microvirgula Medium (DSMZ Medium 957) Composition per liter: Na-succinate.................................................................................. 1.5g KNO3 ............................................................................................ 1.5g (NH4)2SO4 ..................................................................................... 1.0g KH2PO4 ....................................................................................... 0.82g K2HPO4 ......................................................................................... 0.7g MgSO4·7H2O ................................................................................ 0.5g Yeast extract................................................................................ 0.25g pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. The final pH should be 7.0.

Preparation of Medium: To 100.0mL of Middlebrook 7H9 broth,

Use: For the cultivation of Mycobacterium species from the blood of patients suspected of having mycobacteremia.

Middlebrook ADC Enrichment (Middlebrook Albumin Dextrose Catalase Enrichment) Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g Catalase..................................................................................... 0.003g

Source: This medium is available as a prepared enrichment from BD Diagnostic Systems. Preparation of Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Use: For use as a supplement to other Middlebrook media for the isolation, cultivation, and maintenance of Mycobacterium species. Also used as a supplement to other Middlebrook media for determining the antimicrobial susceptibility of mycobacteria.

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Middlebrook 7H9 Broth with Middlebrook ADC Enrichment

Middlebrook 7H9 Broth with Middlebrook ADC Enrichment Composition per liter: Na2HPO4 ....................................................................................... 2.5g KH2PO4 ......................................................................................... 1.0g Monosodium glutamate ................................................................ 0.5g (NH4)2SO4 ..................................................................................... 0.5g Sodium citrate ............................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Middlebrook ADC enrichment ..............................................100.0mL Glycerol .....................................................................................2.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook ADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g Catalase ...................................................................................... 3.0mg

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook ADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/deionized water and add remaining components, except Middlebrook ADC enrichment. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook ADC enrichment. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H9 Broth with Middlebrook OADC Enrichment Composition per liter: Na2HPO4 ....................................................................................... 2.5g KH2PO4 ......................................................................................... 1.0g Monosodium glutamate ................................................................ 0.5g (NH4)2SO4 ..................................................................................... 0.5g Sodium citrate ............................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................2.0mL pH 6.6 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook OADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl............................................................................................ 0.85g Oleic acid .................................................................................... 0.05g Catalase...................................................................................... 4.0mg

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook OADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/deionized water and add remaining components, except Middlebrook OADC enrichment. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H9 Broth with Middlebrook OADC Enrichment and Triton™ WR 1339 Composition per liter: Na2HPO4 ....................................................................................... 2.5g KH2PO4......................................................................................... 1.0g Monosodium glutamate ................................................................ 0.5g (NH4)2SO4 .................................................................................... 0.5g Sodium citrate............................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Middlebrook OADC enrichment with Triton™ WR 1339...................................................100.0mL Glycerol .....................................................................................2.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook OADC Enrichment with Triton™ WR 1339: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl............................................................................................ 0.85g Triton™ WR 1339 ...................................................................... 0.25g Oleic acid .................................................................................... 0.05g Catalase...................................................................................... 4.0mg

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Middlebrook 7H10 Agar with Middlebrook OADC Enrichment Preparation of Middlebrook OADC Enrichment with Triton™ WR 1339: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/deionized water and add remaining components, except Middlebrook OADC enrichment with Triton™ WR 1339. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment with Triton™ WR 1339. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H9 Broth, Supplemented Composition per liter: Na2HPO4 ....................................................................................... 2.5g KH2PO4 ......................................................................................... 1.0g Monosodium glutamate ................................................................ 0.5g (NH4)2SO4 ..................................................................................... 0.5g Tween™ 80 ................................................................................... 0.5g Sodium citrate ............................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g Mycobactin J.............................................................................. 2.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Dubos oleic albumin complex ...............................................100.0mL Glycerol .....................................................................................2.0mL pH 6.6 ± 0.2 at 25°C

Source: Mycobactin J is available from Allied Laboratories, Inc. Dubos Oleic Albumin Complex: Composition per 100.0mL: Bovine serum albumin, fraction V................................................ 5.0g Oleic acid, sodium salt................................................................ 0.05g NaCl (0.85% solution) ...........................................................100.0mL

Preparation of Dubos Oleic Albumin Complex: Add bovine serum albumin and oleic acid to 100.0mL of NaCl solution. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except Dubos oleic albumin complex, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile Dubos oleic albumin complex. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Mycobacterium avium. Middlebrook 7H10 Agar with Glycerol See: Middlebrook 7H10 Agar with Middlebrook ADC Enrichment

Middlebrook 7H10 Agar with Middlebrook ADC Enrichment Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g © 2010 by Taylor and Francis Group, LLC

1161

KH2PO4......................................................................................... 1.5g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid............................................................................. 0.5g Sodium citrate............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O.............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook ADC enrichment ..............................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Middlebrook ADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g Catalase..................................................................................... 0.003g

Source: The medium and enrichment are available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook ADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H10 Agar with Middlebrook OADC Enrichment (Middlebrook and Cohn 7H10 Agar) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4......................................................................................... 1.5g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid............................................................................. 0.5g Sodium citrate............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O.............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

1162

Middlebrook 7H10 Agar with Middlebrook OADC Enrichment and Hemin

Middlebrook OADC Enrichment: Composition per 100.0mL:

Hemin Solution: Composition per 100.0mL:

Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl ............................................................................................ 0.85g Oleic acid .................................................................................... 0.05g Catalase ...................................................................................... 4.0mg

Hemin ........................................................................................... 1.0g NaOH (1N solution).................................................................20.0mL

Preparation of Hemin Solution: Add hemin to 20.0mL of 1N

Source: This enrichment is available as a prepared enrichment from

Preparation of Medium: Add glycerol to 891.1mL of distilled/de-

BD Diagnostic Systems.

ionized water and add remaining components, except Middlebrook OADC enrichment. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Middlebrook OADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add glycerol to 900.0mL of distilled/deionized water and add remaining components, except Middlebrook OADC enrichment. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H10 Agar with Middlebrook OADC Enrichment and Hemin (Hemin Medium for Mycobacterium) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g (NH4)2SO4 ..................................................................................... 0.5g L-Glutamic acid ............................................................................. 0.5g Sodium citrate ............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................5.0mL Hemin solution...........................................................................3.9mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook OADC Enrichment: Composition per 100.0mL:

NaOH solution. Mix thoroughly. Bring volume to 100.0mL with distilled/ deionized water.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. For the cultivation and maintenance of Mycobacterium haemophilum. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H10 Agar with Middlebrook OADC Enrichment and Triton™ WR 1339 Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4......................................................................................... 1.5g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid............................................................................. 0.5g Sodium citrate............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook OADC enrichment with Triton™ WR 1339....100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Source: This enrichment is available as a prepared enrichment from

BD Diagnostic Systems.

Preparation of Middlebrook OADC Enrichment: Add com-

ionized water and add remaining components, except Middlebrook OADC enrichment with Triton™ WR 1339. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–

Preparation of Middlebrook OADC Enrichment with Triton™ WR 1339: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/de-

Middlebrook 7H11 Agar with Middlebrook ADC Enrichment

121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment with Triton™ WR 1339. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation, cultivation, and maintenance of Mycobacterium species, including Mycobacterium tuberculosis. Also used for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook 7H10 Agar with Streptomycin Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g (NH4)2SO4 ..................................................................................... 0.5g L-Glutamic acid ............................................................................. 0.5g Sodium citrate ............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Malachite Green...................................................................... .0.25mg Glycerol .....................................................................................5.0mL Streptomycin .......................................................................... 100.0mg pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add glycerol to 1.0L of distilled/deionized water and add remaining components. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically add streptomycin. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation, cultivation, and maintenance of Mycobacterium kansasii.

Middlebrook 7H11 Agar, Selective Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g Pancreatic digest of casein ............................................................ 1.0g (NH4)2SO4 ..................................................................................... 0.5g L–Glutamic acid ............................................................................ 0.5g Sodium citrate ............................................................................... 0.4g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine .................................................................................. 1.0mg ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg CaCl2·2H2O................................................................................ 0.5mg Malachite Green....................................................................... 0.25mg D–Biotin.......................................................................................0.5μg Middlebrook OADC enrichment ...........................................100.0mL Antibiotic solution ...................................................................10.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

1163

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook OADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Antibiotic Solution: Composition per 10.0mL: Carbenicillin ............................................................................ 0.05mg Trimethoprim lactate................................................................ 0.02mg Amphotericin B ....................................................................... 0.01mg Polymyxin B ........................................................................ 200,000U

Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add glycerol to 890.0mL of distilled/deionized water and add remaining components, except Middlebrook OADC enrichment and antibiotic solution. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment and 10.0mL of sterile antibiotic solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation and cultivation of pathogenic mycobacteria from specimens potentially contaminated with bacteria and fungi.

Middlebrook 7H11 Agar with Middlebrook ADC Enrichment (Mycobacteria 7H11 Agar with Middlebrook ADC Enrichment) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4......................................................................................... 1.5g Pancreatic digest of casein............................................................ 1.0g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid ............................................................................ 0.5g Sodium citrate............................................................................... 0.4g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine.................................................................................. 1.0mg Malachite Green....................................................................... 0.25mg D-Biotin....................................................................................... 0.5μg Middlebrook ADC enrichment ..............................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook ADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g

1164

Middlebrook 7H11 Agar with Middlebrook OADC Enrichment

Glucose ......................................................................................... 2.0g Catalase ..................................................................................... 0.003g

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook ADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Use: For the cultivation of drug-resistant (isoniazid [INH]) strains of Mycobacterium tuberculosis. For the cultivation of particularly fastidious strains of tubercle bacilli that occur following treatment of tuberculosis patients with secondary antitubercular drugs. Generally, these strains fail to grow on 7H10 medium.

Middlebrook 7H11 Agar with Middlebrook OADC Enrichment (Mycobacteria 7H11 Agar with Middlebrook OADC Enrichment) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g Pancreatic digest of casein ............................................................ 1.0g (NH4)2SO4 ..................................................................................... 0.5g L-Glutamic acid............................................................................. 0.5g Sodium citrate ............................................................................... 0.4g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine .................................................................................. 1.0mg Malachite Green....................................................................... 0.25mg D-Biotin.......................................................................................0.5μg Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Middlebrook OADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl ............................................................................................ 0.85g Oleic acid .................................................................................... 0.05g Catalase ...................................................................................... 4.0mg

Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Middlebrook 7H11 Agar with Middlebrook OADC Enrichment and Triton™ WR 1339 (Mycobacteria 7H11 Agar with Middlebrook OADC Enrichment and Triton™ WR 1339) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4......................................................................................... 1.5g Pancreatic digest of casein............................................................ 1.0g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid ............................................................................ 0.5g Sodium citrate............................................................................... 0.4g MgSO4·7H2O .............................................................................. 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine.................................................................................. 1.0mg Malachite Green....................................................................... 0.25mg D-Biotin....................................................................................... 0.5μg Middlebrook OADC enrichment with Triton™ WR 1339...................................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Preparation of Middlebrook OADC Enrichment with Triton™ WR 1339: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/de-

Preparation of Middlebrook OADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

ionized water and add remaining components, except Middlebrook OADC enrichment with Triton™ WR-1339. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook OADC enrichment with Triton™ WR-1339. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Medium: Add glycerol to 900.0mL of distilled/de-

Use: For the cultivation of drug-resistant (isoniazid [INH]) strains of

ionized water and add remaining components, except Middlebrook OADC enrichment. Mix thoroughly. Gently heat and bring to boiling.

Mycobacterium tuberculosis. For the cultivation of particularly fastidious strains of tubercle bacilli that occur following treatment of tuberculosis

Source: This enrichment is available as a prepared enrichment from BD Diagnostic Systems.

Middlebrook 7H12 Medium

1165

patients with secondary antitubercular drugs. Generally, these strains fail to grow on 7H10 medium.

Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Middlebrook 7H11 HiVeg Agar Base with Middlebrook ADC Enrichment

Source: This medium, without glycerol and Middlebrook OADC en-

Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g Plant hydrolysate........................................................................... 1.0g L-Glutamic acid............................................................................. 0.5g (NH4)2SO4 ..................................................................................... 0.5g Sodium citrate ............................................................................... 0.4g MgSO4 ........................................................................................ 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine .................................................................................. 1.0mg Malachite green.......................................................................... 1.0mg Biotin ......................................................................................... 0.5mg Middlebrook ADC enrichment ..............................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium, without glycerol and Middlebrook ADC enrichment, is available as a premixed powder from HiMedia.

Preparation of Middlebrook ADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Middlebrook 7H11 HiVeg Agar Base with Middlebrook OADC Enrichment Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g Plant hydrolysate........................................................................... 1.0g L-Glutamic acid............................................................................. 0.5g (NH4)2SO4 ..................................................................................... 0.5g Sodium citrate ............................................................................... 0.4g MgSO4 ........................................................................................ 0.05g Ferric ammonium citrate............................................................. 0.04g Pyridoxine .................................................................................. 1.0mg Malachite green.......................................................................... 1.0mg Biotin ......................................................................................... 0.5mg © 2010 by Taylor and Francis Group, LLC

richment, is available as a premixed powder from HiMedia.

Preparation of Middlebrook OADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Middlebrook 7H12 Medium Composition per 102.5mL: Bovine serum albumin.................................................................. 0.5g Casein hydrolyslate....................................................................... 0.1g Catalase..................................................................................... 4800U 14 C-Palmitic acid ..................................................................... 100μCi Middlebrook 7H9 broth .........................................................100.0mL Antibiotic solution .....................................................................2.5mL pH 6.8 ± 0.1 at 25°C

Preparation of Middlebrook 7H9 Broth: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Antibiotic Solution: Composition per 5.0mL: Nalidixic acid................................................................................ 0.2g Azlocillin ...................................................................................... 0.1g Amphotericin B .......................................................................... 0.05g

1166

Middlebrook Medium

Trimethoprim .............................................................................. 0.05g Polymyxin B ........................................................................ 500,000U

Use: For the isolation, cultivation, and maintenance of Mycobacterium species.

Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize.

Middlebrook Medium with Mycobactin (DSMZ Medium 780)

Preparation of Medium: To 100.0mL of Middlebrook 7H9 broth,

Composition per liter:

add remaining components, except antibiotic solution. Mix thoroughly. Filter sterilize. Aseptically distribute into bottles in 4.0mL volumes. Prior to inoculation, aseptically add 0.1mL of antibiotic solution to each bottle. Mix thoroughly.

Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4......................................................................................... 1.5g (NH4)2SO4 .................................................................................... 0.5g L-Glutamic acid............................................................................. 0.5g Sodium citrate............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g Mycobactin J.............................................................................. 2.0mg ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine.................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O ............................................................................... 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook ADC enrichment ..............................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Use: For the cultivation of Mycobacterium species from the blood of patients suspected of having mycobacteremia.

Middlebrook and Cohn 7H10 Agar See: Middlebrook 7H10 Agar with Middlebrook OADC Enrichment

Middlebrook Medium (DSMZ Medium 645) Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4 ....................................................................................... 1.5g KH2PO4 ......................................................................................... 1.5g (NH4)2SO4 ..................................................................................... 0.5g L-Glutamic acid ............................................................................. 0.5g Sodium citrate ............................................................................... 0.4g Ferric ammonium citrate............................................................. 0.04g MgSO4·7H2O ............................................................................ 0.025g ZnSO4·7H2O .............................................................................. 1.0mg CuSO4·5H2O .............................................................................. 1.0mg Pyridoxine .................................................................................. 1.0mg Biotin ......................................................................................... 0.5mg CaCl2·2H2O................................................................................ 0.5mg Malachite Green...................................................................... .0.25mg Middlebrook OADC enrichment ...........................................100.0mL Glycerol .....................................................................................5.0mL pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-

Source: Mycobactin J is available from Allied Laboratories, Inc. Middlebrook ADC Enrichment: Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g Catalase..................................................................................... 0.003g Distilled water........................................................................100.0mL

Source: This medium and enrichment is available from BD Diagnostic Systems. Preparation of Middlebrook ADC Enrichment: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add glycerol to 900.0mL of distilled/de-

Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g Catalase ..................................................................................... 0.003g Distilled water........................................................................100.0mL

ionized water and add remaining components, except Middlebrook ADC enrichment and mycobactin. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 100.0mL of sterile Middlebrook ADC enrichment and mycobactin. The mycobactin is dissolved in 2.0mL ethanol. Be sure to add all of the mycobactin; wash with additional 2.0mL ethanol if needed. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Source: This enrichment is available as a prepared enrichment from

Use: For the cultivation of Mycobacterium avium subsp. paratubercu-

agnostic Systems.

Middlebrook OADC Enrichment: Composition per 100.0mL:

BD Diagnostic Systems.

losis.

Middlebrook OADC Enrichment (Middlebrook Oleic Albumin Dextrose Catalase Enrichment) Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl............................................................................................ 0.85g Oleic acid .................................................................................... 0.05g Catalase...................................................................................... 4.0mg

Milk HiVeg Agar Source: This enrichment is available as a prepared enrichment from

1167

BD Diagnostic Systems.

to boiling. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Enrichment: Add components to distilled/deion-

Use: For the cultivation and differentiation of members of the Enter-

ized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

obacteriaceae on the basis of motility, lysine decarboxylase activity, lysine deaminase activity, and indole production.

Use: For use as a supplement to other Middlebrook media for the iso-

Milk Agar See: Skim Milk Agar

lation, cultivation, and maintenance of Mycobacterium species. Also used as a supplement to other Middlebrook media for determining the antimicrobial susceptibility of mycobacteria.

Milk Agar Middlebrook OADC Enrichment with Triton™ WR 1339 (Middlebrook Oleic Albumin Dextrose Catalase Enrichment with Triton™ WR 1339) Composition per 100.0mL: Bovine albumin fraction V ........................................................... 5.0g Glucose ......................................................................................... 2.0g NaCl ............................................................................................ 0.85g Triton™ WR 1339 ...................................................................... 0.25g Oleic acid .................................................................................... 0.05g Catalase ...................................................................................... 4.0mg

Composition per liter: Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g Yeast extract.................................................................................. 3.0g Milk (solids or............................................................................... 1.0g fresh milk) ........................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add components to distilled/deionized

BD Diagnostic Systems.

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Enrichment: Add components to distilled/deion-

Use: For the cultivation of microorganisms from dairy and water sam-

ized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

ples.

Source: This enrichment is available as a prepared enrichment from

Milk Agar

Use: For use as a supplement to other Middlebrook media for the isolation, cultivation, and maintenance of Mycobacterium species. Also used as a supplement to other Middlebrook media for determining the antimicrobial susceptibility of mycobacteria.

Middlebrook Oleic Albumin Dextrose Catalase Enrichment See: Middlebrook OADC Enrichment Middlebrook Oleic Albumin Dextrose Catalase Enrichment with Triton™ WR 1339 See: Middlebrook OADC Enrichment with Triton™ WR 1339

MIL Medium (Motility Indole Lysine Medium) Composition per liter: Peptone........................................................................................ 10.0g Pancreatic digest of casein .......................................................... 10.0g L-Lysine·HCl ............................................................................... 10.0g Yeast extract.................................................................................. 3.0g Agar .............................................................................................. 2.0g Dextrose ........................................................................................ 1.0g Ferric ammonium citrate............................................................... 0.5g Bromcresol Purple ...................................................................... 0.02g pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder and prepared

Composition per liter: Mixture A...............................................................................500.0mL Mixture B...............................................................................500.0mL

Mixture A: Composition per 500.0mL: Instant nonfat milk.................................................................... 100.0g

Preparation of Mixture A: Add instant nonfat milk to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool rapidly to 55°C.

Mixture B: Composition per 500.0mL: Agar ............................................................................................ 15.0g Nutrient broth.............................................................................. 12.5g NaCl.............................................................................................. 2.5g

Preparation of Mixture B: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool rapidly to 55°C.

Preparation of Medium: Aseptically combine cooled, sterile mixture A with cooled, sterile mixture B. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes.

Use: For the cultivation and estimation of the numbers of Pseudomonas aeruginosa in water by the membrane filter method.

Milk HiVeg Agar

medium from BD Diagnostic Systems.

Composition per liter:

Preparation of Medium: Add components to distilled/deionized

1168

Milk HiVeg Agar

Yeast extract.................................................................................. 3.0g Milk solids .................................................................................... 1.0g pH 7.2 ± 0.2 at 25°C

Plant peptones............................................................................. 10.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Media.

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of microorganisms from dairy and water samples.

Milk HiVeg Agar (Brown and Scott Modified) Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation of Bacillus subtilis and Escherichia coli.

Miller Luria Bertani HiVeg Broth (Luria Bertani HiVeg Broth, Miller) Composition per liter:

Instant nonfat milk .................................................................... 100.0g Agar ............................................................................................ 15.0g Plant peptone................................................................................. 5.0g NaCl .............................................................................................. 5.0g Plant extract .................................................................................. 1.5g Yeast extract.................................................................................. 1.5g pH 7.2 ± 0.2 at 25°C

NaCl............................................................................................ 10.0g Plant peptones............................................................................. 10.0g Yeast extract.................................................................................. 5.0g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Media.

Preparation of Medium: Add components, except milk, to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Separately add nonfat milk to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 5 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically combine the two sterile solutions. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Use: For the confirmation of Pseudomonas aeruginosa in swimming pool waters.

Milk Protein Hydrolysate Agar See: MPH Agar

Milk Salt HiVeg Agar Base Composition per liter: NaCl ............................................................................................ 65.0g Agar ............................................................................................ 15.0g Plant extract .................................................................................. 3.0g Plant peptone................................................................................. 5.0g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation of halophilic microorganisms.

Miller Luria Bertani HiVeg Agar (Luria Bertani HiVeg Agar, Miller) Composition per liter: Agar ............................................................................................ 15.0g NaCl ............................................................................................ 10.0g © 2010 by Taylor and Francis Group, LLC

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized

Use: For the cultivation of Bacillus subtilis and Escherichia coli.

Mineral Agar Composition per liter: NH4Cl ........................................................................................... 0.5g Na2HPO4·7H2O...................................................................... 670.0mg KH2PO4.................................................................................. 340.0mg MgSO4·7H2O ......................................................................... 112.0mg CaCl2 ........................................................................................ 14.0mg ZnSO4·7H2O .............................................................................. 5.0mg Na2MoO4·2H2O ......................................................................... 2.5mg FeCl3 ........................................................................................ 0.13mg 1,4-Dichlorobenzene............................................................... variable pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except 1,4-dichlorobenzene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. After inoculation, place Petri dishes or tubes into a desiccator. Add a few crystals of 1,4-dichlorobenzene to the desiccator.

Use: For the cultivation of dichlorobenzene-degrading Pseudomonas species.

Mineral Base E for Autotrophic Growth Composition per liter: Noble agar................................................................................... 15.0g K2HPO4......................................................................................... 1.2g KH2PO4..................................................................................... 0.624g (NH4)2SO4 .................................................................................... 0.5g NaCl.............................................................................................. 0.1g CaCl2·6H2O solution................................................................10.0mL MgSO4·7H2O solution .............................................................10.0mL

Mineral Lactate Medium

Mineral solution .........................................................................1.0mL p-Aminobenzoic acid solution ...................................................1.0mL

CaCl2·6H2O Solution: Composition per liter: CaCl2·6H2O................................................................................... 5.0g

Preparation of CaCl2·6H2O Solution: Add CaCl2·6H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

MgSO4·7H2O Solution: Composition per liter: MgSO4·7H2O .............................................................................. 20.0g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

p-Aminobenzoic Acid Solution: Composition per 10.0.mL: p-Aminobenzoic acid............................................................. 100.0mg

Preparation of p-Aminobenzoic Acid Solution: Add p-aminobenzoic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Mineral Solution: Composition per 1000.0mL: Disodium EDTA ......................................................................... 1.58g ZnSO4·7H2O ................................................................................ 0.7g MnSO4·4H2O .............................................................................. 0.18g FeSO4·7H2O................................................................................ 0.16g CoCl2·6H2O .............................................................................. 0.052g Na2MoO4·2H2O ........................................................................ 0.047g CuSO4·5H2O ............................................................................. 0.047g

Preparation of Medium: Add components, except CaCl2·6H2O solution, MgSO4·7H2O solution, and p-aminobenzoic acid solution, to distilled/deionized water and bring volume to 979.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add in the following order: 10.0mL of sterile CaCl2·6H2O solution, 10.0mL of sterile MgSO4·7H2O solution, and 1.0mL of sterile p-aminobenzoic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Incubate inoculated tubes in 50% CO2. Use: For the autotrophic cultivation and maintenance of Pseudomonas thermocarboxydovorans.

Mineral Base E for Heterotrophic Growth Composition per liter: Noble agar................................................................................... 15.0g K2HPO4 ......................................................................................... 1.2g KH2PO4 ..................................................................................... 0.624g (NH4)2SO4 ..................................................................................... 0.5g NaCl .............................................................................................. 0.1g CaCl2·6H2O solution................................................................10.0mL MgSO4·7H2O solution .............................................................10.0mL Sodium pyruvate solution ........................................................10.0mL Mineral solution .........................................................................1.0mL p-Aminobenzoic acid solution ...................................................1.0mL

1169

Preparation of CaCl2·6H2O Solution: Add CaCl2·6H2O to dis-

tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

MgSO4·7H2O Solution: Composition per liter: MgSO4·7H2O .............................................................................. 20.0g

Preparation of MgSO4·7H2O Solution: Add MgSO4·7H2O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Sodium Pyruvate Solution: Composition per 10.0mL: Sodium pyruvate........................................................................... 2.0g

Preparation of Sodium Pyruvate Solution: Add sodium pyruvate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. p-Aminobenzoic Acid Solution: Composition per 10.0mL: p-Aminobenzoic acid............................................................. 100.0mg

Preparation of p-Aminobenzoic Acid Solution: Add p-aminobenzoic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Mineral Solution: Composition per 100.0mL: Disodium EDTA ......................................................................... 1.58g ZnSO4·7H2O ................................................................................ 0.7g MnSO4·4H2O .............................................................................. 0.18g FeSO4·7H2O................................................................................ 0.16g CoCl2·6H2O .............................................................................. 0.052g Na2MoO4·2H2O ........................................................................ 0.047g CuSO4·5H2O............................................................................. 0.047g

Preparation of Medium: Add components, except CaCl2·6H2O solution, MgSO4·7H2O solution, sodium pyruvate solution, and p-aminobenzoic acid solution, to distilled/deionized water and bring volume to 969.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add in the following order: 10.0mL of the sterile CaCl2·6H2O solution, 10.0mL of the sterile MgSO4·7H2O solution, 10.0mL of sterile sodium pyruvate solution, and 1.0mL of sterile p-aminobenzoic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the heterotrophic cultivation and maintenance of Pseudomonas thermocarboxydovorans.

Mineral Base Medium with Acetate See: MBM Acetate Medium

Mineral Lactate Medium Composition per liter: K2HPO4·3H2O ............................................................................ 1.13g NaCl.............................................................................................. 1.0g NH4Cl ........................................................................................... 1.0g KH2PO4....................................................................................... 0.88g MgSO4·7H2O ............................................................................... 0.5g Sodium lactate .............................................................................. 0.5g CaCl2·2H2O ............................................................................... 5.0mg Trace elements solution .............................................................1.2mL pH 7.0 ± 0.2 at 25°C

1170

Mineral Medium

Trace Elements Solution: Composition per liter: Disodium EDTA ......................................................................... 50.0g ZnSO4·7H2O ............................................................................... 22.0g CaCl2·2H2O................................................................................. 5.54g MnCl2·4H2O................................................................................ 5.06g FeSO4·7H2O.................................................................................. 5.0g CoCl2·6H2O ................................................................................ 1.61g CuSO4·5H2O ............................................................................... 1.57g (NH4)6Mo7O24·4H2O .................................................................... 1.1g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0 with KOH. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Mineral Medium Composition per liter: NH4Cl ........................................................................................... 0.5g Yeast extract.................................................................................. 0.2g 1,4-Dichlorobenzene..................................................................... 0.1g Na2HPO4·7H2O...................................................................... 670.0mg KH2PO4.................................................................................. 340.0mg MgSO4·7H2O ......................................................................... 112.0mg CaCl2 ........................................................................................ 14.0mg ZnSO4·7H2O .............................................................................. 5.0mg Na2MoO4·2H2O ......................................................................... 2.5mg FeCl3 ........................................................................................ 0.13mg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of dichlorobenzene-degrading Pseudomonas species.

Use: For the cultivation and maintenance of Pseudomonas species and Spirillum species.

Mineral Medium Composition per liter: Yeast extract.................................................................................. 2.0g Mineral base 5X.....................................................................200.0mL Trace elements solution SL-6 ....................................................1.0mL Thiamine·HCl .............................................................................3.0μg Biotin ..........................................................................................0.2μg pH 6.8 ± 0.2 at 25°C

Mineral Base 5X: Composition per liter: NaCl .............................................................................................. 5.0g NH4Cl ........................................................................................... 2.0g KH2PO4 ....................................................................................... 1.35g MgSO4·7H2O ................................................................................ 1.0g K2HPO4 ....................................................................................... 0.87g CaCl2 ........................................................................................... 0.05g FeCl3·6H2O .............................................................................. 1.25mg

Preparation of Mineral Base 5X: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Mineral Medium (DSMZ Medium 994) Composition per liter: D-Glucose...................................................................................... 3.6g KH2PO4......................................................................................... 1.0g NH4Cl ........................................................................................... 0.6g NaCl.............................................................................................. 0.4g MgSO4·7H2O ................................................................................ 0.4g CaCl2·2H2O ............................................................................... 0.5mg Vitamin solution.........................................................................5.0mL Trace elements solution SL-4 ....................................................1.0mL pH 6.8 ± 0.2 at 25°C

Vitamin Solution: Composition per liter: Thiamine-HCl·2H2O................................................................ 50.0mg Riboflavin ................................................................................ 50.0mg Vitamin B12 .............................................................................. 50.0mg D-Ca-pantothenate ................................................................... 50.0mg p-Aminobenzoic acid............................................................... 50.0mg Lipoic acid ............................................................................... 50.0mg Nicotinic acid........................................................................... 25.0mg Niconinamide........................................................................... 25.0mg Biotin ....................................................................................... 20.0mg Folic acid ................................................................................. 20.0mg Pyridoxine-HCl........................................................................ 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly for several hours. Filter sterilize. Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Mineral Medium with Antipyrin

NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

1171

Mineral Medium with 3-Aminophenol (DSMZ Medium 465f)

Preparation of Trace Elements Solution SL-6: Add components

Composition per liter:

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Na2HPO4·2H2O............................................................................. 3.5g KH2PO4......................................................................................... 1.0g (NH4)2SO4 .................................................................................... 0.5g MgCl2·6H2O ................................................................................. 0.1g Ca(NO3)2·4H2O .......................................................................... 0.05g Aminophenol solution .............................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except vitamin and trace elements solutions, to distilled/deionized water and bring volume to 994.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Aseptically add sterile trace elements and vitamin solutions. Mix thoroughly. Adjust pH to 6.8. Aseptically dispense into tubes, flasks, or bottles. Use: For the cultivation of Stenotrophomonas maltophilia.

Mineral Medium (DSMZ Medium 1007) Composition per liter: KNO3 .......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.05g CaCl2·2H2O................................................................................. 0.01g Methanol ..................................................................................75.0mL Trace elements solution .............................................................1.0mL pH 5.7 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: EDTA ............................................................................................ 5.0g FeSO4·7H2O................................................................................. 2.0g CoCl2·6H2O .................................................................................. 0.2g CuCl2·2H2O .................................................................................. 0.1g ZnSO4·7H2O ................................................................................ 0.1g MnCl2·4H2O................................................................................ 0.03g H3BO3 ......................................................................................... 0.03g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except trace elements solution, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add sterile trace elements solution. Mix thoroughly. Adjust pH to 5.7. Aseptically dispense into tubes, flasks, or bottles. Note: Methane can be substituted for methanol for the growth of some strains.

Use: For the cultivation of Methylocella tundrae and Methylocystis heyeri.

Mineral Medium A Composition per liter: (NH4)2SO4 ..................................................................................... 1.0g K2HPO4 ......................................................................................... 1.0g

Preparation of Medium: Add components to tap water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Saccharobacterium ovale. © 2010 by Taylor and Francis Group, LLC

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Aminophenol Solution: Add 100.0mL boiling water to 1.0g aminophenol crystals. Stir the solution to mix thoroughly. Cool to room temperature. Sterilize by filtration.

Preparation of Medium: Add components, except aminophenol solution, to 990.0mL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL aminophenol solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks. Use: For the cultivation of Arthrobacter sp. and other aminophenol-utilizing bacteria.

Mineral Medium with Antipyrin Composition per liter: Antipyrin....................................................................................... 1.0g Na2HPO4·12H2O........................................................................... 0.7g (NH4)2HPO4.................................................................................. 0.7g KH2PO4......................................................................................... 0.3g (NH4)H2PO4.................................................................................. 0.3g MgSO4·7H2O .............................................................................. 0.25g (NH4)2SO4 .................................................................................... 0.1g CaCl2·6H2O ................................................................................ 0.05g H3BO3 ........................................................................................ 0.5mg MnSO4·4H2O ............................................................................. 0.4mg ZnSO4·7H2O .............................................................................. 0.4mg FeCl3·6H2O................................................................................ 0.2mg

1172

Mineral Medium with Atrazine

(NH4)6Mo7O24·4H2O ................................................................. 0.2mg KI ............................................................................................... 0.1mg CuSO4·5H2O ............................................................................ 0.04mg Vitamin solution.......................................................................20.0mL pH 6.8–7.0 at 25°C

Preparation of Medium: Add components, except atrazine solution, to 990.0mLL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL atazine solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Vitamin Solution: Composition per 20.0mL:

Use: For the cultivation of Pseudomonas sp. and other atrazine-utilizing bacteria.

Biotin ......................................................................................... 0.1mg Vitamin B12 .............................................................................. 0.03mg

Preparation of Vitamin Solution: Add biotin and vitamin B12 to 20.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 6.8–7.0 with 1N NaOH. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add the sterile vitamin solution. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Phenylobacterium immobile.

Mineral Medium with Atrazine (DSMZ Medium 465i) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g Na-citrate ...................................................................................... 1.0g KH2PO4 ......................................................................................... 1.0g MgCl2·6H2O.................................................................................. 0.1g CaCl2 ........................................................................................... 0.05g Atrazine solution......................................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter:

Mineral Medium with Benzylcyanide (DSMZ Medium 465d) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4......................................................................................... 1.0g MgCl2·6H2O ................................................................................. 0.1g Ca(NO3)2·4H2O .......................................................................... 0.05g Benzylcyanide solution............................................................10.0mL Glucose solution ......................................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution SL-6: Composition per liter:

Glucose Solution: Composition per 10.0mL:

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Atrazine Solution: Composition per 10.0mL: Atrazine..................................................................................... 100mg

Preparation of Atrazine Solution: Add (2-chloro-4(ethylamino)6-(isopropylamino)-1,3,5-triazine) to 10.0mL methanol. Mix thoroughly. Shortly sonicate to reduce particle size. © 2010 by Taylor and Francis Group, LLC

Glucose ......................................................................................... 1.8g

Preparation of Glucose Solution: Add glucose to 10.0mL distilled/deionized water. Mix thoroughly. Filter sterilize.

Benzylcyanide Solution: Composition per 100.0mL: Benzylcyanide............................................................................. 0.12g

Preparation of Benzylcyanide Solution: Add benzylcyanide to 10.0mL distilled/deionized water. Mix thoroughly. Do not sterilize.

Preparation of Medium: Add components, except benzylcyanide solution and glucose solution, to 980.0mL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL glucose solution and 10.0mL benzylcyanide solution to the medium. Mix thoroughly. Aseptically distribute the medium to sterile tubes or flasks.

Use: For the cultivation of Pseudomonas sp., Rhodococcus erythropolis, and other benzylcyanide-utilizing bacteria.

Mineral Medium with Chloridazon

Mineral Medium, Brunner

1173

Mineral Medium for Chemolithotrophic Growth

Composition per liter:

Composition per 985.0mL:

Agar ............................................................................................ 15.0g Na2HPO4·2H2O............................................................................. 2.9g KH2PO4......................................................................................... 2.3g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaHCO3 ........................................................................................ 0.5g CaCl2·2H2O ................................................................................ 0.01g Ferric ammonium citrate solution............................................20.0mL Trace elements solution SL-6 ....................................................5.0mL pH 6.8 ± 0.2 at 25°C

Ferric Ammonium Citrate Solution: Composition per 20.0mL:

Trace Elements Solution SL-6: Composition per liter:

Preparation of Ferric Ammonium Citrate Solution: Add fer-

MnCl2·4H2O.................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and maintenance of Alcaligenes species,

Ferric ammonium citrate............................................................. 0.05g ric ammonium citrate to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C.

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except ferric ammoni-

Bacillus benzoevorans, Bacillus gordonae, Comamonas acidovorans, Hyphomicrobium species, Moraxella species, Nocardia species, Pseudomonas species, Rhodococcus species, Sphingomonas species, and Xanthobacter species.

um citrate solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 20.0mL of sterile ferric ammonium citrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Mineral Medium with Camphor

Use: For the chemolithotrophic growth and cultivation of a wide variety of bacteria.

Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4 ......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 .......................................................................................10.0μg Camphor crumbs..................................................................... variable pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except camphor crumbs, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Inoculate tubes or flasks and place in a dessicator jar in which crumbs of camphor will be evaporated.

Mineral Medium with Chloridazon Composition per liter: Chloridazon................................................................................... 1.0g Na2HPO4·12H2O........................................................................... 0.7g (NH4)2HPO4.................................................................................. 0.7g KH2PO4......................................................................................... 0.3g (NH4)H2PO4.................................................................................. 0.3g MgSO4·7H2O .............................................................................. 0.25g (NH4)2SO4 .................................................................................... 0.1g CaCl2·6H2O ................................................................................ 0.05g H3BO3 ........................................................................................ 0.5mg MnSO4·4H2O ............................................................................. 0.4mg ZnSO4·7H2O .............................................................................. 0.4mg FeCl3·6H2O................................................................................ 0.2mg (NH4)6Mo7O24·4H2O ................................................................. 0.2mg KI ............................................................................................... 0.1mg CuSO4·5H2O............................................................................ 0.04mg Vitamin solution.......................................................................20.0mL pH 6.8–7.0 at 25°C

1174

Mineral Medium with 2-Chlorobenzoate

Vitamin Solution: Composition per 20.0mL: Biotin ......................................................................................... 0.1mg Vitamin B12 .............................................................................. 0.03mg

Preparation of Vitamin Solution: Add biotin and vitamin B12 to 20.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Mineral Medium with 2-Chlorobenzoate (DSMZ Medium 457a) Composition per liter: Na2HPO4 ..................................................................................... 2.44g KH2PO4 ....................................................................................... 1.52g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g Tween 80....................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.05g Trace elements solution SL-4 ..................................................10.0mL Chlorobenzoate solution ..........................................................10.0mL pH 7.4 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Chlorobenzoate Solution: Composition per liter: 2-Chlorobenzoic acid .................................................................. 78.3g

Preparation of Chlorobenzoate Solution: Add 2-chlorobenzoic acid to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Slowly add concentrated NaOH to adjust pH to 7.4. Filter sterilize. Preparation of Medium: Add components, except chlorobenzoate solution, to 990.0mL distilled/deionized water. Adjust pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL chlorobenzoate solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Mineral Medium with Crude Oil Composition per 100.0mL: K2HPO4....................................................................................... 0.45g (NH4)2SO4 .................................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.02g NaCl............................................................................................ 0.01g CaCl2 ........................................................................................... 0.01g FeCl3 ......................................................................................... 0.002g Crude oil ....................................................................................5.0mL pH 7.2 ± 0.3 at 25°C

Preparation of Medium: Add components, except crude oil, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 5.0mL of filter-sterilized crude oil. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Acinetobacter baumannii.

Mineral Medium with Cyanuric Acid as Nitrogen Source (DSMZ Medium 465g) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4......................................................................................... 1.0g MgCl2·6H2O ................................................................................. 0.1g Ca(NO3)2·4H2O .......................................................................... 0.05g Cyanuric acid solution .............................................................10.0mL Vitamin solution.......................................................................10.0mL Glycerol solution .....................................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Vitamin Solution: Composition per liter: Vitamin B12.............................................................................. 50.0mg Pantothenic acid....................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg Alpha-lipoic acid ..................................................................... 50.0mg p-Aminobenzoic acid............................................................... 50.0mg Thiamine-HCl·2H2O................................................................ 50.0mg Nicotinic acid........................................................................... 25.0mg

Mineral Medium with Dichloromethane

1175

Nicotinamide............................................................................ 25.0mg Biotin ....................................................................................... 20.0mg Folic acid.................................................................................. 20.0mg Pyridoxamine-HCl ................................................................... 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.

2,4-Dichlorobenzoate Solution: Composition per 10.0mL:

Cyanuric Acid Solution: Composition per 10.0mL:

Preparation of 2,4-Dichlorobenzoate Solution: Add 2,4-di-

Cyanuric acid ......................................................................... 645.0mg

Preparation of Cyanuric Acid Solution: Add cyanuric acid to

2,4-Dichlorobenzoate................................................................. 5.0mg chlorobenzoate to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,4-dichlo-

Glycerol Solution: Composition per 10.0mL:

robenzoate solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Adjust pH to 6.8 with 1N KOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add the sterile 2,4-dichlorobenzoate solution. Mix thoroughly. Distribute into sterile tubes or flasks.

Glycerol ........................................................................................ 5.5g

Use: For the cultivation and maintenance of Actinomyces viscosus.

10.0mL distilled/deionized water. Mix thoroughly. Adjust pH to 7.0. Filter sterilize.

Preparation of Glycerol Solution: Add glycerol to distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except cyanuric acid solution, glycerol solution, and vitamin solution, to 1.0L distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL cyanuric acid solution, 10.0mL glycerol solution, and 10.0mL vitamin solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Use: For the cultivation of Gordonia rubripertincta=Rhodococcus rubropertinctus and other cyanuric acid-utilizing bacteria.

Mineral Medium with Dichlorobenzoate Composition per liter: Na2HPO4 ..................................................................................... 2.78g KH2PO4 ....................................................................................... 2.78g (NH4)2SO4 ..................................................................................... 1.0g Hutner’s mineral base ..............................................................20.0mL 2,4-Dichlorobenzoate solution.................................................10.0mL pH 6.8 ± 0.2 at 25°C

Hutner’s Mineral Base: Composition per liter: MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O................................................................................. 3.34g FeSO4·7H2O............................................................................. 99.0mg (NH4)2MoO4 ............................................................................ 9.25mg Metals “44” ..............................................................................50.0mL

Preparation of Hutner’s Mineral Base: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Readjust pH to 7.2 with H2SO4 or KOH. Add distilled/deionized water to 1.0L.

Mineral Medium with Dichloromethane (DSMZ Medium 465c) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4......................................................................................... 1.0g (NH4)2SO4 .................................................................................... 0.5g MgCl2·6H2O ................................................................................. 0.1g Ca(NO3)2·4H2O .......................................................................... 0.05g Bromthymol blue ..................................................................... 50.0mg Methanol ..................................................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL Dichloromethane..................................................................... variable pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components, except methanol and dichloromethane, to 1.0L distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Inoculate the medium and supply methanol via incubation atmosphere (up to 10.0mL methanol per liter medium). Subsequently feed small amounts of dichloromethane (toxic to bacteria at 2 mmol per liter or less) via incubation atmosphere. Readjust pH of the culture with sterile 1M NaOH as necessary

1176

Mineral Medium with 2,4-Dichlorophenoxyacetic Acid

Use: For the cultivation of Methylophilus leisingeri, Rhizobium radiobacter, and other dichloromethane-utilizing bacteria.

Mineral Medium with 2,4-Dichlorophenoxyacetic Acid Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4 ......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 .......................................................................................10.0μg 2,4-Dichlorophenoxyacetic acid solution ................................10.0mL pH 7.0 ± 0.2 at 25°C

2,4-Dichlorophenoxyacetic Acid Solution: Composition per 10.0mL: 2,4-Dichlorophenoxyacetic acid ................................................... 0.5g

Preparation of 2,4-Dichlorophenoxyacetic Acid Solution: Add 2,4-dichlorophenoxyacetic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,4-dichlorophenoxyacetic acid solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile 2,4-dichlorophenoxyacetic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of bacteria that can utilize 2,4-dichlorophenoxyacetic acid as sole carbon source.

Mineral Medium with 2,4-Dichlorotoluene Composition per liter: NH4Cl ........................................................................................... 0.5g Yeast extract.................................................................................. 0.1g 2,4-Dichlorotoluene ...................................................................... 0.1g Na2HPO4·7H2O...................................................................... 670.0mg KH2PO4 .................................................................................. 340.0mg MgSO4·7H2O ......................................................................... 112.0mg CaCl2 ........................................................................................ 14.0mg ZnSO4·7H2O .............................................................................. 5.0mg Na2MoO4·2H2O ......................................................................... 2.5mg FeCl3 ........................................................................................ 0.13mg pH 7.0 ± 0.2 at 25°C

Solution B ................................................................................10.0mL Solution A..................................................................................5.0mL pH 6.8 ± 0.2 at 25°C

Solution A: Composition per 100.0mL: Ferric ammonium citrate............................................................... 1.0g CaCl2 ............................................................................................. 0.1g

Preparation of Solution A: Add ferric ammonium citrate and CaCl2 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Solution B: Composition per 100.0mL: Glucose ....................................................................................... 10.0g

Preparation of Solution B: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except solution A and solution B, to distilled/deionized water and bring volume to 985.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add sterile solution A and sterile solution B. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation and maintenance of Alcaligenes latus.

Mineral Medium H-3 Composition per liter: Agar (if needed).......................................................................... 20.0g Na2HPO4·2H2O ............................................................................ 2.9g KH2PO4 ........................................................................................ 2.3g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O................................................................................ 0.5g NaHCO3........................................................................................ 0.5g CaCl2·2H2O ................................................................................ 0.01g Ferric ammonium citrate solution............................................20.0mL Trace elements solution .............................................................5.0mL

Ferric Ammonium Citrate Solution: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

Use: For the cultivation of 2,4-dichlorotoluene-degrading Pseudomonas species.

H3BO3 ........................................................................................... 0.3g CoCl3·6H2O.................................................................................. 0.2g ZnSO4·7H2O................................................................................. 0.1g MnCl2·4H2O ............................................................................... 0.03g Na2MoO4·2H2O.......................................................................... 0.03g CuCl2·2H2O................................................................................ 0.01g NiCl2·6H2O............................................................................... 0.002g

Mineral Medium with Glucose

Preparation of Trace Elements Solution: Add components to

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Composition per liter:

distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Agar ............................................................................................ 20.0g Na2HPO4 ....................................................................................... 4.8g KH2PO4 ......................................................................................... 4.4g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g

Preparation of Medium: Add components, except ferric ammoni-

Mineral Medium M9

1177

ly. Pour into sterile Petri dishes or distribute into sterile tubes. Incubate cultures in 60% H2 + 28% N2 + 10% CO2 + 2% O2.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Use: For the chemolithotrophic growth of Alcaligenes eutrophus.

Hydroxybiphenyl Solution: Composition per 100.0mL:

Mineral Medium for Hydrogen Bacteria Composition per liter: Agar ............................................................................................ 15.0g Na2HPO4·2H2O............................................................................. 2.9g KH2PO4 ......................................................................................... 2.3g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaHCO3 ........................................................................................ 0.5g CaCl2·2H2O................................................................................. 0.01g Ferric ammonium citrate solution............................................20.0mL

Ferric Ammonium Citrate Solution: Composition per 20.0mL: Ferric ammonium citrate............................................................. 0.05g

Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to 20.0mL of distilled/deionized water. Filter sterilize. Preparation of Medium: Add components, except ferric ammonium citrate solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add the sterile ferric ammonium citrate solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Incubate inoculated medium at 30°C under 60% H2 + 25% N2 + 10% CO2 + 5% O2.

Use: For the cultivation and maintenance of Alcaligenes eutrophus, Hydrogenophaga flava, and Hydrogenophaga pseudoflava.

Mineral Medium with 2-Hydroxybiphenyl (DSMZ Medium 465a) Composition per1010.0mL: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 0.5g MgCl2·6H2O.................................................................................. 0.1g Ca(NO3)2·4H2O........................................................................... 0.05g Hydroxybiphenyl solution .......................................................10.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

2-Hydroxybiphenyl....................................................................... 5.0g

Preparation of Hydroxybiphenyl Solution: Add 2-hydroxybiphenyl to 100.0mL ethanol. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except hydroxybiphenyl solution, to 1.0L distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add hydroxybiphenyl solution to a sterile culture vessel so that the final concentration of 2-hydroxybiphenyl will be 0.5g/L medium. Let the ethanol evaporate under sterile conditions. Aseptically add the liquid medium to the culture vessel to achieve the appropriate concentration of biphenyl.

Use: For the cultivation of Pseudomonas sp. and other hydroxybiphenyl-utilizing bacteria.

Mineral Medium M9 Composition per 1013.0mL: Proline...................................................................................... 20.0mg 10X M9 salts..........................................................................100.0mL Glucose solution ......................................................................10.0mL CaCl2·2H2O solution..................................................................1.0mL MgSO4 solution..........................................................................1.0mL Thiamine·HCl solution ..............................................................1.0mL pH 7.4 ± 0.2 at 25°C

10X M9 Salts: Composition per liter: Na2HPO4 ..................................................................................... 60.0g KH2PO4....................................................................................... 30.0g NH4Cl ......................................................................................... 10.0g NaCl.............................................................................................. 5.0g

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. CaCl2·2H2O Solution: Composition per 100.0mL: CaCl2·2H2O ................................................................................ 1.47g

Preparation of CaCl2·2H2O Solution: Add CaCl2·2H2O to dis-

Trace Elements Solution SL-6: Composition per liter:

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

MgSO4 Solution: Composition per 100.0mL:

Preparation of Trace Elements Solution SL-6: Add components to

Thiamine·HCl Solution: Composition per 10.0mL:

MgSO4 ...................................................................................... 12.04g

Preparation of MgSO4 Solution: Add MgSO4 to distilled/deion-

ized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Thiamine·HCl ............................................................................. 3.37g

1178

Mineral Medium with 6-Methylnicotinate

Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add proline and 10X M9 salts to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile glucose solution, 1.0mL of sterile CaCl2·2H2O solution, 1.0mL of sterile MgSO4 solution, and 1.0mL of sterile thiamine·HCl solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Escherichia coli JM strains.

Mineral Medium with 6-Methylnicotinate (DSMZ Medium 465h) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 0.5g MgCl2·6H2O.................................................................................. 0.1g Ca(NO3)2·4H2O........................................................................... 0.05g Nicotinate solution ...................................................................10.0mL Selenite-tungstate solution .........................................................1.0mL Vitamin solution.........................................................................1.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter: EDTA ............................................................................................ 0.5g FeSO4·7H2O.................................................................................. 0.2g Trace elements solution SL-6 ................................................100.0mL

Trace Elements Solution SL-6: Composition per liter: H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Vitamin Solution: Composition per liter: Vitamin B12 .............................................................................. 50.0mg Pantothenic acid ....................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg Alpha-lipoic acid...................................................................... 50.0mg p-Aminobenzoic acid ............................................................... 50.0mg Thiamine-HCl·2H2O................................................................ 50.0mg Nicotinic acid ........................................................................... 25.0mg Nicotinamide............................................................................ 25.0mg Biotin ....................................................................................... 20.0mg Folic acid.................................................................................. 20.0mg Pyridoxamine-HCl ................................................................... 10.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Selenite-Tungstate Solution Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Nicotinate Solution: Composition per 10.0mL: 6-Methylnicotinate........................................................................ 5.0g

Preparation of Nicotinate Solution: Add nicotinate to 10.0mL distilled/deionized wate. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Medium: Add components, except nicotinate solution, selenite-tungstate solution, and vitamin solution, to 9898.0mL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 10.0mL nicotinate solution, 1.0mL selenite-tungstate solution, and 1.0mL vitamin solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Use: For the cultivation of Paenibacillus sp. and other nicotinate-utilizing bacteria.

Mineral Medium (N4)

Composition per liter:

HEPES buffer ........................................................................... 11.92g NaCl.............................................................................................. 0.5g (NH4)2SO4 .................................................................................... 0.5g KH2PO4 ........................................................................................ 0.2g MgSO4·7H2O.............................................................................. 0.04g CaCl2·2H2O ................................................................................ 0.02g FeSO4/EDTA solution .............................................................10.0mL Phenol Red (0.04%)...................................................................1.0mL

FeSO4/EDTA Solution: Composition per 100.0mL: EDTA ........................................................................................ 0.019g FeSO4·7H2O ............................................................................. 0.018g

Preparation of FeSO4/EDTA Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Bacillus species.

Mineral Medium, Nagel and Andreesen (DSM Medium 461) Composition per liter: MgSO4·7H2O ................................................................................ 0.5g NH4Cl ........................................................................................... 0.3g NaCl............................................................................................ 0.05g CaCl2 ........................................................................................... 0.01g MnSO4 ........................................................................................ 0.01g Phosphate solution ...................................................................50.0mL

Mineral Medium with o-Nitrophenol

Vitamin solution.........................................................................5.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.5 ± 0.2 at 25°C

Phosphate Solution: Composition per 50.0mL: Na2HPO4·2H2O........................................................................... 1.45g KH2PO4 ....................................................................................... 0.25g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C.

Vitamin Solution: Composition per liter: Folic acid..................................................................................... 20.0g α-Lipoic acid............................................................................ 50.0mg p-Aminobenzoic acid............................................................... 50.0mg Pantothenic acid ....................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg Thamine·HCl............................................................................ 50.0mg Vitamin B12 .............................................................................. 50.0mg Nicotinamide............................................................................ 25.0mg Biotin ....................................................................................... 20.0mg Nicotinic acid ........................................................................... 20.0mg Pyridoxamine·HCl ................................................................... 10.0mg

1179

MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 ....................................................................................... 10.0μg Naphthalene crumbs ............................................................... variable pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except naphthalene crumbs, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Inoculate tubes or flasks and place in a dessicator jar in which crumbs of naphthalene will be evaporated. Use: For the cultivation of bacteria that can utilize naphthalene as sole carbon source.

Mineral Medium with Nicotinic Acid Composition per liter:

deionized water and bring volume to 1.0L Mix thoroughly. Stir for 2 hr. Filter sterilize.

Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 ....................................................................................... 10.0μg Nicotinic acid solution.............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter:

Nicotinic Acid Solution: Composition per 10.0mL:

Preparation of Vitamin Solution: Add components to distilled/

FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components, except phosphate solution, vitamin solution, and trace elements solution SL-10, to distilled/ deionized water and bring volume to 944.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 50.0mL of sterile phosphate solution, 5.0mL of sterile vitamin solution, and 1.0mL of sterile trace elements solution SL-10. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Arthrobacter species, Mycobacterium species, Paracoccus denitrificans, Pseudomonas putida, and Rhodococcus species.

Mineral Medium with Naphthalene Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4 ......................................................................................... 1.5g © 2010 by Taylor and Francis Group, LLC

Nicotinic acid................................................................................ 0.5g

Preparation of Nicotinic Acid Solution: Add nicotinic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except nicotinic acid solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile nicotinic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of bacteria that can utilize nicotinic acid as sole carbon source.

Mineral Medium with o-Nitrophenol (DSMZ Medium 461c) Composition per liter: MgSO4·7H2O ................................................................................ 0.5g NH4Cl ........................................................................................... 0.3g NaCl............................................................................................ 0.05g CaCl2 .......................................................................................... 0.01g MnSO4 ........................................................................................ 0.01g o-Nitrophenol solution...........................................................200.0mL Phosphate solution .................................................................100.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

1180

Mineral Medium with PCP

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Pentachlorophenol Solution: Composition per liter:

Phosphate Solution: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 1.45g KH2PO4 ....................................................................................... 0.25g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. o-Nitrophenol Solution: Composition per liter: o-Nitrophenol................................................................................ 0.5g

Preparation of o-Nitrophenol Solution: Dissolve o-nitrophenol in phosphate buffer (50 mM, pH 7.5) and bring volume to 1.0L. Sterilize by filtration.

Preparation of Medium: Add components, except phosphate solution and o-nitrophenol solution, to distilled/deionized water and bring volume to 700.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 100.0mL phosphate solution and 200.0mL o-nitrophenol solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of o-nitrophenol-utilizing bacteria.

Mineral Medium with PCP (DSMZ Medium 465b) Composition per liter:

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Pentachlorophenol ........................................................................ 1.0g

Preparation of Pentachlorophenol Solution: Add pentachlorophenol to 1.0L 0.1M NaOH. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except pentachlorophenol solution, to 900.0mL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 100.0mL sterile pentachlorophenol solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks.

Use: For the cultivation of Sphingobium chlorophenolicum=Sphingomonas chlorophenolica and other pentachlorophenol-utilizing bacteria.

Mineral Medium, pH 7.25 Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4......................................................................................... 1.0g (NH4)2SO4 .................................................................................... 0.5g MgCl2·6H2O ................................................................................. 0.1g Ca(NO3)2·4H2O .......................................................................... 0.05g Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Na2HPO4·2H2O............................................................................. 3.5g KH2PO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 0.5g MgCl2·6H2O.................................................................................. 0.1g Ca(NO3)2·4H2O........................................................................... 0.05g Pentachlorophenol solution....................................................100.0mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution SL-4: Composition per liter:

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to distilled/deionized

Trace Elements Solution SL-6: Composition per liter:

Use: For the cultivation of Azotobacter species, Pseudomonas species,

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g © 2010 by Taylor and Francis Group, LLC

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. and Sphingomonas species.

Mineral Medium with Phenol Composition per liter: Phenol ........................................................................................... 1.0g K2HPO4......................................................................................... 1.0g NH4NO3 ........................................................................................ 1.0g (NH4)2SO4 .................................................................................... 0.5g

Mineral Medium with Quinoline

MgSO4 .......................................................................................... 0.5g KH2PO4 ......................................................................................... 0.5g NaCl .............................................................................................. 0.5g CaCl2 ........................................................................................... 0.02g FeSO4 .......................................................................................... 0.02g Wolfe’s mineral solution ..........................................................10.0mL

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Preparation of Medium: Add components, except phenol, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add the phenol. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Pseudomonas putida.

Mineral Medium with Phenylacetate Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4 ......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 .......................................................................................10.0μg Phenylacetic acid solution .......................................................10.0mL pH 7.0 ± 0.2 at 25°C

1181

Mineral Medium with Pyrrolic Acid (DSMZ Medium 461b) Composition per liter: MgSO4·7H2O ................................................................................ 0.5g NH4Cl ........................................................................................... 0.3g NaCl............................................................................................ 0.05g CaCl2 .......................................................................................... 0.01g MnSO4 ........................................................................................ 0.01g Phosphate solution .................................................................100.0mL Pyrrolic acid solution...............................................................10.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................................................................................... 1.5g H3BO3 .................................................................................... 300.0mg CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O.............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)....................................................................7.7mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Phosphate Solution: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 1.45g KH2PO4....................................................................................... 0.25g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Pyrrolic Acid Solution: Composition per liter: Pyrrolic acid................................................................................ 10.0g

Preparation of Pyrrolic Acid Solution: Dissolve pyrrolic acid in phosphate buffer (50 mM, pH 7.5) and bring volume to 1.0L. Sterilize by filtration.

Preparation of Medium: Add components, except phosphate solu-

Phenylacetic acid .......................................................................... 0.5g

tion and pyrrolic acid solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 100.0mL phosphate solution and 10.0mL pyrrolic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks

Preparation of Phenylacetic Acid Solution: Add phenylacetic

Use: For the cultivation of pyrrolic acid-utilizing bacteria.

Phenylacetic Acid Solution: Composition per 10.0mL:

acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except phenylacetic acid solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL of sterile phenylacetic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Mineral Medium with Quinoline (DSMZ Medium 461a) Composition per liter: MgSO4·7H2O ................................................................................ 0.5g NH4Cl ........................................................................................... 0.3g NaCl............................................................................................ 0.05g CaCl2 .......................................................................................... 0.01g MnSO4 ........................................................................................ 0.01g

1182

Mineral Medium S with 1% Sucrose

Phosphate solution .................................................................100.0mL Quinoline emulsion................................................................100.0mL Vitamin solution.........................................................................5.0mL Trace elements solution SL-10 ..................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution SL-10: Composition per liter:

add 100.0mL phosphate solution, 5.0mL vitamin solution, and 100.0mL quinoline emulsion. Mix thoroughly. Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Rhodococcus rhodochrous (Rhodococcus roseus) and other quinoline-utilizing bacteria.

Mineral Medium S with 1% Sucrose

FeCl2·4H2O ................................................................................... 1.5g H3BO3 .................................................................................... 300.0mg CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ....................................................................7.7mL

Sucrose........................................................................................ 10.0g NH4H2PO4 .................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2 ............................................................................................. 0.1g KCl................................................................................................ 0.1g FeCl3 ......................................................................................... 0.005g NaOH (1N solution).................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add

Preparation of Medium: Add components to distilled/deionized

FeCl2·4H2O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/ deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Vitamin Solution: Composition per liter: Vitamin B12 .............................................................................. 50.0mg Pantothenic acid ....................................................................... 50.0mg Riboflavin ................................................................................ 50.0mg Alpha-lipoic acid...................................................................... 50.0mg p-Aminobenzoic acid ............................................................... 50.0mg Thiamine-HCl·2H2O................................................................ 50.0mg Nicotinic acid ........................................................................... 25.0mg Nicotinamide............................................................................ 25.0mg Biotin ....................................................................................... 20.0mg Folic acid.................................................................................. 20.0mg Pyridoxamine-HCl ................................................................... 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Phosphate Solution: Composition per 100.0mL: Na2HPO4·2H2O........................................................................... 1.45g KH2PO4 ....................................................................................... 0.25g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Quinoline Emulsion: Composition per liter: Quinoline ...................................................................................... 3.0g

Preparation of Quinoline Emulsion: Prepare an emulsion of quinoline in 50 mM phosphate buffer by stirring or ultrasonication. Add quinoline acid to phosphate buffer (50 mM, pH 7.5) and bring volume to 1.0L. Stir vigorously or sonicate to form emulsion. Autoclave for 15 min at 15 psi pressure–121°C in a gas tight vessel. Cool to room temperature.

Preparation of Medium: Add components, except phosphate solution, vitamin solution, and quinoline emulsion to distilled/deionized water and bring volume to 795.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically © 2010 by Taylor and Francis Group, LLC

Composition per liter:

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Saccharobacterium acuminatum.

Mineral Medium with Salicylate Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 ....................................................................................... 10.0μg Salicylic acid solution..............................................................10.0mL pH 7.0 ± 0.2 at 25°C

Salicylic Acid Solution: Composition per 10.0mL: Salicylic acid................................................................................. 0.5g

Preparation of Salicylic Acid Solution: Add salicylic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except salicylic acid solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile salicylic acid solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of bacteria that can utilize salicylic acid as sole carbon source.

Mineral Medium with Santonin Composition per liter: K2HPO4......................................................................................... 6.3g α-Santonin .................................................................................... 4.0g KH2PO4....................................................................................... 1.82g NH4NO3 ........................................................................................ 1.0g CaCl2·2H2O ................................................................................ 0.75g MgSO4·7H2O ................................................................................ 0.1g FeSO4·7H2O................................................................................ 0.06g

Mineral Medium with Vitamins

MnSO4 (anhydrous) ................................................................600.0μg Na2MoO4·2H2O ......................................................................600.0μg pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Pseudomonas species.

Mineral Medium with Sulfobenzoic Acid (DSMZ Medium 465e) Composition per liter: Na2HPO4·2H2O............................................................................. 3.5g KH2PO4 ......................................................................................... 1.0g (NH4)2SO4 ..................................................................................... 0.5g MgCl2·6H2O.................................................................................. 0.1g Ca(NO3)2·4H2O........................................................................... 0.05g 2-Sulfobenzoic acid .................................................................50.0mL Vitamin solution.........................................................................2.5mL Trace elements solution SL-4 ....................................................1.0mL pH 7.25 ± 0.2 at 25°C

Trace Elements Solution SL-4: Composition per liter:

1183

Preparation of Medium: Add components, except vitamin solution and 2-sulfobenzoic acid solution, to 947.5mL distilled/deionized water. Adjust pH to 7.25. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 2-sulfobenzoic acid solution and vitamin solution. Mix thoroughly. Aseptically distribute to sterile tubes or flasks. Use: For the cultivation of sulfobenzoic acid-utilizing bacteria.

Mineral Medium with Toluene Composition per liter: Na2HPO4·12H2O........................................................................... 9.0g Ferric ammonium citrate............................................................... 5.0g MnSO4·H2O .................................................................................. 3.0g NH4Cl ........................................................................................... 2.0g KH2PO4......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.2g ZnSO4·7H2O ................................................................................. 0.2g Titriplex I ................................................................................. 10.0mg CoSO4 ....................................................................................... 10.0μg Toluene ...................................................................................... 1 drop pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except toluene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Inoculate tubes or flasks and place in a dessicator jar in which 1 drop of toluene will be evaporated.

Trace Elements Solution SL-6: Composition per liter:

Use: For the cultivation of bacteria that can utilize toluene as sole carbon source.

H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................. 0.1g MnCl2·4H2O................................................................................ 0.03g Na2MoO4·H2O ............................................................................ 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2··2H2O................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4.

Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Vitamin Solution: Composition per 100.0mL: Pyridoxamine ............................................................................. 5.0mg Vitamin B12 ................................................................................ 2.0mg Nicotinic acid ............................................................................. 2.0mg Thiamine-HCl·2H2O .................................................................. 1.0mg p-Aminobenzoate....................................................................... 1.0mg Ca-pantothenate ......................................................................... 0.5mg Biotin ......................................................................................... 0.2mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

2-Sulfobenzoic Acid Solution: Composition per 100.0mL: 2-Sulfobenzoic acid ...................................................................... 2.0g

Mineral Medium with Vitamins Composition per 1002.5mL: Mineral medium...................................................................1000.0mL Schlegel’s vitamin solution........................................................2.5mL

Mineral Medium: Composition per 1010.0mL: Na2HPO4 ..................................................................................... 2.44g KH2PO4....................................................................................... 1.52g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O ................................................................................ 0.05g Trace elements solution SL-4 ..................................................10.0mL pH 6.9 ± 0.2 at 25°C

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

1184

Mineral Salts Agar

Preparation of Trace Elements Solution SL-4: Add components

Preparation of Medium: Add components to distilled/deionized

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Schlegel’s Vitamin Solution: Composition per 100.0mL: Nicotinic acid ................................................................................ 2.0g Pyridoxamine ............................................................................. 5.0mg Cyanocobalamin ........................................................................ 2.0mg p-Aminobenzoate....................................................................... 1.0mg Thiamine .................................................................................... 1.0mg Calcium DL-pantothenate........................................................... 0.5mg Biotin ......................................................................................... 0.2mg

Preparation of Schlegel’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Filter sterilize. Preparation of Medium: Add components, except Schlegel’s vitamin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 2.5mL of sterile Schlegel’s vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Pseudomonas chlororaphis and Rhodococcus species.

Mineral Pectin 5 Medium See: MP 5 Medium Mineral Pectin 7 Medium See: MP 7 Medium

Mineral Salts Agar Composition per liter: Agar ............................................................................................ 15.0g NaNO3........................................................................................... 2.0g K2HPO4 ......................................................................................... 1.2g MgSO4 .......................................................................................... 0.5g KCl................................................................................................ 0.5g KH2PO4 ....................................................................................... 0.14g Yeast extract................................................................................ 0.02g Fe2(SO4)3·H2O ............................................................................ 0.01g pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Distribute into tubes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position. Add a strip of sterile filter paper onto cooled slant. Inoculate organisms on filter paper.

Use: For the cultivation and maintenance of Cytophaga aurantiaca and Sporocytophaga myxococcoides.

Mineral Salts Enrichment Medium Composition per liter: KH2PO4 ....................................................................................... 1.36g (NH4)2SO4 ..................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g CaCl2·2H2O................................................................................. 0.01g FeSO4·7H2O............................................................................... 5.0mg MnSO4·7H2O ............................................................................. 2.5mg Na2MoO4·2H2O ......................................................................... 2.5mg Na2HPO4 .................................................................................. 2.13mg pH 7.2 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Use: For the enrichment and cultivation of Caulobacter species.

Mineral Salts with Butane Composition per liter of tap water: (NH4)2HPO4.................................................................................. 8.0g Na2HPO4·12H2O........................................................................... 2.5g KH2PO4......................................................................................... 2.0g MgSO4·7H2O ................................................................................ 0.5g Yeast extract........................................................................... 100.0mg CaCl2·2H2O ............................................................................. 60.0mg FeSO4·7H2O............................................................................. 30.0mg MnCl2·4H2O ............................................................................. 60.0μg CuSO4·5H2O ............................................................................. 15.0μg pH 7.1 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Pseudomonas butanovora.

Mineral Salts Medium Composition per liter: Na2HPO4 ....................................................................................... 4.0g KH2PO4......................................................................................... 1.5g NH4Cl ........................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.2g Ferric ammonium citrate............................................................ 5.0mg Modified Hoagland trace elements solution ..............................1.0mL pH 7.0 ± 0.2 at 25°C

Modified Hoagland Trace Elements Solution: Composition per 3.6L: H3BO3 ......................................................................................... 11.0g MnCl2·4H2O ................................................................................. 7.0g AlCl3 ............................................................................................. 1.0g CoCl2 ............................................................................................ 1.0g CuCl2 ............................................................................................ 1.0g KI .................................................................................................. 1.0g NiCl2 ............................................................................................. 1.0g ZnCl2 ............................................................................................. 1.0g BaCl2 ............................................................................................. 0.5g KBr ............................................................................................... 0.5g LiCl ............................................................................................... 0.5g Na2MoO4 ...................................................................................... 0.5g SeCl4 ............................................................................................. 0.5g SnCl2·2H2O................................................................................... 0.5g NaVO3·H2O .................................................................................. 0.1g

Preparation of Modified Hoagland Trace Elements Solution: Prepare each component as a separate solution. Dissolve each salt in approximately 100.0mL of distilled/deionized water. Adjust the pH of each solution to below 7.0. Combine all the salt solutions and bring the volume to 3.6L with distilled/deionized water. Adjust the pH to 3–4. A yellow precipitate may form after mixing. After a few days, it will turn into a fine white precipitate. Mix the solution thoroughly before using.

Mineral Salts Peptonized Milk Agar

1185

Preparation of Medium: Add components to distilled/deionized

Preparation of Medium: Add components, except methanol, to

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Filter sterilize methanol. Aseptically add sterile methanol to cooled, sterile basal medium. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Rhodococcus rhodochrous.

Use: For the cultivation and maintenance of Pseudomonas species.

Mineral Salts Medium with Methanol Composition per liter: NaNH4HPO4·4H2O ..................................................................... 1.74g NaH2PO4·H2O............................................................................. 0.54g MgSO4·7H2O ................................................................................ 0.2g KCl.............................................................................................. 0.04g FeSO4·7H2O............................................................................... 5.0mg Methanol ....................................................................................5.0mL Trace minerals solution..............................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Minerals Solution: Composition per liter: H3BO3 ......................................................................................... 2.86g MnCl2·4H2O................................................................................ 1.81g ZnSO4·7H2O ............................................................................... 0.22g CuSO4·5H2O ............................................................................... 0.08g CoCl2·6H2O................................................................................. 0.06g Na2MoO4·2H2O ....................................................................... 25.0mg

Preparation of Trace Minerals Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Filter sterilize methanol. Aseptically add sterile methanol to cooled, sterile basal medium.

Use: For the cultivation and maintenance of Rhodococcus rhodochrous.

Mineral Salts Medium with Methanol and Yeast Extract Composition per liter: NaNH4HPO4·4H2O ..................................................................... 1.74g NaH2PO4·H2O............................................................................. 0.54g MgSO4·7H2O ................................................................................ 0.2g Yeast extract.................................................................................. 0.2g KCl.............................................................................................. 0.04g FeSO4·7H2O............................................................................... 5.0mg Methanol ....................................................................................5.0mL Trace minerals solution..............................................................1.0mL pH 7.2 ± 0.2 at 25°C

Mineral Salts Medium for Thermophiles Composition per liter: NaNO3 ........................................................................................ 0.25g NH4Cl ......................................................................................... 0.25g Na2HPO4 ................................................................................ 210.0mg MgSO4·7H2O ......................................................................... 200.0mg NaH2PO4 .................................................................................. 90.0mg KCl........................................................................................... 40.0mg CaCl2 ........................................................................................ 15.0mg FeSO4 ......................................................................................... 1.0mg Trace minerals solution............................................................10.0mL n-Heptadecane ...........................................................................1.0mL

Trace Minerals Solution: Composition per liter: ZnSO4·7H2O .............................................................................. 7.0mg H3BO4 ........................................................................................ 1.0mg MoO3 ......................................................................................... 1.0mg CuSO4·5H2O........................................................................... 500.0μg CoSO4·7H2O............................................................................. 18.0μg MnSO4·5H2O .............................................................................. 7.0μg

Preparation of Trace Minerals Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Bacillus thermoleovorans.

Mineral Salts Peptonized Milk Agar (SPMA) Composition per liter: Agar ............................................................................................ 15.0g Milk, peptonized ........................................................................... 1.0g Mineral solution.....................................................................100.0mL

Mineral Solution: Composition per 100.0mL: MgSO4·7H2O ................................................................................ 0.5g CaCl2 ........................................................................................... 0.25g K2HPO4....................................................................................... 0.25g (NH4)2SO4 .................................................................................... 0.1g FeCl3·6H2O................................................................................. 0.01g MnCl2......................................................................................... 0.1mg

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except mineral solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 100.0mL of sterile mineral solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

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Mineral Salts for Thermophiles

Use: For the cultivation of freshwater Myxobacterium species.

Minerals Modified Medium Composition per liter:

Mineral Salts for Thermophiles (L Salts for Thermophiles) Composition per liter: NaNO3......................................................................................... 0.25g NH4Cl ......................................................................................... 0.25g Na2HPO4 ..................................................................................... 0.21g MgSO4·7H2O ................................................................................ 0.2g NaH2PO4 ..................................................................................... 0.09g KCl.............................................................................................. 0.04g CaCl2 ........................................................................................... 0.02g FeSO4 ......................................................................................... 1.0mg Trace minerals solution............................................................10.0mL n-Heptadecane ...........................................................................1.0mL

Trace Minerals Solution: Composition per liter: ZnSO4·7H2O .............................................................................. 7.0mg H3BO4 ........................................................................................ 1.0mg MoO3.......................................................................................... 1.0mg CuSO4·5H2O ...........................................................................500.0μg CoSO4·7H2O .............................................................................18.0μg MnSO4·5H2O ..............................................................................7.0μg

Preparation of Trace Minerals Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except n-heptadecane, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add the nheptadecane. Mix thoroughly. Distribute into sterile tubes or flasks.

Use: For the cultivation and maintenance of Bacillus thermoleovorans.

Minerals Modified Glutamate Agar Composition per liter: Agar ............................................................................................ 15.0g Lactose ........................................................................................ 10.0g Sodium glutamate ....................................................................... 6.35g NH4Cl ........................................................................................... 2.5g K2HPO4 ......................................................................................... 0.9g Sodium formate........................................................................... 0.25g MgSO4·7H2O ................................................................................ 0.1g L-Aspartic acid .......................................................................... 0.024g L-Arginine ................................................................................... 0.02g L-Cystine ..................................................................................... 0.02g Bromcresol Purple ...................................................................... 0.01g CaCl2·2H2O................................................................................. 0.01g Ferric ammonium citrate............................................................. 0.01g Nicotinic acid ............................................................................. 1.0mg Pantothenic acid ......................................................................... 1.0mg Thiamine .................................................................................... 1.0mg pH 6.7 ± 0.2 at 25°C

Lactose........................................................................................ 20.0g Sodium glutamate ....................................................................... 12.7g NH4Cl ........................................................................................... 5.0g K2HPO4......................................................................................... 1.8g Sodium formate ............................................................................ 0.5g MgSO4·7H2O ................................................................................ 0.2g L-Aspartic acid .......................................................................... 0.048g L-Cystine ..................................................................................... 0.04g L-Arginine ................................................................................... 0.04g Ferric ammonium citrate............................................................. 0.02g CaCl2·2H2O ................................................................................ 0.02g Bromcresol Purple ...................................................................... 0.02g Thiamine .................................................................................... 2.0mg Nicotinic acid............................................................................. 2.0mg Pantothenic acid......................................................................... 2.0mg pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Preparation of Medium: Add NH4Cl to distilled/deionized water

and bring volume to 800.0mL. Add remaining components and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.7. Distribute into tubes or flasks. Autoclave for 10 min at 10 psi pressure–116°C. Check pH after autoclaving. This medium is double strength.

Use: For the enumeration of coliform bacteria in water.

Minimal Agar I Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g (NH4)2SO4 .................................................................................... 5.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl.............................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.04mg Folic acid .................................................................................... 2.0μg Biotin .......................................................................................... 2.0μg Growth supplement solution....................................................10.0mL pH 5.5 ± 0.2 at 25°C

Growth Supplement Solution: Composition per 10.0mL: L-Tryptophan ........................................................................... 20.0mg Uracil ....................................................................................... 20.0mg L-Histidine·HCl........................................................................ 20.0mg

Minimal Agar, Davis Preparation of Growth Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except growth supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile growth supplement solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Saccharomyces cerevisiae.

Minimal Agar II Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g (NH4)2SO4 ..................................................................................... 5.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl .............................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.04mg Folic acid.....................................................................................2.0μg Biotin ..........................................................................................2.0μg Growth supplement solution ....................................................10.0mL pH 5.5 ± 0.2 at 25°C

(NH4)2SO4 .................................................................................... 5.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl.............................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O............................................................................ 0.04mg Folic acid .................................................................................... 2.0μg Biotin .......................................................................................... 2.0μg Growth supplement solution....................................................10.0mL pH 5.5 ± 0.2 at 25°C

Growth Supplement Solution: Composition per 10.0mL: L-Leucine ................................................................................. 30.0mg L-Tryptophan ........................................................................... 20.0gm Uracil ....................................................................................... 20.0mg

Preparation of Growth Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except growth supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile growth supplement solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of Saccharomyces cerevisiae.

Growth Supplement Solution: Composition per 10.0mL: Adenine sulfate ........................................................................ 20.0mg L-Arginine·HCl ........................................................................ 20.0mg L-Histidine·HCl........................................................................ 20.0mg

Use: For the cultivation of Saccharomyces cerevisiae.

Minimal Agar III Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g © 2010 by Taylor and Francis Group, LLC

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Minimal Agar, Davis Composition per liter: Agar ............................................................................................ 15.0g K2HPO4......................................................................................... 7.0g KH2PO4......................................................................................... 2.0g (NH4)2SO4 .................................................................................... 1.0g Glucose ......................................................................................... 1.0g Sodium citrate............................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to cold distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation, cultivation, and characterization of nutritional mutants of Escherichia coli.

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Minimal Broth I

Minimal Broth I Composition per liter: Glucose ....................................................................................... 20.0g (NH4)2SO4 ..................................................................................... 5.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl .............................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid ................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.04mg Folic acid.....................................................................................2.0μg Biotin ..........................................................................................2.0μg Growth supplement solution ....................................................10.0mL pH 5.5 ± 0.2 at 25°C

Growth Supplement Solution: Composition per 10.0mL: L-Tryptophan............................................................................ 20.0mg

Uracil ....................................................................................... 20.0mg L-Histidine·HCl........................................................................ 20.0mg

Preparation of Growth Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except growth supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL of sterile growth supplement solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Saccharomyces cerevisiae.

Minimal Broth II Composition per liter: Glucose ....................................................................................... 20.0g (NH4)2SO4 ..................................................................................... 5.0g KH2PO4 ......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl .............................................................................................. 0.1g CaCl2·2H2O................................................................................... 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid ................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg © 2010 by Taylor and Francis Group, LLC

FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.04mg Folic acid .................................................................................... 2.0μg Biotin .......................................................................................... 2.0μg Growth supplement solution....................................................10.0mL pH 5.5 ± 0.2 at 25°C

Preparation of Growth Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except growth supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL of sterile growth supplement solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Saccharomyces cerevisiae.

Minimal Broth III Composition per liter: Agar ............................................................................................ 20.0g Glucose ....................................................................................... 20.0g (NH4)2SO4 .................................................................................... 5.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O ................................................................................ 0.5g NaCl.............................................................................................. 0.1g CaCl2·2H2O .................................................................................. 0.1g Inositol ....................................................................................... 2.0mg KI ............................................................................................... 1.0mg H3BO3 ........................................................................................ 0.5mg ZnSO4·7H2O .............................................................................. 0.4mg MnSO4·4H2O ............................................................................. 0.4mg Thiamine·HCl ............................................................................ 0.4mg Pyroxidine·HCl .......................................................................... 0.4mg Niacin......................................................................................... 0.4mg Calcium pantothenate ................................................................ 0.4mg p-Aminobenzoic acid................................................................. 0.2mg Riboflavin .................................................................................. 0.2mg FeCl3 .......................................................................................... 0.2mg Na2MoO4·4H2O ......................................................................... 0.2mg CuSO4·5H2O ............................................................................ 0.04mg Folic acid .................................................................................... 2.0μg Biotin .......................................................................................... 2.0μg Growth supplement solution....................................................10.0mL pH 5.5 ± 0.2 at 25°C

Growth Supplement Solution: Composition per 10.0mL: L-Leucine ................................................................................. 30.0mg L-Tryptophan ........................................................................... 20.0mg Uracil ....................................................................................... 20.0mg

Minimum Essential Medium with Bicarbonate, Serum, and Antibiotics

1189

990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 10.0mL of sterile growth supplement solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

K2HPO4·3H2O ............................................................................ 0.87g KH2PO4....................................................................................... 0.54g

Use: For the cultivation of Saccharomyces cerevisiae.

water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Minimal Broth, Davis Composition per liter: K2HPO4 ......................................................................................... 7.0g KH2PO4 ......................................................................................... 2.0g (NH4)2SO4 ..................................................................................... 1.0g Glucose ......................................................................................... 1.0g Sodium citrate ............................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to cold distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the isolation, cultivation, and characterization of nutritional mutants of Escherichia coli. Also recommended for the isolation and characterization of nutritional mutants from wild-type strains of Bacillus subtilis when used in conjunction with minimal agar Davis and antibiotic medium 3.

Minimal F-Top Agar Composition per liter: NaCl .............................................................................................. 8.0g Agar .............................................................................................. 4.5g K2HPO4 ......................................................................................... 2.1g KH2PO4 ........................................................................................ .0.6g (NH4)2SO4 .................................................................................... .0.3g Glucose ......................................................................................... 0.3g Sodium citrate ............................................................................. 0.15g MgSO4·7H2O .............................................................................. 0.03g pH 7.0 ± 0.2 at 25°C

Preparation of Minimal Agar: Add components to cold distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the distribution of bacteriophage or bacterial cells evenly in a thin layer over the surface of a plate. Minimal Lactate Medium See: ML Medium

Minimal Medium for Denitrifying Bacteria Composition per liter: Solution A ..............................................................................980.0mL Solution B ................................................................................10.0mL Solution C ................................................................................10.0mL

Solution A: Composition per 980.0mL: KNO3 ............................................................................................ 5.0g Carbon source ............................................................................... 4.0g (NH4)2SO4 ..................................................................................... 1.0g © 2010 by Taylor and Francis Group, LLC

Preparation of Solution A: Add components to distilled/deionized

Solution B: Composition per 100.0mL: MgSO4·7H2O ................................................................................ 2.0g

Preparation of Solution B: Add MgSO4·7H2O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution C: Composition per 100.0mL: CaCl2·2H2O .................................................................................. 0.2g FeSO4·7H2O.................................................................................. 0.1g MnSO4·H2O ................................................................................ 0.05g CuSO4·5H2O............................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g HCl (0.1N solution) ...............................................................100.0mL

Preparation of Solution C: Combine components. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Aseptically combine 980.0mL of cooled sterile solution A, 10.0mL of cooled sterile solution B, and 10.0mL of cooled sterile solution C. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the isolation and cultivation of denitrifying bacteria.

Minimal Medium for Penicillium Interspecific Hybrids Composition per liter: Glucose ....................................................................................... 40.0g NaNO3 .......................................................................................... 3.0g KH2PO4......................................................................................... 1.0g KCl................................................................................................ 0.5g MgSO4·7H2O ................................................................................ 0.5g FeSO4·7H2O................................................................................ 0.01g pH 6.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of genetic variants of Penicillium species.

Minimum Essential Medium with Bicarbonate, Serum, and Antibiotics Composition per 1100.0mL: Minimum essential medium ..................................................950.0mL Fetal bovine serum, heat inactivated .....................................100.0mL NaHCO3 solution.....................................................................40.0mL Penicillin-streptomycin solution..............................................10.0mL pH 7.2 ± 0.2 at 25°C

Minimum Essential Medium (MEM): Composition per liter: Inorganic salt solution............................................................400.0mL Other component solution......................................................400.0mL Amino acid solution...............................................................100.0mL Vitamin solution.....................................................................100.0mL

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Minimum Salts with HiVeg Acid Hydrolysate

Inorganic Salt Solution: Composition per 400.0mL:

NaHCO3 Solution: Composition per 40.0mL:

NaCl .............................................................................................. 6.8g KCl................................................................................................ 0.4g CaCl2, anhydrous .......................................................................... 0.2g NaH2PO4·H2O............................................................................. 0.14g MgSO4, anhydrous................................................................. 97.67mg

NaHCO3 ........................................................................................ 2.0g

Preparation of Inorganic Salt Solution: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Other Component Solution: Composition per 400.0mL: D-Glucose...................................................................................... 1.0g

Phenol Red ............................................................................... 10.0mg

Preparation of Other Component Solution: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Amino Acid Solution: Composition per 100.0mL: L-Glutamine ........................................................................... 292.0mg L-Arginine·HCl ...................................................................... 126.1mg L-Lysine·HCl............................................................................ 72.5mg

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

ionized water and bring volume to 40.0mL. Mix thoroughly. Filter sterilize.

Penicillin-Streptomycin Solution Composition per 10.0mL: Penicillin ..................................................................................... 0.01g Streptomycin............................................................................... 0.01g

Preparation of Penicillin-Streptomycin Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Aseptically combine 950.0mL of sterile minimum essential medium, 100.0mL of sterile heat inactivated fetal bovine serum, 40.0mL of sterile NaHCO3 solution, and 10.0mL of sterile penicillin-streptomycin solution. Adjust pH to 7.2 with humidified 10% CO2 in 90% air. Use: For the cultivation of Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Naegleria fowleri, and Nosema corneum.

L-Isoleucine.............................................................................. 52.0mg L-Leucine ................................................................................. 52.0mg L-Tyrosine,

disodium salt......................................................... 52.0mg L-Threonine.............................................................................. 48.0mg L-Valine .................................................................................... 46.0mg L-Histidine·HCl·H2O................................................................ 42.0mg L-Phenylalanine........................................................................ 32.0mg L-Cysteine·2HCl....................................................................... 31.0mg L-Methionine............................................................................ 15.0mg L-Glutamic acid........................................................................ 14.7mg L-Aspartic acid ......................................................................... 13.3mg L-Asparagine·H2O.................................................................... 13.2mg L-Proline................................................................................... 11.5mg L-Serine .................................................................................... 10.5mg L-Tryptophan............................................................................ 10.0mg L-Alanine.................................................................................... 8.9mg Glycine....................................................................................... 7.5mg

Preparation of Amino Acid Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Minimum Salts with HiVeg Acid Hydrolysate Composition per liter: Na2HPO4 ....................................................................................... 6.8g Glucose ......................................................................................... 4.0g Plant acid hydrolysate................................................................... 4.0g KH2PO4......................................................................................... 3.0g NH4Cl ........................................................................................... 1.0g NaCl.............................................................................................. 0.5g MgSO4 ........................................................................................ 0.24g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Heat gently and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation of Escherichia coli strains used for genetic and molecular studies.

Vitamin Solution: Composition per 100.0mL: i-Inositol..................................................................................... 2.0mg D-Ca pantothenate ...................................................................... 1.0mg Choline chloride......................................................................... 1.0mg Folic acid.................................................................................... 1.0mg Niacinamide ............................................................................... 1.0mg Pyridoxal·HCl ............................................................................ 1.0mg Thiamine·HCl ............................................................................ 1.0mg Riboflavin .................................................................................. 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

MIO HiVeg Medium (Motility Indole Ornithine HiVeg Medium) Composition per liter: Plant hydrolysate ........................................................................ 10.0g Plant peptone .............................................................................. 10.0g L-Ornithine hydrochloride ............................................................ 5.0g Yeast extract.................................................................................. 3.0g Agar .............................................................................................. 2.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.6 ± 0.2 at 25°C

Preparation of Minimum Essential Medium (MEM): Asepti-

Source: This medium is available as a premixed powder from Hi-

cally combine 400.0mL of sterile inorganic salt solution, 400.0mL of sterile other component solution, 100.0mL of sterile amino acid solution, and 100.0mL of sterile vitamin solution.

Preparation of Medium: Add components to distilled/deionized

Media. water and bring to 1.0L. Mix thoroughly. Gently heat and bring to boil-

Mitsuokella dentalis Medium

ing. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the differentiation of Gram-negative enteric bacteria based on their motility, indole production, and ornithine decarboxylase activity. MIO Medium See: Motility Indole Ornithine Medium

Mist Agar Composition per liter: Cow manure, dry......................................................................... 50.0g Agar ............................................................................................ 15.0g

Preparation of Medium: Add cow manure to 1.0L of tap water. Boil for 1 hr. Filter through cheesecloth. Filter through paper. Add agar to filtrate and bring volume to 1.0L with tap water. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Streptomyces fragmentosporus.

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K2HPO4......................................................................................... 4.0g Glucose ......................................................................................... 1.0g Trypan Blue .............................................................................. 0.075g Crystal Violet ............................................................................. 0.8mg Na2TeO3 solution .......................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without tellurite, is available as a premixed powder from HiMedia.

Na2TeO3 Solution: Composition per 10.0mL: Na2TeO3 ........................................................................................ 0.1g

Preparation of Na2TeO3 Solution: Add Na2TeO3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Caution: Potassium tellurite is toxic. Preparation of Medium: Add components to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool medium to 50°–55°C. Aseptically add 1.0mL of the sterile Na2TeO3 solution to the cooled basal medium. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation of Streptococcus mitis, Streptococcus

Mitis Salivarius Agar Composition per liter: Sucrose........................................................................................ 50.0g Agar ............................................................................................ 15.0g Enzymatic digest of protein ........................................................ 10.0g Proteose peptone ......................................................................... 10.0g K2HPO4 ......................................................................................... 4.0g Dextrose ........................................................................................ 1.0g Trypan Blue ................................................................................ 0.08g Crystal Violet ............................................................................. 0.8mg Na2TeO3 solution .......................................................................1.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Na2TeO3 Solution: Composition per 10.0mL: Na2TeO3 ........................................................................................ 0.1g

Preparation of Na2TeO3 Solution: Add Na2TeO3 to 10.0mL of

salivarius, and other viridans streptococci and enterococci.

Mitsuokella dentalis Medium Composition per 1003.3mL: Yeast extract................................................................................ 10.0g Beef extract................................................................................... 5.0g Glucose ......................................................................................... 5.0g Trypticase™.................................................................................. 5.0g L-Cysteine·HCl ............................................................................. 0.5g (NH4)2SO4 .................................................................................... 0.5g Resazurin ................................................................................... 1.0mg Bovine serum ...........................................................................50.0mL Mineral solution.......................................................................40.0mL Hemin solution...........................................................................1.0mL Vitamin K1 solution....................................................................0.2mL pH 6.9 ± 0.2 at 25°C

Bovine Serum: Composition per 50.0mL:

distilled/deionized water. Mix thoroughly. Filter sterilize.

Bovine serum ...........................................................................50.0mL

Caution: Potassium tellurite is toxic.

Preparation of Bovine Serum: Incubate 50.0mL of bovine serum in a GasPak™ container overnight to make anaerobic.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool medium to 50°–55°C. Aseptically add 1.0mL of the sterile Na2TeO3 solution to the cooled basal medium. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the selective isolation of Streptococcus mitis, Streptococcus salivarius, and other viridans streptococci and enterococci.

Mitis Salivarius HiVeg Agar Base with Tellurite Composition per liter: Sucrose........................................................................................ 50.0g Agar ............................................................................................ 15.0g Plant hydrolysate......................................................................... 15.0g Plant peptone................................................................................. 5.0g © 2010 by Taylor and Francis Group, LLC

Mineral Solution: Composition per liter: NaHCO3 ...................................................................................... 10.0g NaCl.............................................................................................. 2.0g K2HPO4......................................................................................... 1.0g KH2PO4......................................................................................... 1.0g MgSO4·7H2O .............................................................................. 0.48g CaCl2·2H2O .................................................................................. 0.3g

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly.

Hemin Solution: Composition per 100.0mL: Hemin ........................................................................................ 5.0mg NaOH (0.002% solution) ...........................................................1.0mL

1192

Mixed Cereal Agar

Preparation of Hemin Solution: Add hemin to 1.0mL of NaOH solution. Mix thoroughly.

Vitamin K1 Solution: Composition per 100.0mL: Vitamin K1 .................................................................................. 1.09g

Preparation of Vitamin K1 Solution: Add vitamin K1 to 99.0mL of absolute ethanol. Store in the dark at 4°C.

Preparation of Medium: Add components, except L-cysteine·HCl and bovine serum, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% CO2. Add L-cysteine·HCl. Mix thoroughly. Adjust pH to 6.0 with 8N NaOH. After pH has been reached, change sparging gas to 100% N2. Anaerobically distribute into bottles under 100% N2. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 50.0mL of bovine serum. Mix thoroughly.

Use: For the cultivation and maintenance of Mitsuokella dentalis.

Mixed Cereal Agar Composition per liter: Gerber™ mixed cereal (oats, wheat, corn, barley).......................................................................... 50.0g Agar ............................................................................................ 15.0g Sucrose........................................................................................ 15.0g Thiamine·HCl ............................................................................ 5.0mg

Use: For the cultivation of Nitrobacter vulgaris.

Mixotrophic Nitrobacter Medium (LMG Medium 246) Composition per liter: NaNO2 .......................................................................................... 2.0g Yeast extract................................................................................ 1.50g Peptone ....................................................................................... 1.50g Na-pyruvate ................................................................................ 0.55g Stock solution ........................................................................100.0mL Trace elements solution .............................................................1.0mL pH 7.4 ± 0.2 at 25°C

Stock Solution: Composition per liter: NaCl.............................................................................................. 5.0g KH2PO4......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.5g CaCO3 ......................................................................................... 0.07g

Preparation of Medium: Add components to distilled/deionized

Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution: Composition per liter:

Use: For the cultivation and maintenance of Tilletia caries.

Mixotrophic Nitrobacter Medium (DSMZ Medium 756a) Composition per liter: NaNO2........................................................................................... 2.0g Yeast extract.................................................................................. 1.5g Peptone.......................................................................................... 1.5g Na-pyruvate ................................................................................ 0.55g Stock solution.........................................................................100.0mL Trace elements solution .............................................................1.0mL pH 7.4 ± 0.2 at 25°C

Stock Solution: Composition per liter: NaCl .............................................................................................. 5.0g KH2PO4 ......................................................................................... 1.5g MgSO4·7H2O ................................................................................ 0.5g CaCO3 ........................................................................................ 0.07g

Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

(NH4)Mo7O2........................................................................ 437.10mg FeSO4·7H2O........................................................................... 97.30mg ZnSO4·7H2O .......................................................................... 43.10mg H3BO3 .................................................................................... 39.40mg MnSO4·H2O ........................................................................... 33.80mg CuSO2·5H2O .......................................................................... 25.00mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Use: For the cultivation of mixotrophic Nitrobacter spp.

Mixotrophic Nitrobacter Medium, 10% (DSMZ Medium 756b) Composition per liter: NaNO2 .......................................................................................... 2.0g Yeast extract................................................................................ 0.15g Peptone ....................................................................................... 0.15g Na-pyruvate .............................................................................. 0.055g Stock solution ........................................................................100.0mL Trace elements solution .............................................................1.0mL pH 8.6 ± 0.2 at 25°C

MJ Medium

MgSO4·7H2O ................................................................................ 0.5g CaCO3 ........................................................................................ 0.07g

Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 8.6. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation of Nitrobacter hamburgensis and Nitrobacter vulgaris.

MJ Medium (DSMZ Medium 1011) Composition per liter: NaCl ........................................................................................... 30.0g MgCl2·6H2O................................................................................ 4.18g MgSO4·7H2O ................................................................................ 3.4g KCl.............................................................................................. 0.33g NH4Cl ......................................................................................... 0.25g KH2PO4 ....................................................................................... 0.14g CaCl2·2H2O............................................................................... 0.014g Fe(NH4)2(SO4)2·6H2O ............................................................... 0.01g NiCl2·6H2O ................................................................................ 0.5mg Na2SeO3·5H2O........................................................................... 0.5mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Thiosulfate solution .................................................................10.0mL Bicarbonate solution ................................................................10.0mL pH 6.7 ± 0.2 at 25°C

Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.5g

Preparation of Bicarbonate Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O................................................................................ 1.5g

Preparation of Thiosulfate Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

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CoSO4·7H2O............................................................................... 0.18g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g H3BO3 ......................................................................................... 0.01g Na2MoO4·4H2O .......................................................................... 0.01g CuSO4·5H2O............................................................................... 0.01g Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Add components, except thiosulfate, bicarbonate, and vitamin solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 1 min. Cool to room temperature while sparging with a gas mixture of 80% N2 + 20% CO2. Dispense under the same atmosphere into culture vessels. Fill up to a volume of 20% volume of vessel. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature under an atmosphere of 0% N2 + 20% CO2. Aseptically add sterile thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 6.7. Aseptically dispense into tubes, flasks, or bottles. After inoculation pressurize vessels to 0.5 bar overpressure with 80% N2 + 20% CO2 gas mixture. Add sterile air in an amount that is equivalent to a volume of 20% of the headspace. After inoculation reduce medium with 10–20mg sodium dithionite per liter medium, added from a 5% solution freshly prepared under N2 and filter sterilized. Pressurize vessels to 2 bar overpressure with 80% H2 and 20% CO2.

Use: For the cultivation of Sulfurimonas autotrophica, Thiomicrospira thermophila, and Desulfothermus okinawensis.

1194

MJ Medium for Thiobacter subterraneus

NiCl2·6H2O ................................................................................ 0.5mg Na2SeO3·5H2O........................................................................... 0.5mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Thiosulfate solution .................................................................10.0mL Bicarbonate solution ................................................................10.0mL Pyruvate solution .....................................................................10.0mL Yeast extract solution ...............................................................10.0mL Lactate soltuion........................................................................10.0mL pH 6.7 ± 0.2 at 25°C

Pyruvate Solution: Composition per 10.0mL: Sodium pyruvate ........................................................................... 0.5g

Preparation of Pyruvate Solution: Add sodium pyruvate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Lactate Solution: Composition per 10.0mL: Sodium lactate............................................................................... 0.5g

Preparation of Lactate Solution: Add sodium lactate to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Yeast Extract Solution: Composition per 10.0mL: Yeast extract.................................................................................. 0.1g

Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.5g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O................................................................................ 1.5g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Add components, except lactate, pyruvate, yeast extract, thiosulfate, bicarbonate, and vitamin solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 1 min. Cool to room temperature while sparging with a gas mixture of 80% H2 + 20% CO2. Dispense under the same atmosphere into culture vessels. Fill up to a volume of 20% volume of vessel. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature under an atmosphere of 80% H2 + 20% CO2. Aseptically add sterile lactate, pyruvate, yeast extract, thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 6.7. Aseptically dispense into tubes, flasks, or bottles. After inoculation pressurize vessels to 0.5 bar overpressure with 80% H2 + 20% CO2 gas mixture. Add sterile air in an amount that is equivalent to a volume of 20% of the headspace. After inoculation reduce medium with 10–20 mg sodium dithionite per liter medium, added from a 5% solution freshly prepared under N2 and filter sterilized. Pressurize vessels to 2 bar overpressure with 80% H2 and 20% CO2.

Use: For the cultivation of Desulfothermus okinawensis.

MJ Medium for Thiobacter subterraneus (DSMZ Medium 1011a) Composition per liter: MnCl2·6H2O ............................................................................... 4.18g NaCl ............................................................................................. 3.0g MgSO4·7H2O .............................................................................. 0.34g KCl.............................................................................................. 0.33g NH4Cl ......................................................................................... 0.25g KH2PO4....................................................................................... 0.14g CaCl2·2H2O .............................................................................. 0.014g Fe(NH4)2(SO4)2·6H2O ............................................................... 0.01g NiCl2·6H2O ................................................................................ 0.5mg Na2SeO3·5H2O........................................................................... 0.5mg Trace elements solution ...........................................................10.0mL Vitamin solution.......................................................................10.0mL Thiosulfate solution .................................................................10.0mL Bicarbonate solution ................................................................10.0mL pH 6.7 ± 0.2 at 25°C

Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.5g

MJANHOX-NO3 Medium with Supplement Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Thiosulfate Solution: Composition per 10.0mL: NaS2O3·5H2O................................................................................ 1.5g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature.

Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................................ 10.0mg Thiamine-HCl·2H2O .................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid ............................................................................. 5.0mg D-Ca-pantothenate...................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid.................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Preparation of Medium: Add components, except thiosulfate, bicarbonate, and vitamin solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 1 min. Cool to room temperature while sparging with a gas mixture of 80% N2 + 20% CO2. Dispense under the same atmosphere into culture vessels. Fill up to a volume of 20% volume of vessel. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature under an atmosphere of 80% N2 + 20% CO2. Aseptically add sterile thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 6.7. Aseptically dispense into tubes, flasks, or bottles. After inoculation pressurize vessels to 0.5 bar overpressure with 80% N2 + 20% CO2 gas mixture. Add sterile air in an amount that is equivalent to a volume of 50% of the headspace. After inoculation reduce medium with 10– 20mg sodium dithionite per liter medium, added from a 5% solution © 2010 by Taylor and Francis Group, LLC

1195

freshly prepared under N2 and filter sterilized. Pressurize vessels to 2 bar overpressure with 80% H2 and 20% CO2.

Use: For the cultivation of Thiobacter subterraneus.

MJANHOX-NO3 Medium with Supplement (DSMZ Medium 1000) Composition per liter: NaCl ............................................................................................. 3.0g MgCl2·6H2O ............................................................................. 0.418g Na2SiO3......................................................................................... 0.5g NH4Cl ........................................................................................... 0.4g MgSO4·7H2O .............................................................................. 0.34g KH2PO4....................................................................................... 0.14g KCl.............................................................................................. 0.033 CaCl2·2H2O .............................................................................. 0.014g Fe2(SO4)3·7H2O ........................................................................ 0.005g NiCl2·6H2O............................................................................ 0.005mg Na2SeO3·5H2O....................................................................... 0.005mg Bicarbonate solution ................................................................10.0mL Nitrate solution ........................................................................10.0mL Thiosulfate solution .................................................................10.0mL Trace elements solution .............................................................1.0mL pH 7.7 ± 0.2 at 25°C

Thiosulfate Solution: Composition per 10.0mL: Na2S2O3·5H2O .............................................................................. 2.4g

Preparation of Thiosulfate Solution: Add Na2S2O3·5H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 1.5g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Nitrate Solution: Composition per 10.0mL: NaNO3 .......................................................................................... 2.0g

Preparation of Nitrate Solution: Add NaNO3 to distilled/deion-

ized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

1196

ML Medium

distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Adjust the pH to 7.7. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add thiosulfate solution, bicarbonate solution, nitrate solution, and vitamin solution. Sparge with a gas mixture of 80% H2 + 20% CO2 for 5 min. Compress gas mixture into gas phase (> 80% volume of the tube or bottle) at 3 atm.

Use: For the cultivation of Sulfurihydrogenibium subterraneum.

M-Lauryl Sulfate HiVeg Broth Composition per liter: Plant special peptone .................................................................. 39.0g Lactose........................................................................................ 30.0g Yeast extract.................................................................................. 6.0g Sodium lauryl sulfate.................................................................... 1.0g Phenol Red.................................................................................... 0.2g pH 7.4 ± 0.2 at 25°C

m-Kleb Agar See: Kleb Agar

Source: This medium is available as a premixed powder from Hi-

m-Klebsiella Medium See: Klebsiella Medium

Preparation of Medium: Add components to distilled/deionized

ML Medium (Minimal Lactate Medium) Composition per liter: Sodium lactate............................................................................... 5.0g MgSO4·7H2O ................................................................................ 2.0g NH4Cl ........................................................................................... 1.0g Na2SO4 .......................................................................................... 1.0g Yeast extract.................................................................................. 1.0g K2HPO4 ......................................................................................... 0.5g L-Cysteine ..................................................................................... 0.5g CaCl2·6H2O................................................................................... 0.1g Resazurin ................................................................................... 1.0mg NaHCO3 solution .....................................................................25.0mL FeSO4·7H2O solution...............................................................25.0mL pH 6.8 ± 0.2 at 25°C

NaHCO3 Solution: Composition per 25.0mL:

Media. water and bring volume to 1.0L. Mix thoroughly. Distribute into bottles or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and enumeration of coliform bacteria, especially Escherichia coli, in water by the membrane filter method.

MLCB Agar (Mannitol Lysine Crystal Violet-Brilliant Green Agar) Composition per liter: Agar ............................................................................................ 15.0g Peptone ....................................................................................... 10.0g Yeast extract.................................................................................. 5.0g L-Lysine·HCl ................................................................................. 5.0g NaCl.............................................................................................. 4.0g Na2S2O3 ........................................................................................ 4.0g Mannitol........................................................................................ 3.0g Beef extract................................................................................... 2.0g Ferric ammonium citrate............................................................... 1.0g Crystal Violet .............................................................................. 0.01g Brilliant Green ......................................................................... 12.5mg pH 6.8 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from Oxoid

NaHCO3 ........................................................................................ 4.0g

Unipath.

Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/de-

Preparation of Medium: Add components to distilled/deionized

ionized water and bring volume to 25.0mL. Mix thoroughly. Filter sterilize. Gas with O2-free 97% N2 + 3% H2. Cap with a rubber stopper.

FeSO4·7H2O Solution: Composition per 25.0mL: FeSO4·7H2O............................................................................... 4.0mg

Preparation of FeSO4·7H2O Solution: Add FeSO4·7H2O to dis-

tilled/deionized water and bring volume to 25.0mL. Mix thoroughly. Filter sterilize. Gas with O2-free 97% N2 + 3% H2. Cap with a rubber stopper.

Preparation of Medium: Add components, except NaHCO3 solu-

water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Do not autoclave. Cool to 50°C. Pour into sterile Petri dishes in 20.0mL volumes.

Use: For the selective isolation and cultivation of Salmonella species from fecal material and foods. m-LES, Endo Agar See: Endo Agar, LES

MM10 Agar (Modified Medium 10 Agar)

tion and FeSO4·7H2O solution, to distilled/deionized water and bring volume to 1.0L. Gently heat and bring to boiling under O2-free 97% N2 + 3% H2. Adjust pH to 6.8. Continue boiling until resazurin becomes colorless, indicating reduction. Distribute anaerobically under O2-free 97% N2 + 3% H2 into tubes in 10.0mL volumes. Cap with rubber stoppers. Place tubes in a press. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Prior to inoculation, add 0.25mL of sterile NaHCO3 solution and 0.25mL of sterile FeSO4·7H2O solution to each test tube containing 10.0mL of sterile basal medium.

Composition per liter:

Use: For the cultivation and maintenance of Desulfovibrio species.

Agar ............................................................................................ 15.0g Casein peptone.............................................................................. 2.0g Glucose ......................................................................................... 1.0g Sodium formate ............................................................................ 1.0g

Base........................................................................................954.0mL Dithiothreitol solution..............................................................20.0mL Sheep blood .............................................................................20.0mL Na2CO3 solution........................................................................5.0mL Menadione solution ...................................................................1.0mL pH 7.2 ± 0.2 at 25°C

Base: Composition per 954.0mL:

MMB Medium

KNO3 ............................................................................................ 0.5g Yeast extract.................................................................................. 0.5g Hemin.......................................................................................... 0.01g Mineral salt solution 1 .............................................................38.0mL Mineral salt solution 2 .............................................................38.0mL Sodium lactate solution..............................................................4.0mL

Preparation of Base: Add components to distilled/deionized water and bring volume to 954.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Mineral Salt Solution 1: Composition per 100.0mL: K2HPO4 ......................................................................................... 0.6g

Preparation of Mineral Salt Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Mineral Salt Solution 2: Composition per 100.0mL:

1197

MMA Salts Medium Composition per liter: Agar, noble.................................................................................. 20.0g K2HPO4......................................................................................... 1.2g KH2PO4....................................................................................... 0.62g (NH4)2SO4 .................................................................................... 0.5g MgSO4·7H2O ................................................................................ 0.2g NaCl.............................................................................................. 0.1g CaCl2·6H2O ................................................................................ 0.05g ZnSO4·7H2O ............................................................................. 70.0μg H3BO3 ....................................................................................... 10.0μg MnSO4·5H2O ............................................................................ 10.0μg Na2MoO4·2H2O ........................................................................ 10.0μg CoCl2·6H2O ................................................................................ 5.0μg CuSO4·5H2O............................................................................... 5.0μg FeCl3·6H2O................................................................................ 1.0mg Monomethylamine solution .....................................................10.0mL pH 7.0 ± 0.2 at 25°C

NaCl .............................................................................................. 1.2g (NH4)2SO4 ..................................................................................... 1.2g KH2PO4 ......................................................................................... 0.6g CaCl2 ........................................................................................... 0.12g

Monomethylamine Solution: Composition per 10.0mL:

Preparation of Mineral Salt Solution 2: Add components to dis-

ylamine to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Sodium Lactate Solution: Composition per 100.0mL: Sodium lactate............................................................................. 60.0g

Preparation of Sodium Lactate Solution: Add sodium lactate to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Dithiothreitol Solution: Composition per 100.0mL: Dithiothreitol................................................................................. 1.0g

Preparation of Dithiothreitol Solution: Add dithiothreitol to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Monomethylamine........................................................................ 1.0g

Preparation of Monomethylamine Solution: Add monometh-

Preparation of Medium: Add components, except monomethylamine solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL of sterile methylamine solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Hyphomicrobium species and Methylophilus methylotrophus.

MMB Medium Composition per liter:

Preparation of Menadione Solution: Add vitamin K1 to 100.0mL of ethanol. Mix thoroughly. Filter sterilize.

Glucose ......................................................................................... 1.0g (NH4)2SO4 .................................................................................. 0.25g Peptone ....................................................................................... 0.15g Yeast extract................................................................................ 0.15g Glucose solution ......................................................................20.0mL Hutner's basal salts solution.....................................................20.0mL Vitamin solution.......................................................................10.0mL pH 7.2 ± 0.2 at 25°C

Na2CO3 Solution: Composition per 100.0mL:

Glucose Solution: Composition per 20.0mL:

Na2CO3 ........................................................................................ 8.0g

Glucose ......................................................................................... 1.5g

Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize.

Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: To 954.0mL of sterile cooled base, aseptically add 20.0mL of sterile dithiothreitol solution, 20.0mL of sterile, defibrinated sheep blood, 5.0mL of sterile Na2CO3 solution, and 1.0mL of sterile menadione solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile screw-capped tubes.

Hutner’s Basal Salts Solution: Composition per liter:

Menadione Solution: Composition per 100.0mL: Vitamin K1 (phytomenadione) .................................................... 0.05g Ethanol (95% solution) ..........................................................100.0mL

MgSO4·7H2O .............................................................................. 29.7g Nitrilotriacetic acid ..................................................................... 10.0g CaCl2·2H2O .............................................................................. 3.335g FeSO4·7H2O............................................................................. 99.0mg (NH4)6MoO7O24·4H2O ............................................................ 9.25mg "Metals 44" ..............................................................................50.0mL

1198

MMJS Medium (Modified)

"Metals 44": Composition per 100.0mL: ZnSO4·7H2O ............................................................................. 1.095g FeSO4·7H2O.................................................................................. 0.5g Sodium EDTA............................................................................. 0.25g MnSO4·H2O.............................................................................. 0.154g CuSO4·5H2O ............................................................................ 39.2mg Co(NO3)2·6H2O ....................................................................... 24.8mg Na2B4O7·10H2O....................................................................... 17.7mg

Preparation of “Metals 44”: Add sodium EDTA to distilled/deionized water and bring volume to 90.0mL. Mix thoroughly. Add a few drops of concentrated H2SO4 to retard precipitation of heavy metal ions. Add remaining components. Mix thoroughly. Bring volume to 100.0mL with distilled/deionized water. Preparation of Hutner’s Basal Salts Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Adjust pH to 6.8.

Vitamin Solution: Composition per 100.0mL: Vitamin B12 .............................................................................. 0.01mg Calcium DL-pantothenate........................................................... 0.5mg Nicotinamide.............................................................................. 0.5mg Pyridoxine·HCl .......................................................................... 0.5mg Riboflavin .................................................................................. 0.5mg Thiamine·HCl ............................................................................ 0.5mg Biotin ......................................................................................... 0.2mg Folic acid.................................................................................... 0.2mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except glucose solution and vitamin solution, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile glucose solution and 10.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Angulomicrobium tetraedrale, Labrys monachus, Prosthecomicrobium polyspheroidum, and Aquabacter spiritensis.

MMJS Medium (Modified) (DSMZ Medium 1121) Composition per liter: NaCl ........................................................................................... 20.0g MgSO4·7H2O ................................................................................ 4.0g Sulfur, elemental .......................................................................... 3.0g MgCl2·6H2O.................................................................................. 3.0g Na2S2O3·5H2O .............................................................................. 2.0g NH4Cl ........................................................................................ 1.25g CaCl2·2H2O................................................................................... 0.8g KCl ............................................................................................. 0.33g K2HPO4 ....................................................................................... 0.09g KH2PO4 ....................................................................................... 0.07g Fe2(SO4)3·7H2O .......................................................................... 0.01g Na2SeO3·5H2O........................................................................... 1.0mg H2WO4 ...................................................................................... 1.0mg NiCl2·6H2O ................................................................................ 1.0mg Bicarbonate solution ................................................................10.0mL © 2010 by Taylor and Francis Group, LLC

Trace elements solution ..........................................................10.0mL Vitamin solution.........................................................................1.0mL pH 6.8 ± 0.2 at 25°C

Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ....................................................................... 1.5g MnSO4·2H2O ................................................................................ 0.5g CoSO4·7H2O ................................................................................. 0.5g ZnSO4·7H2O ............................................................................... 0.18g CaCl2·2H2O .................................................................................. 0.1g FeSO4·7H2O.................................................................................. 0.1g NiCl2·6H2O ............................................................................... 0.025g KAl(SO4)2·12H2O....................................................................... 0.02g CuSO4·5H2O ............................................................................... 0.01g H3BO3 ......................................................................................... 0.01g NaBr............................................................................................ 0.01g SrCl2·6H2O ................................................................................. 0.01g KI ................................................................................................ 0.01g Na2MoO4·4H2O ......................................................................... 1.0mg Na2SeO3·5H2O........................................................................... 0.3mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO3 ........................................................................................ 2.0g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except trace elements solution, bicarbonate solution, and vitamin solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Adjust the pH to 6.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the trace elements solution, bicarbonate solution, and vitamin solution. Sparge wtih a gas mixture of 80% N2 + 20% CO2 for 5 min. Compress gas mixture of 79% N2 + 20% CO2 + 1% O2 into gas phase (> 80% volume of the tube or bottle) at 2 atm.

Use: For the cultivation Sulfurivirga caldicuralii.

MMN Agar Composition per liter: Agar ............................................................................................ 15.0g D-Glucose.................................................................................... 10.0g

MN Marine Medium

Malt extract ................................................................................... 3.0g KH2PO4 ......................................................................................... 0.5g Ammonium tartrate..................................................................... 0.25g MgSO4·7H2O .............................................................................. 0.15g CaCl2 ........................................................................................... 0.05g NaCl .......................................................................................... 0.025g Thiamine·HCl ............................................................................ 0.1mg FeCl3 solution ............................................................................1.2mL

FeCl3 Solution: Composition per 100.0mL: FeCl3 ............................................................................................. 1.0g

Preparation of Medium: Prepare and dispense medium under 80% N2 and 20% CO2. Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5 with H2SO4. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the cultivation and maintenance of Thermotoga neapolitana.

Preparation of FeCl3 Solution: Add FeCl3 to distilled/deionized

MN See: Melin–Norkrans Medium

Preparation of Medium: Add components to distilled/deionized

Mn Agar No. 1 See: Manganese Agar No. 1

water and bring volume to 100.0mL. Mix thoroughly.

Use: For the cultivation and maintenance of Cenococcum geophilum, Cortinarius species, Gyrodon lividus, Hebeloma crustuliniforme, Hebeloma pusillum, Hygrophorous purpurascens, Hygrophorus russula, Laccaria bicolor, Laccaria laccata, Lyophyllum fumosum, Lyophyllum shimeji, Macrolepiota rhacodes, Obolarina dryophila, Paxillus atromentosus, Phaeolepiota aurea, Pisolithus tinctoruis, Rhizopogon colossus, Rhizopogon ellenae, many Rhizopogon species, Sarcodon aspratu, Scleroderma albidum, Scleroderma aurantium, many Suillus species, Tricholoma flavovirens, and many Tricholoma species.

MMS Medium for Thermotoga neapolitana Composition per liter: NaCl ............................................................................................ 6.93g Starch ............................................................................................ 5.0g MgSO4·7H2O .............................................................................. 1.75g MgCl2·6H2O................................................................................ 1.38g KH2PO4 ......................................................................................... 0.5g Na2S·9H2O .................................................................................... 0.5g CaCl2 ........................................................................................... 0.38g KCl.............................................................................................. 0.16g NaBr......................................................................................... 25.0mg H3BO3 ........................................................................................ 7.5mg SrCl2·6H2O................................................................................. 3.8mg (NH4)2Ni(SO4)2.......................................................................... 2.0mg Resazurin ................................................................................... 1.0mg KI ........................................................................................... 0.025mg Wolfe’s mineral solution ..........................................................15.0mL pH 6.5 ± 0.2 at 25°C

Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ................................................................................ 3.0g Nitrilotriacetic acid ....................................................................... 1.5g NaCl .............................................................................................. 1.0g MnSO4·H2O .................................................................................. 0.5g FeSO4·7H2O.................................................................................. 0.1g CoCl2·6H2O .................................................................................. 0.1g CaCl2 ............................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g CuSO4·5H2O ............................................................................... 0.01g AlK(SO4)2·12H2O....................................................................... 0.01g H3BO3 ......................................................................................... 0.01g Na2MoO4·2H2O .......................................................................... 0.01g © 2010 by Taylor and Francis Group, LLC

1199

Mn Agar No. 2 See: Manganese Agar No. 2

Mn HiVeg Agar Base with Manganese Composition per liter: Agar ............................................................................................ 12.0g KH2PO4......................................................................................... 2.0g MnCO3 .......................................................................................... 2.0g Plant extract .................................................................................. 1.0g Fe(NH4)2SO4 .............................................................................. 0.15g Sodium citrate............................................................................. 0.15g Yeast extract.............................................................................. 0.075g Cyanocobalamin solution ........................................................10.0mL pH 7.0 ± 0.2 at 25°C

Cyanocobalamin Solution: Composition per 10.0mL: Cyanocobalamin ........................................................................ 5.0mg

Preparation of Cyanocobalamin: Add cyanocarbalamin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except cyanocarbalamin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0mL sterile cyanocarbolamin solution. Mix thoroughly. Pour into sterile Petri dishes or aseptically distribute into tubes. Use: For the cultivation of Leptothrix spp. and detection of Leptothrix by its ability to oxidize manganous ions.

MN Marine Medium Composition per liter: Noble agar................................................................................... 10.0g NaNO3 ........................................................................................ 0.75g MgSO4·7H2O .............................................................................. 0.04g CaCl2·2H2O ................................................................................ 0.02g K2HPO4·3H2O ............................................................................ 0.02g Na2CO3 ....................................................................................... 0.02g Citric acid................................................................................... 3.0mg Ferric ammonium citrate............................................................ 3.0mg

1200

MN Marine Medium with Vitamin B12

Disodium potassium EDTA ....................................................... 0.5mg Trace metal mix A-5 ..................................................................1.0mL pH 8.5 ± 0.2 at 25°C

A-5 Trace Metal Mix: Composition per liter: H3BO3 ......................................................................................... 2.86g MnCl2·4H2O................................................................................ 1.81g ZnSO4·7H2O ............................................................................. 0.222g CuSO4·5H2O ............................................................................. 0.079g Na2MoO4·2H2O ........................................................................ 0.039g Co(NO3)2·6H2O ........................................................................ 0.049g

Preparation of A-5 Trace Metal Mix: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to 750.0mL of seawater and bring volume to l.0L with glass-distilled water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. After autoclaving, adjust pH to 8.5 with KOH.

Use: For the cultivation and maintenance of marine cyanobacteria.

MN Marine Medium with Vitamin B12

Composition per liter:

Noble agar................................................................................... 10.0g NaNO3......................................................................................... 0.75g MgSO4·7H2O .............................................................................. 0.04g CaCl2·2H2O................................................................................. 0.02g K2HPO4·3H2O............................................................................. 0.02g Na2CO3 ....................................................................................... 0.02g Citric acid................................................................................... 3.0mg Ferric ammonium citrate............................................................ 3.0mg Disodium potassium EDTA ....................................................... 0.5mg Vitamin B12 ...............................................................................20.0μg Trace metal mix A-5 ..................................................................1.0mL pH 8.5 ± 0.2 at 25°C

Preparation of A-5 Trace Metal Mix: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components to 750.0mL of seawater and bring volume to 1.0L with glass-distilled water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. After autoclaving, adjust pH to 8.5 with KOH.

Use: For the cultivation and maintenance of Dermocarpa species, Dermocarpella species, Myxosarcina species, Phormidium species, Pleurocapsa species, Synechococcus species, Synechocystis species, and Xenococcus species.

Mobiluncus Agar (LMG Medium 117)

NaCl.............................................................................................. 5.0g Resazurin .................................................................................. 10.0µg Rabbit serum............................................................................20.0mL pH 7.1–7.5

Source: Special peptone and Agar No. 1 are available from Oxoid Unipath.

Preparation of Medium: Add components, except rabbit serum, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 20.0mL sterile rabbit serum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Mobiluncus curtisii subsp. holmesii and Mobiluncus mulieris.

Moderate Halophilic Medium (HM) Composition per liter: NaCl............................................................................................ 81.0g Agar ............................................................................................ 20.0g Yeast extract................................................................................ 10.0g MgSO4·7H2O................................................................................ 9.6g MgCl2·6H2O ................................................................................. 7.0g Proteose peptone No. 3 ................................................................. 5.0g KCl................................................................................................ 2.0g Glucose ......................................................................................... 1.0g CaCl2·2H2O ............................................................................... .0.36g NaHCO3...................................................................................... 0.06g NaBr.......................................................................................... 0.026g pH 7.2 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Bacillus halophilus, Halococcus saccharolyticus, Marinococcus albus, Marinococcus halophilus, and Marinococcus hispanicus.

Modified AEA Sporulation Medium Base Composition 1070.0mL: Biopeptone.................................................................................. 10.0g Yeast extract................................................................................ 10.0g Na2HPO4 ..................................................................................... 4.36g Ammonium acetate....................................................................... 1.5g KH2PO4....................................................................................... 0.25g MgSO4·7H2O ................................................................................ 0.2g Raffinose solution ....................................................................40.0mL Carbonate solution ...................................................................10.0mL Cobalt chloride solution...........................................................10.0mL Sodium ascorbate solution.......................................................10.0mL pH 7.8 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Raffinose Solution: Composition per 40.0mL:

Composition per liter:

Raffinose....................................................................................... 4.0g

Special peptone ........................................................................... 23.0g Agar No. 1................................................................................... 10.0g Starch, soluble............................................................................. 10.0g

Preparation of Raffinose Solution: Add raffinose to distilled/de-

ionized water and bring volume to 40.0mL. Mix thoroughly. Filter sterilize.

Modified Buffered Charcoal HiVeg Agar Base with Cysteine

1201

Carbonate Solution: Composition per 10.0mL:

Trace Elements Solution SL-7: Composition per 1001.0mL:

Na2CO3 ......................................................................................... 0.7g

CoCl2......................................................................................... 0.032g

CoCl2·6H2O ........................................................................... 200.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg H3BO3 ...................................................................................... 60.0mg Na2MoO4·2H2O ....................................................................... 40.0mg CuCl2·2H2O ............................................................................. 20.0mg NiCl2·6H2O.............................................................................. 20.0mg HCl (25%)..................................................................................1.0mL

Preparation of Cobalt Chloride Solution: Add CoCl2 to dis-

Preparation of Trace Elements Solution SL-7: Add components

Preparation of Carbonate Solution: Add Na2CO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Cobalt Cloride Solution: Composition per 10.0mL:

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Sodium Ascorbate Solution: Composition per 10.0mL: Sodium ascorbate ........................................................................ 0.15g

Preparation of Sodium Ascorbate Solution: Add sodium ascorbate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except raffinose, carbonate, cobalt chloride, and sodium ascorbate solutions, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute 15.0mL aliquots into screw capped tubes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Add 0.6L raffinose solution and 0.2mL each of the carbonate and cobalt chloride solutions dropwise to the medium in each of the tubes. Just before using, steam the medium for 10 min. Cool to room temperature. Aseptically add 0.2mL of freshly prepared sodium ascorbate solution to each tube of the medium.

Use: For the early sporulation of Clostridium perfringens from foods.

Modified Biebl and Pfennig's Medium (DSMZ Medium 1069)

to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Preparation of Medium: Add components, except vitamin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust the pH to 6.8. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Aseptically add the vitamin solution. Mix thoroughly. Aseptically dispense into culture vessels.

Use: For the cultivation of Rhodovulum imhoffii.

Modified Bile Esculin Azide Agar Composition per liter: Casein enzymic hydrolysate ....................................................... 17.0g Agar ............................................................................................ 13.5g Oxgall ......................................................................................... 10.0g Yeast extract.................................................................................. 5.0g NaCl.............................................................................................. 5.0g Peptic digest of animal tissue ....................................................... 3.0g Sodium citrate............................................................................... 1.0g Esculin .......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g NaN3 ........................................................................................... 0.25g pH 7.1 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

Composition per liter:

Caution: Sodium azide has a tendency to form explosive metal azides

NaCl ............................................................................................ 20.0g Malate/pyruvate ........................................................................... 3.0g MgSO4·7H2O ................................................................................ 2.0g KH2PO4 ......................................................................................... 0.5g Yeast extract ................................................................................. 0.4g NH4Cl ........................................................................................ 0.34g CaCl2·2H2O................................................................................. 0.15g Ferric citrate solution .................................................................5.0mL Trace elements solution SL-7 ....................................................1.0mL Vitamin solution.........................................................................1.0mL pH 6.8 ± 0.2 at 25°C

with plumbing materials. It is advisable to use enough water to flush off the disposables.

Vitamin Solution: Composition per 10.0ml: Vitamin B12 ................................................................................ 0.1mg

Preparation of Vitamin Solution: Add vitamin B12 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Ferric Citrate Solution: Composition per 10.0mL:

Use: For the selective isolation and enumeration of group D streptococci.

Modified Buffered Charcoal HiVeg Agar Base with Cysteine Composition per liter: Agar ............................................................................................ 17.0g ACES buffer ............................................................................... 10.0g Plant peptone No. 3..................................................................... 10.0g Charcoal, activated ....................................................................... 2.0g α-Ketoglutarate monopotassium salt............................................ 1.0g L-Cysteine solution ....................................................................4.0mL pH 6.9 ± 0.2 at 25°C

Source: This medium, without L-cysteine solution, is available as a

Ferric citrate ................................................................................ 0.01g

premixed powder from HiMedia.

Preparation of Ferric Citrate Solution: Add ferric citrate to dis-

L-Cysteine

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

L-cysteine·HCl·H2O ...................................................................... 0.4g

Solution: Composition per 10.0mL:

1202

Modified Campylobacter Blood-Free Selective Agar Base

L-Cysteine

Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

zone solution, 10.0mL of sterile amphotericin B solution, and 10.0mL of sterile rifampicin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Preparation of Medium: Add components, except L-cysteine solu-

Use: For the cultivation of Campylobacter species. For the recovery of

tion, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust medium to pH 6.9 with 1N KOH. Heat gently and bring to boiling for 1 min. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 4.0mL of L-cysteine solution. Mix thoroughly. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.

injured Campylobacter spp. from foods.

Use: For the isolation, cultivation, and maintenance of Legionella pneumophila and other Legionella species from environmental and clinical specimens.

Modified Campylobacter Blood-Free Selective Agar Base (Modified Campylobacter Charcoal Differential Agar) (Modified CCDA) (BAM M30a) Composition per 1012.0mL: Agar ............................................................................................ 12.0g Beef extract ................................................................................. 10.0g Peptone........................................................................................ 10.0g NaCl .............................................................................................. 5.0g Charcoal ........................................................................................ 4.0g Casein hydrolysate ........................................................................ 3.0g Yeast extract.................................................................................. 2.0g Sodium deoxycholate.................................................................... 1.0g FeSO4 .......................................................................................... 0.25g Sodium pyruvate ......................................................................... 0.25g Cefoperazone solution ...............................................................4.0mL Amphotericin B solution............................................................4.0mL Rifampicin solution....................................................................4.0mL pH 7.4 ± 0.2 at 25°C

Cefoperazone Solution: Composition per 10.0mL:

Modified CM + YE Agar See: CM plus YE Agar, Modified

Modified CPLM HiVeg Medium Base with Horse Serum (Trichomonas Modified CPLM HiVeg Medium Base) Composition per liter: Plant peptone .............................................................................. 32.0g Liver digest ................................................................................. 20.0g L-Cysteine·HCl ............................................................................. 2.4g Maltose ......................................................................................... 1.6g Ringer's salt solution, 1/4X...........................................................1.0L Horse serum ...........................................................................100.0mL pH 6.0 ± 0.2 at 25°C

Ringer's Salt Solution, 1/4X: Composition per 400.0mL: NaCl.............................................................................................. 9.0g KCl............................................................................................ 0.042g CaCl2 ......................................................................................... 0.024g

Preparation of Ringer's Salt Solution, 1/4X: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Preparation of Medium: Add components, except horse serum, to 1.0L Ringer’s salt solution, 1/4X. Mix thoroughly. Adjust pH to 6.0. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 100.0mL of sterile, heat-inactivated horse serum. Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks. Use: For the cultivation of Trichomonas vaginalis.

Cefoperazone ............................................................................ 0.037g

Preparation of Cefoperazone Solution: Add cefoperazone to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Rifampicin Solution: Composition per 100.0mL: Rifampicin .................................................................................. 0.25g Ethanol, absolute......................................................................50.0mL

Preparation of Rifampicin Solution: Add rifampicin to 50.0mL of ethanol. Mix thoroughly. Bring volume to 100.0mL with distilled/ deionized water. Filter sterilize. Amphotericin B Solution: Composition per 10.0mL: Amphotericin B......................................................................... 0.005g

Preparation of Amphotericin B Solution: Add amphotericin B to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Can be stored for 1 year at –20°C.

Preparation of Medium: Add components, except cefoperazone solution, amphotericin B solution, and rifampicin solution, to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile cefopera© 2010 by Taylor and Francis Group, LLC

Modified CPLM HiVeg Medium Base with Horse Serum, Penicillin, Streptomycin, and Nystatin (Trichomonas Modified CPLM HiVeg Medium Base) Composition per liter: Plant peptone .............................................................................. 32.0g Liver digest ................................................................................. 20.0g L-Cysteine·HCl ............................................................................. 2.4g Maltose ......................................................................................... 1.6g Ringer's salt solution, 1/4X...........................................................1.0L Horse serum ...........................................................................100.0mL Penicllin-streptomycin solution ...............................................10.0mL Nystatin solution......................................................................10.0mL pH 6.0 ± 0.2 at 25°C

Preparation of Ringer's Salt Solution, 1/4X: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly.

Modified Fungal HiVeg Agar Base

Penicllin-Streptomycin Solution: Composition per 10.0mL: Streptomycin ................................................................................. 0.1g Penicillin ........................................................................... 1,000,000U

Preparation of Penicllin-Streptomycin Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Adjust pH to 7.8 with filter-sterilized 0.66M Na2CO3. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and induction of sporulation of Clostridium perfringens.

Nystatin Solution: Composition per 10.0mL: Nystatin.................................................................................. 50,000U

Preparation of Nystatin Solution: Add nystatin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize

Preparation of Medium: Add components, except horse serum, penicillin-streptomycin solution, and nystatin solution, to 1.0L of Ringer’s salt solution, 1/4X. Mix thoroughly. Adjust pH to 6.0. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure– 121°C. Cool to 25°C. Aseptically add 100.0mL of sterile, heat-inactivated horse serum, 10.0mL sterile penicllin-streptomycin solution, and 10.0mL sterile nystatin solution. . Mix thoroughly. Aseptically distribute into sterile, screw-capped tubes or flasks. Use: For the selective cultivation of Trichomonas vaginalis.

Modified Differential Clostridial Broth Composition per liter: Meat extract .................................................................................. 8.0g Casein enzymic hydrolysate ......................................................... 5.0g Meat peptone................................................................................. 5.0g Sodium acetate .............................................................................. 5.0g Yeast extract.................................................................................. 1.0g Starch ............................................................................................ 1.0g Glucose ......................................................................................... 1.0g L-Cysteine hydrochloride.............................................................. 0.5g NaHSO3 ........................................................................................ 0.5g Ammonium ferric citrate .............................................................. 0.5g Resazurin .................................................................................. 0.002g pH 7.2 ± 0.2 at 25°C

Use: For the detection of Clostridium spp. from foods by the MPN technique.

Modified Duncan Strong HiVeg Medium (DS HiVeg Medium) Composition per liter: Plant peptone No. 3..................................................................... 15.0g Na2HPO4 ..................................................................................... 10.0g Raffinose ....................................................................................... 4.0g Yeast extract.................................................................................. 4.0g Na-thioglycollate .......................................................................... 1.0g pH 7.8 ± 0.2 at 25°C

1203

Modified Duncan Strong Medium (DS Medium) Composition per liter: Proteose peptone......................................................................... 15.0g Na2HPO4 ..................................................................................... 10.0g Raffinose....................................................................................... 4.0g Yeast extract.................................................................................. 4.0g Na-thioglycollate .......................................................................... 1.0g pH 7.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Adjust pH to 7.8 with filter-sterilized 0.66M Na2CO3. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and induction of sporulation of Clostridium perfringens.

Modified Fungal Agar Base (Modified Inhibitory Mold Agar) Composition per liter: Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g Casein enzymic hydrolysate ......................................................... 2.5g Peptic digest of animal tissue ....................................................... 2.5g Yeast extract.................................................................................. 5.0g Na2HPO4 ....................................................................................... 3.5g KH2PO4......................................................................................... 3.4g NH4Cl ........................................................................................... 1.4g NaCO3 ........................................................................................... 1.0g Chloramphenicol........................................................................... 0.1g MgSO4·7H2O .............................................................................. 0.06g Polysorbate 80 .........................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium is available from HiMedia. Preparation of Medium: Add components, except polysorbate 80, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Add polysorbate 80. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the detection and enumeration of molds in cosmetics and toiletries.

Modified Fungal HiVeg Agar Base (Modified Inhibitory Mold HiVeg Agar Base) Composition per liter: Glucose ....................................................................................... 20.0g Agar ............................................................................................ 15.0g

1204

Modified FWM Medium

Yeast extract.................................................................................. 5.0g Na2HPO4 ....................................................................................... 3.5g KH2PO4 ......................................................................................... 3.4g Plant hydrolysate........................................................................... 2.5g Plant peptone................................................................................. 2.5g NH4Cl ........................................................................................... 1.4g Na2CO3 ......................................................................................... 1.0g Chloramphenicol........................................................................... 0.1g MgSO4 ........................................................................................ 0.06g Polysorbate 80..........................................................................20.0mL pH 7.0 ± 0.2 at 25°C

Source: This medium, without polysorbate 80, is available as a premixed powder from HiMedia.

Use: For the isolation of pathogenic fungi.

Modified FWM Medium (DSMZ Medium 503) Composition per 1013.0mL: Solution A ..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G ................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D ..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas atmosphere. Add components to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

lution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with

80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Solution D: Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Solution F: Composition per 10.0mL: Na-(D,L)-3-hydroxybutyrate ......................................................... 1.5g

Preparation of Solution F: Add Na-(D,L)-3-hydroxybutyrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G, to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Clostridium homopropionicum DSM 5847.

Modified FWM Medium

Solution G ................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D ..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C

Solution A: Composition per 940.0mL: NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

atmosphere. Add components to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize. Solution D: Composition per liter: NaOH ............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g © 2010 by Taylor and Francis Group, LLC

1205

Preparation of Solution E: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution F: Composition per 10.0mL: Fructose......................................................................................... 2.0g

Preparation of Solution F: Add fructose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Clostridium homopropionicum DSM 5847.

Modified FWM Medium (DSMZ Medium 503) Composition per 1013.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

1206

Modified FWM Medium

H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid ............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Solution F: Composition per 10.0mL: Na2-succinate ................................................................................ 2.5g

Preparation of Solution F: Add Na2-succinate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Modified FWM Medium (DSMZ Medium 503) Composition per 1013.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Modified FWM Medium Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize. Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Solution F: Composition per 10.0mL: Na-(D/L)-3-hydroxybutyrate ......................................................... 1.5g

Preparation of Solution F: Add Na-(D/L)-3-hydroxybutyrate to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Desulfococcus biacutus DSM 5651.

Modified FWM Medium (DSMZ Medium 503)

1207

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Preparation of Solution D: Add components to distilled/deionized

Composition per 1013.0mL: Solution A ..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G ................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D ..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C

water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution A: Composition per 940.0mL:

Solution F: Composition per 10.0mL:

NaCl .............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O.................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4 ......................................................................................... 0.2g CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 0.5mg

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution E: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Xylan............................................................................................. 2.0g

Preparation of Solution F: Add xylan to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C.

1208

Modified FWM Medium

Preparation of Medium: Prepare and dispense medium under 80%

Preparation of Solution C: Add components to distilled/deionized

N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Use: For the cultivation of Cytophaga xylanolytica DSM 6779.

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Solution F: Composition per 10.0mL: Xylose ........................................................................................... 2.0g

Preparation of Solution F: Add xylose to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution B: Composition per liter:

FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid ............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg © 2010 by Taylor and Francis Group, LLC

Preparation of Medium: Prepare and dispense medium under 80%

Use: For the cultivation of Cytophaga xylanolytica DSM 6779.

Modified FWM Medium (DSMZ Medium 503) Composition per 1013.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g

Modified FWM Medium

1209

CaCl2·2H2O................................................................................. 0.15g Resazurin ................................................................................... 0.5mg

N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL:

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Solution F: Composition per 10.0mL: Pivalic acid.................................................................................... 1.0g NaNO3......................................................................................... 0.85g

Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Thavera pivalivorans DSM 14691.

Modified FWM Medium (DSMZ Medium 503) Composition per 1013.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

1210

Modified FWM Medium

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid ............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Solution F: Composition per 10.0mL: Glucose ......................................................................................... 5.0g Yeast extract.................................................................................. 2.0g

Preparation of Solution F: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution G: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Opitutus sp. DSM 14424.

Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C

Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution E: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with

Modified FWM Medium

100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution F: Composition per 10.0mL: Na2-fumarate................................................................................. 3.2g Na-acetate ..................................................................................... 0.8g

1211

H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter:

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O.............................................................. 200.0mg Nicotinic acid......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin............................................................................... 20.0mg

Preparation of Medium: Prepare and dispense medium under 80%

Preparation of Solution C: Add components to distilled/deionized

Solution G: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.125g

water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Use: For the cultivation of Geovibrio thiophilus DSM 11263.

Solution D: Composition per liter:

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution E: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution A: Composition per 940.0mL:

Solution F: Composition per 10.0mL:

Na-glycolate.................................................................................. 2.0g Yeast extract.................................................................................. 1.0g

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg © 2010 by Taylor and Francis Group, LLC

Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Clostridium sp. DSM 11261.

1212

Modified FWM Medium

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid ............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Preparation of Solution E: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution F: Composition per 10.0mL: Na-3-hydroxybutyrate................................................................... 3.0g Yeast extract.................................................................................. 1.0g

Solution G: Composition per 10.0mL: Na2S·9H2O................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of unclassified bacterium DSM 11262.

Modified FWM Medium (DSMZ Medium 503) Composition per 1023.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................20.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 940.0mL: Na2SO4 ......................................................................................... 2.8 g NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

atmosphere. Add components to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% N2

Modified FWM Medium

+ 20% CO2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution B: Composition per liter: FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O........................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution) ..................................................................10.0mL

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Solution C: Composition per liter: Pyridoxine hydrochloride ...................................................... 300.0mg Thiamine-HCl·2H2O .............................................................. 200.0mg Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Solution F: Composition per 10.0mL: Syringic acid ................................................................................ 0.6 g

Preparation of Solution F: Add syringic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Adjust pH to 7.0. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

1213

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 20.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4. Prior to inoculation the completed medium should equilibrate overnight.

Use: For the cultivation of Parasporobacterium paucivorans DSM 15970.

Modified FWM Medium (DSMZ Medium 503) Composition per 1023.0mL: Solution A..............................................................................940.0mL Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution H................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL pH 6.5–7.0 at 25°C Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

1214

Modified Gorodkowa Agar

Nicotinic acid ......................................................................... 200.0mg Vitamin B12 ............................................................................ 100.0mg Calcium pantothenate ............................................................ 100.0mg p-Aminobenzoic acid ............................................................... 80.0mg D(+)-Biotin ............................................................................... 20.0mg

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Solution F: Composition per 10.0mL: Trimethylamine hydrochloride ..................................................... 2.0g Methanol ....................................................................................... 0.6g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution H: Composition per 10.0mL: FeSO4·6H2O................................................................................ 0.02g

Preparation of Solution H: Add FeSO4·6H2O to distilled/deion-

ized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, and 10.0mL solution G to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 6.5–7.0. After inoculation aseptically inject 0.1mL of solution H for each 10.0mL of medium.

Modified Gorodkowa Agar Composition per liter: Agar ............................................................................................ 20.0g Peptic digest of animal tissue ..................................................... 10.0g NaCl.............................................................................................. 5.0g Glucose ......................................................................................... 1.0g pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For promoting sporulation of yeasts.

Modified ISP5 Medium (DSMZ Medium 993b) Composition per liter: KCl.............................................................................................. 100.0 Agar ............................................................................................ 20.0g Glycerol ...................................................................................... 10.0g Yeast extract ................................................................................. 5.0g L-asparagine, anhydrous ............................................................... 1.0g K2HPO4, anhydrous ...................................................................... 1.0g Trace elements solution .............................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O ................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Use: For the cultivation of Nesterenkonia xinjiangensis and Microbacterium halotolerans.

Modified ISP5 Medium with Sodium Chloride (DSMZ Medium 993c) Composition per liter: NaCl.......................................................................................... 100.0g Agar ............................................................................................ 20.0g Glycerol ...................................................................................... 10.0g L-asparagine, anhydrous ............................................................... 1.0g K2HPO4, anhydrous ...................................................................... 1.0g Trace elements solution .............................................................1.0mL pH 7.2 ± 0.2 at 25°C

Trace Elements Solution: Composition per 100.0mL: FeSO4·7H2O.................................................................................. 0.1g MnCl2·4H2O ................................................................................. 0.1g ZnSO4·7H2O ................................................................................. 0.1g

Modified Marine Broth Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly.

Use: For the cultivation of Nesterenkonia lutea.

Modified Letheen HiVeg Agar (Letheen HiVeg Agar, Modified) Composition per liter:

1215

Plant hydrolysate .......................................................................... 5.0g Plant peptone ................................................................................ 5.0g NaCl.............................................................................................. 5.0g Plant extract .................................................................................. 3.0g Phenyl ethanol .............................................................................. 2.5g LiCl ............................................................................................... 0.5g Sheep blood, defibrinated ........................................................50.0mL Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Agar ............................................................................................ 20.0g Plant peptone............................................................................... 20.0g Plant extract .................................................................................. 5.0g Plant hydrolysate........................................................................... 5.0g NaCl .............................................................................................. 5.0g Polysorbate 80............................................................................... 5.0g Yeast extract.................................................................................. 2.0g Lecithin ......................................................................................... 0.7g NaHSO3 ........................................................................................ 0.1g pH 7.2 ± 0.2 at 25°C

Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-

Source: This medium is available as a premixed powder from Hi-

lective supplement solution, to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood and 10.0mL selective supplement solution. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes.

Media.

Use: For the screening cosmetic products for microbial contamination.

Modified Letheen HiVeg Broth (Letheen HiVeg Broth, Modified) Composition per liter: Plant peptone............................................................................... 20.0g Plant extract .................................................................................. 5.0g Plant hydrolysate........................................................................... 5.0g NaCl .............................................................................................. 5.0g Polysorbate 80............................................................................... 5.0g Yeast extract.................................................................................. 2.0g Lecithin ......................................................................................... 0.7g NaHSO3 ........................................................................................ 0.1g pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the screening of cosmetic products for microbial contamination.

Modified McBride Listeria HiVeg Agar Base with Blood and Cycloheximide Composition per liter: Agar ............................................................................................ 15.0g Glycine anhydride....................................................................... 10.0g © 2010 by Taylor and Francis Group, LLC

mation and inhalation.

Selective Supplement Solution: Composition per 10.0mL: Cycloheximide.............................................................................. 0.2g

Preparation of Selective Supplement Solution: Add cycloheximide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except blood and se-

Use: For the selective isolation of Listeria monocytogenes from clinical and nonclinical specimens containing mixed flora.

Modified Marine Broth (Modified Medium 514) (DSMZ Medium 1173) Composition per liter: NaCl.......................................................................................... 39.45g Peptone ....................................................................................... 10.0g MgCl2............................................................................................ 8.8g Yeast extract.................................................................................. 5.0g Na2SO3 ........................................................................................ 3.24g CaCl2 ............................................................................................. 1.8g KCl.............................................................................................. 0.55g NaHCO3 ...................................................................................... 0.16g Ferric citrate.................................................................................. 0.1g KBr ............................................................................................. 0.08g SrCl2............................................................................................ 0.03g H3BO3 ......................................................................................... 0.02g Na2HPO4 .................................................................................... 8.0mg Na2SiO3...................................................................................... 4.0mg NaF ............................................................................................ 2.4mg NH4NO3 ..................................................................................... 1.6mg pH 7.6 ± 0.2 at 25°C

Use: For the cultivation of Shewanella atlantica.

1216

Modified MYP Agar Base

Modified MYP Agar Base Composition per liter: Agar ............................................................................................ 12.0g Peptic digest of animal tissue...................................................... 10.0g D-Mannitol .................................................................................. 10.0g NaCl ............................................................................................ 10.0g Meat extract .................................................................................. 1.0g Phenol Red ................................................................................ 0.025g Egg yolk emulsion .................................................................100.0mL Selective supplement solution .................................................10.0mL pH 7.1 ± 0.2 at 25°C

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Selective Supplement Solution: Composition per 10.0mL: Polymyxin B ............................................................................. 1000U

Preparation of Selective Supplement Solution: Add polymyxin B to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except egg yolk emulsion and selective supplement solution, to distilled/deionized water and bring volume to 890.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add egg yolk emulsion and selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the isolation and identification of Bacillus species and pathogenic staphylococci.

Modified MYP HiVeg Agar Base with Egg Yok and Polymyxin B Composition per liter: Agar ............................................................................................ 12.0g D-Mannitol .................................................................................. 10.0g Plant peptone............................................................................... 10.0g NaCl ............................................................................................ 10.0g Plant extract No. 1 ........................................................................ 1.0g Phenol red ................................................................................. 0.025g Egg yolk emulsion, 20% ..........................................................10.0mL Polymyxin B solution ................................................................1.0mL pH 7.1 ± 0.2 at 25°C

Source: This medium, without egg yolk emulsion and polymyxin B solution, is available as a premixed powder from HiMedia.

Egg Yolk Emulsion, 20%: Composition per 100.0mL: Chicken egg yolks............................................................................ 11 Whole chicken egg............................................................................. 1 NaCl (0.9% solution) ...............................................................80.0mL © 2010 by Taylor and Francis Group, LLC

Preparation of Polymyxin B Solution: Add polymyxin B to distilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components—except egg yolk emulsion, 20%, and polymyxin B solution—to distilled/deionized water and bring volume to 989.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile egg yolk emulsion, 20%, and 1.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes.

Use: For the cultivation and maintenance of Bacillus cereus. For the isolation and identification of Bacillus species and pathogenic staphylococci.

Modified Oxford Listeria Selective Agar (Oxford Agar, Modified) (Listeria Selective Agar, Modified Oxford) (MOX Agar) (BAM M103a) Composition per 1002.0mL: Special peptone........................................................................... 23.0g LiCl ............................................................................................. 15.0g Agar ............................................................................................ 12.0g NaCl.............................................................................................. 5.0g Cornstarch..................................................................................... 1.0g Esculin .......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Buffered colistin methane sulfonate solution ............................1.0mL Buffered moxalactam solution...................................................2.0mL pH 7.2 ± 0.2 at 25°C

Buffered Colistin Methane Sulfonate Solution: Composition per 100.0mL: Colistin methane sulfonate ........................................................... 1.0g Potassium phosphate buffer, 0.1M. pH 6.0............................100.0mL

Preparation of Buffered Colistin Methane Sulfonate Solution: Add colistin methane sulfonate to 100.0mL 0.1M potassium phosphate buffer. Mix thoroughly. Adjust pH to 6.0. Filter sterilize. Store at –20°C.

Buffered Moxalactam Solution: Composition per 100.0mL: Sodium or ammonium moxalactam.............................................. 1.0g

Preparation of Buffered Moxalactam Solution: Add sodium or ammonium moxalactam to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Store at –20°C. Preparation of Medium: Gradually add components, except buffered moxalactam solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool quick-

Modified Shieh Agar

ly to 46°C. Aseptically add 2.0mL of sterile moxalactam solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and cultivation of Listeria monocytogenes from specimens containing a mixed bacterial flora.

Modified PYNFH Medium

1217

Papaic digest of soybean meal...................................................... 5.0g Sodium acetate.............................................................................. 3.0g Yeast extract.................................................................................. 2.5g Ascorbic acid ................................................................................ 0.5g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

Composition per liter:

Media.

Peptone........................................................................................ 10.0g Yeast extract................................................................................ 10.0g Yeast nucleic acid.......................................................................... 1.0g Folic acid.................................................................................. 15.0mg Hemin......................................................................................... 1.0mg Fetal bovine serum, heat inactivated......................................100.0mL Buffer solution .........................................................................20.0mL pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2 with 2N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the cultivation of lactobacilli from cheese. For the cultivation and enumeration of microorganisms encountered in the dairy industry.

Modified Salt Broth See: Salt Broth, Modified

Buffer Solution: Composition per liter: Na2HPO4 ..................................................................................... 25.0g KH2PO4 ....................................................................................... 18.1g

Preparation of Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Add components, except buffer solution and fetal bovine serum, to distilled/deionized water and bring volume to 880.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Aseptically add 20.0mL of sterile buffer solution and 100.0mL of sterile, heat-inactivated fetal bovine serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks.

Use: For the cultivation of Dexiostoma campyla, Hartmannella vermiformis, Naegleria australiensis, Naegleria fowleri, Naegleria gruberi, Naegleria jadini, Phytomonas davidi, Tetrahymena species, Vahlkampfia avara, and Willaertia magna.

Modified Rappaport Vassiliadis HiVeg Medium

Modified Semisolid Rappaport Vassiliadis Medium (MSRV Medium) Composition per liter: MgCl2, anhydrous..................................................................... 10.93g NaCl............................................................................................ 7.34g Casein hydrolysate...................................................................... 4.59g Tryptose ...................................................................................... 4.59g Agar .............................................................................................. 2.7g KH2PO4....................................................................................... 1.47g Malachite Green oxalate .......................................................... .0.037g Novobiocin solution.................................................................10.0mL pH 5.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath.

Novobiocin Solution: Composition per 10.0mL:

Composition per liter:

Novobiocin ................................................................................. 0.02g

MgCl2·6H2O ............................................................................... 40.0g NaCl .............................................................................................. 8.0g Papaic digest of soybean meal ...................................................... 5.0g KH2PO4 ........................................................................................... 1.6 Malachite Green.......................................................................... 0.04g pH 5.2 ± 0.2 at 25°C

Preparation of Novobiocin Solution: Add novobiocin to 10.0mL

Source: This medium is available as a premixed powder from HiMedia.

Use: For the selective enrichment of Salmonella species from food and environmental specimens.

Modified Rogosa HiVeg Agar (M16 HiVeg Agar) Composition per liter: Agar ............................................................................................ 10.0g Glucose ......................................................................................... 5.0g Plant extract .................................................................................. 5.0g Plant hydrolysate No. 1................................................................. 5.0g © 2010 by Taylor and Francis Group, LLC

of distilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except novobiocin solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat to boiling. Do not autoclave. Cool to 45°– 50°C. Aseptically add 10.0mL of sterile novobiocin solution. Mix thoroughly. Pour into sterile Petri dishes. Air-dry plates for at least 1 hr.

Use: For the isolation and cultivation of motile Salmonella species from food and environmental samples.

Modified Shieh Agar (LMG Medium 215) Composition per liter: Agar ............................................................................................ 15.0g Peptone ......................................................................................... 5.0g Yeast extract.................................................................................. 1.0g MgSO4·7H2O ................................................................................ 0.3g K2HPO4......................................................................................... 0.1g KH2PO4.................................................................................... 50.0mg NaHCO3 ................................................................................... 50.0mg Na-acetate ................................................................................ 10.0mg BaCl2·H2O ............................................................................... 10.0mg

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Modified Skim Milk HiVeg Agar

CaCl2·2H2O............................................................................... 6.7 mg FeSO4·7H2O............................................................................... 1.0mg pH 7.3 ± 0.2 at 25°C

NaCl.............................................................................................. 5.0g Papaic digest of soyabean meal .................................................... 3.0g K2HPO4......................................................................................... 2.5g Glucose ......................................................................................... 2.5g Polysorbate 80 .........................................................................10.0mL pH 7.3 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Flavobacterium spp.,

Source: This medium, without polysorbate 80, is available as a premixed powder from HiMedia.

Flexibacter spp., Chitinophaga pinensis, and Flectobacillus major.

Preparation of Medium: Add components to distilled/deionized

Modified Skim Milk HiVeg Agar Composition per liter: Agar ............................................................................................ 15.0g Plant hydrolysate........................................................................... 5.0g Yeast extract.................................................................................. 2.5g Skim milk powder......................................................................... 1.0g Glucose monohydrate ................................................................... 1.0g pH 7.0 ± 0.1 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the cultivation and differentiation of bacteria based on proteolytic activity. For the cultivation and enumeration of lactic streptococci used in manufacturing of cheddar cheese.

Modified Soyabean Bile Broth Base Composition per liter: Casein enzymic hydrolysate ....................................................... 17.0g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 4.0g Papaic digest of soybean meal ...................................................... 3.0g D-Glucose...................................................................................... 2.5g Bile salts mixture .......................................................................... 1.5g Selective supplement solution .................................................10.0mL pH 7.3 ± 0.2 at 25°C

Source: This medium is available from HiMedia.

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C.

Use: For the microbiological assay of polymyxin B, colistin sulfate, and sodium colistimethate.

Modified TH Agar (DSMZ Medium 1061) Composition per liter: Agar ........................................................................................... 15.0g Tryptone ....................................................................................... 5.0g NaCl ............................................................................................. 5.0g Yeast extract ................................................................................. 3.0g MnSO4·2H2O .............................................................................. 0.01g Trace elements solution SL-6 ....................................................1.0mL pH 7.5 ± 0.2 at 25°C

Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................................................. 0.5g H3BO3 ........................................................................................... 0.3g CoCl2·6H2O .................................................................................. 0.2g ZnSO4·7H2O ................................................................................ 0.1g Na2MoO4·2H2O ......................................................................... 0.03g NiCl2·6H2O ................................................................................. 0.02g CuCl2·2H2O ................................................................................ 0.01g

Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium: Add components to distilled/deionized

Novobiocin............................................................................... 10.0mg

water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into Petri dishes or leave in tubes.

Preparation of Selective Supplement Solution: Add novobio-

Use: For the cultivation of Anoxybacillus voinovskiensis.

Selective Supplement Solution: Composition per 10.0mL:

cin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Modified Soybean HiVeg Agar Composition per liter: Plant hydrolysate......................................................................... 17.0g Agar ............................................................................................ 15.0g © 2010 by Taylor and Francis Group, LLC

Modified Thayer-Martin Agar See: Thayer-Martin Agar, Modified

Modified Thermus Medium (DSMZ Medium 630) Composition per liter: Agar ............................................................................................ 28.0g Yeast extract.................................................................................. 2.5g Tryptone........................................................................................ 2.5g MgCl2·6H2O .......................................................................... 200.0mg Nitrilotriacetic acid ................................................................ 100.0mg CaSO4·2H2O ............................................................................ 40.0mg Phosphate solution .................................................................100.0mL

Modified VWM Medium

Ferric citrate solution .................................................................0.5mL Trace elements solution .............................................................0.5mL pH 7.2 ± 0.2 at 25°C

Phosphate Solution: Composition per liter: Na2HPO4·12H2O......................................................................... 43.0g KH2PO4 ....................................................................................... 5.44g

Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Ferric Citrate Solution: Composition per liter: Ferric citrate .................................................................................. 2.5g

Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ..................................................................... 12.8g FeCl2·4H2O ................................................................................... 1.0g MnCl2·4H2O.................................................................................. 0.5g CoCl2·4H2O .................................................................................. 0.3g Na2MoO4·4H2O ....................................................................... 50.0mg CuCl2·2H2O ............................................................................. 50.0mg NiCl2·6H2O .............................................................................. 20.0mg H3BO3 ...................................................................................... 20.0mg

Preparation of Medium: Add components, except phosphate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 100.0mL phosphate solution. Mix thoroughly. Adjust pH to 7.2. Pour into Petri dishes or aseptically distribute into sterile tubes.

Use: For the cultivation and maintenance of Thermus sp., Rhodothermus marinus, and Albidovulum inexpectatum.

Modified V.P. HiVeg Broth Composition per liter: Plant peptone No. 3....................................................................... 7.0g Glucose ......................................................................................... 5.0g NaCl .............................................................................................. 5.0g pH 6.9 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.9. Distribute into tubes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and differentiation of bacteria based on their ability to produce acetoin.

Modified VWM Medium (DSMZ Medium 503a) Composition per 1013.4mL: Solution A ..............................................................................940.0mL © 2010 by Taylor and Francis Group, LLC

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Solution E ................................................................................50.0mL Solution F.................................................................................10.0mL Solution G................................................................................10.0mL Solution B ..................................................................................1.0mL Solution C ..................................................................................1.0mL Solution D..................................................................................1.0mL Solution H..................................................................................0.4mL pH 7.3 ± 0.2 at 25°C

Solution A: Composition per 940.0mL: NaCl.............................................................................................. 1.0g KCl................................................................................................ 0.5g MgCl2·6H2O ................................................................................. 0.4g NH4Cl ......................................................................................... 0.25g KH2PO4......................................................................................... 0.2g CaCl2·2H2O ................................................................................ 0.15g Resazurin ................................................................................... 0.5mg

Preparation of Solution A: Prepare under 80% N2 + 20% CO2 gas

Preparation of Solution B: Add FeCl2·4H2O to 10.0mL of HCl so-

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

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Moeller Decarboxylase Broth

Moeller Decarboxylase HiVeg Broth Base (Decarboxylase Broth Base, Moeller)

Solution E: Composition per 100.0mL: NaHCO3 ........................................................................................ 5.0g

Composition per liter:

Preparation of Solution E: Add NaHCO3 to distilled/deionized

Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Glucose ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.01g Pyridoxal.................................................................................... 5.0mg Cresol Red ................................................................................. 5.0mg pH 6.0 ± 0.2 at 25°C

water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution F: Composition per 10.0mL: Taurine .......................................................................................... 2.5g

Preparation of Solution F: Add taurine to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% N2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution G: Composition per 10.0mL: Na2S·9H2O ................................................................................ 0.125g

Preparation of Solution G: Add Na2S·9H2O to distilled/deionized

water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Solution H: Composition per 10.0mL: Na-dithionite ................................................................................. 0.5g

Preparation of Solution H: Add Na-dithionite to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Cool to 25°C.

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. Sequentially add 1.0mL solution B, 1.0mL solution C, 1.0mL solution D, 50.0mL solution E, 10.0mL solution F, 10.0mL solution G, and 0.4mL solution H to 940.0mL solution A. Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels. The pH should be 7.2–7.4.

Use: For the cultivation of Desulfonispora thiosulfatigenes.

Moeller Decarboxylase Broth Composition per liter: Amino acid.................................................................................. 10.0g Peptic digest of animal tissue........................................................ 5.0g Beef extract ................................................................................... 5.0g Glucose ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.01g Cresol Red.................................................................................. 5.0mg Pyridoxal .................................................................................... 5.0mg pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Use L-lysine, L-arginine, or L-ornithine. Mix thoroughly. Gently heat until dissolved. Distribute into screw-capped tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. A slight precipitate may form in the ornithine broth.

Use: For the differentiation of Gram-negative enteric bacteria based on the production of arginine dihydrolase, lysine decarboxylase, or ornithine decarboxylase. © 2010 by Taylor and Francis Group, LLC

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until dissolved. Distribute into screw-capped tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: With the addition of amino acid solutions, this medium is used for the differentiation of Gram-negative enteric bacteria based on the production of amino acid decarboxylation reactions.

Moeller Decarboxylase HiVeg Broth Arginine HCl (Decarboxylase Broth Base, Moeller with Arginine) Composition per liter: L-Arginine hydrochloride ........................................................... 10.0g Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Glucose ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.01g Pyridoxal.................................................................................... 5.0mg Cresol Red ................................................................................. 5.0mg pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Use: For the differentiation of Gram-negative enteric bacteria based on the production of arginine dihydrolase.

Moeller Decarboxylase HiVeg Broth with Lysine HCl (Decarboxylase Broth Base, Moeller with Lysine) Composition per liter: L-Lysine

hydrochloride ............................................................... 10.0g Plant extract .................................................................................. 5.0g Plant peptone ................................................................................ 5.0g Glucose ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.01g Pyridoxal.................................................................................... 5.0mg Cresol Red ................................................................................. 5.0mg pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat until dis-

Molybdate Agar

solved. Distribute into screw-capped tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

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Media.

Agar .............................................................................................. 3.0g Na2SO4 .......................................................................................... 1.6g Plant hydrolysate .......................................................................... 1.0g CaCl2 ............................................................................................. 0.9g (NH4)2SO4 .................................................................................... 0.5g Tris hydroxymethyl aminomethane .............................................. 0.5g KCl............................................................................................ 0.275g Yeast extract.................................................................................. 0.1g NaHCO3...................................................................................... 0.08g KBr ............................................................................................. 0.04g SrCl2 ......................................................................................... 0.017g H3BO3 ....................................................................................... 0.011g Phenol red ................................................................................... 0.01g Na2HPO4 .................................................................................... 4.0mg Sodium silicate........................................................................... 2.0mg NaFl ........................................................................................... 1.2mg NH4NO3 ..................................................................................... 0.8mg Carbohydrate solution............................................................100.0mL pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

Source: This medium is available as a premixed powder from Hi-

Use: For the differentiation of Gram-negative enteric bacteria based on the production of lysine decarboxylase.

Moeller Decarboxylase HiVeg Broth with Ornithine HCl Composition per liter: L-Ornithine hydrochloride .......................................................... 10.0g Plant extract .................................................................................. 5.0g Plant peptone................................................................................. 5.0g Glucose ......................................................................................... 0.5g Bromcresol Purple ...................................................................... 0.01g Pyridoxal .................................................................................... 5.0mg Cresol Red.................................................................................. 5.0mg pH 6.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-

water and bring volume to 1.0L. Mix thoroughly. Gently heat until dissolved. Distribute into screw-capped tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the differentiation of Gram-negative enteric bacteria based on the production of ornithine decarboxylase.

Moeller KCN Broth Base Composition per liter: Na2HPO4 ..................................................................................... 5.64g NaCl .............................................................................................. 5.0g Pancreatic digest of casein ............................................................ 1.5g Peptic digest of animal tissue........................................................ 1.5g KH2PO4 ..................................................................................... 0.225g KCN solution ...........................................................................0.15mL pH 7.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

KCN Solution: Composition per 100.0mL:

Media.

Carbohydrate Solution: Composition per 100.0mL: Carbohydrate............................................................................... 10.0g

Preparation of Carbohydrate Solution: Add carbohydrate to distilled/deionized water and bring volume to 100.0mL. Adonitol, arabinose, cellobiose, glucose, dulcitol, fructose, galactose, inositol, lactose, maltose, mannitol, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose, xylose, or other carbohydrates may be used. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except carbohydrate solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat until dissolved. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 100.0mL sterile carbohydrate soltuion. Mix thoroughly. Aseptically distribute into screw-capped tubes in 5.0mL volumes. Use: For the differentiation of marine bacteria on the basis of fermentative and oxidative metabolism of carbohydrates.

Molybdate Agar

KCN .............................................................................................. 0.5g

Composition per101.5mL:

Preparation of KCN Solution: Add KCN to 100.0mL of cold dis-

Base........................................................................................100.0mL Phosphomolybdic acid solution.................................................1.5mL pH 5.3 ± 0.2 at 25°C

tilled/deionized water. Mix thoroughly and cap. Do not mouth pipette.

Caution: Cyanide is toxic. Preparation of Medium: Add components, except KCN solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Prior to use, add 0.15mL of KCN solution. Mix thoroughly. Aseptically distribute into sterile tubes.

Use: For the differentiation of Gram-negative enteric bacteria on the basis of their ability to grow in the presence of cyanide.

MOF HiVeg Medium with Carbohydrate Composition per liter: NaCl .............................................................................................. 9.7g MnCl2 ............................................................................................ 4.4g © 2010 by Taylor and Francis Group, LLC

Base: Composition per liter: Sucrose........................................................................................ 40.0g Agar ............................................................................................ 15.0g Meat peptone .............................................................................. 10.0g

Preparation of Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.6. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C.

Phosphomolybdic Acid Solution: Composition per 100.0mL: P2O5·2OMoO3............................................................................. 12.5g

1222

Monsur Agar

Preparation of Base: Add P2O5·2OMoO3 (phospho-12-molybdic acid, 12–molybdophosphoric acid, or PMA) to sterile distilled/deionized water. Mix thoroughly. Do not adjust pH.

Na2S·9H2O Solution: Composition per 10.0mL:

Preparation of Medium: To 100.0mL of cooled sterile base, add

Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–121°C.

1.5mL of phosphomolybdic acid solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation and presumptive identification of yeast, especially Candida species. Candida albicans appears as smooth, medium olive colonies with medium olive bottoms. Candida stellatoidea appears as shiny, light gray colonies with light gray bottoms. Candida tropicalis appears as smooth, shiny, dark blue/gray colonies with dark blue/gray bottoms. Candida krusei appears as smooth, dull white colonies with white bottoms. Saccharomyces cerevisiae appears as smooth, shiny light blue/dark blue colonies with dark blue/green bottoms.

Monsur Agar (Taurocholate Tellurite Gelatin Agar) Composition per liter: Gelatin......................................................................................... 30.0g Agar ............................................................................................ 15.0g Casein peptone ............................................................................ 10.0g NaCl ............................................................................................ 10.0g Sodium taurocholate ..................................................................... 5.0g Na2CO3·H2O ................................................................................. 1.0g K2TeO3 solution .......................................................................10.0mL pH 8.5 ± 0.2 at 25°C

K2TeO3 Solution: Composition per 10.0mL:

Na2S·9H2O.................................................................................... 0.5g

Selenite-Tungstate Solution: Composition per liter: NaOH............................................................................................ 0.5g Na2WO4·2H2O ........................................................................... 4.0mg Na2SeO3·5H2O........................................................................... 3.0mg

Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N2. Filter sterilize.

Vitamin Solution: Composition per liter: Pyridoxine-HCl........................................................................ 10.0mg Thiamine-HCl·2H2O.................................................................. 5.0mg Riboflavin .................................................................................. 5.0mg Nicotinic acid............................................................................. 5.0mg D-Ca-pantothenate ..................................................................... 5.0mg p-Aminobenzoic acid................................................................. 5.0mg Lipoic acid ................................................................................. 5.0mg Biotin ......................................................................................... 2.0mg Folic acid ................................................................................... 2.0mg Vitamin B12 ................................................................................ 0.1mg

Preparation of K2TeO3 Solution: Add K2TeO3 to 10.0mL of dis-

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H2 + 20% CO2. Filter sterilize.

Caution: Potassium tellurite is toxic.

Trace Elements Solution SL-10: Composition per liter:

K2TeO3 ........................................................................................ 0.02g tilled/deionized water. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except K2TeO3 solu-

tion, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Add 10.0mL of sterile K2TeO3 solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.

Use: For the isolation of Vibrio cholerae from fecal specimens.

Moorella glycerini Medium (DSMZ Medium 793) Composition per liter: NaHCO3 ...................................................................................... 10.0g Yeast extract.................................................................................. 0.5g KH2PO4 ....................................................................................... 0.33g NH4Cl ......................................................................................... 0.33g KCl.............................................................................................. 0.33g MgCl2·6H2O................................................................................ 0.33g CaCl2·2H2O................................................................................. 0.33g Resazurin ................................................................................... 0.5mg Vitamin solution.......................................................................10.0mL Na2S·9H2O solution .................................................................10.0mL Glycerol, 87% ............................................................................3.0mL Trace elements solution SL-10 ..................................................1.0mL Selenite-tungstate solution .........................................................1.0mL pH 6.7± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

FeCl2·4H2O ................................................................................... 1.5g CoCl2·6H2O ........................................................................... 190.0mg MnCl2·4H2O .......................................................................... 100.0mg ZnCl2 ........................................................................................ 70.0mg Na2MoO4·2H2O ....................................................................... 36.0mg NiCl2·6H2O .............................................................................. 24.0mg H3BO3 ........................................................................................ 6.0mg CuCl2·2H2O ............................................................................... 2.0mg HCl (25% solution)..................................................................10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

Preparation of Medium: Prepare and dispense medium under 100% CO2 gas atmosphere. Add components, except NaHCO3, Na2S·9H2O solution, and vitamin solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 5 min. Cool to room temperature while sparging with 100% CO2. Add solid NaHCO3. Adjust pH to 6.7 by adding NaOH and equilibrating with 100% CO2. Distribute into tubes or bottles. Autoclave for 20 min at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL Na2S·9H2O solution and vitamin solution, 0.1mL per 10mL medium. Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes or bottles.

Use: For the cultivation of Moorella glycerini.

Motility Medium

Moraxella Medium (LMG Medium 204) Composition per liter: Special peptone ........................................................................... 23.0g Agar ............................................................................................ 15.0g Glucose ......................................................................................... 5.0g NaCl .............................................................................................. 5.0g Soluble starch................................................................................ 1.0g Cysteine hydrochloride ................................................................. 0.3g Sheep blood, sterile defibrinated .............................................50.0mL pH 7.1 ± 0.2 at 25°C

Source: Special peptone is available from Oxoid Unipath. Preparation of Medium: Add components, except sheep blood, to 950.0mL distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 50.0mL sterile sheep blood. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Moraxella osloensis,

1223

Use: For the cultivation and differentiation of members of the Enterobacteriaceae on the basis of motility, lysine decarboxylase activity, lysine deaminase activity, and indole production.

Motility Indole Ornithine Medium (MIO Medium) Composition per liter: Pancreatic digest of gelatin......................................................... 10.0g Pancreatic digest of casein............................................................ 9.5g L-Ornithine·HCl ............................................................................ 5.0g Yeast extract.................................................................................. 3.0g Agar .............................................................................................. 2.0g Glucose ......................................................................................... 1.5g Bromcresol Purple ...................................................................... 0.02g pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized

Moraxella atlantae, and Cellulomonas hominis.

water and bring to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Motility GI Medium

Use: For the differentiation of Gram-negative enteric bacteria based

Composition per liter: Gelatin......................................................................................... 53.4g Heart infusion broth .................................................................... 25.0g Agar .............................................................................................. 3.0g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to cold distilled/deionized water and bring to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes in 20.0mL volumes or leave in tubes.

Use: For demonstrating the motility of microorganisms and for separating organisms in their motile phase.

Motility Indole Lysine Medium See: MIL Medium

Motility-Indole-Lysine HiVeg Medium (MIL HiVeg Medium) Composition per liter: Plant hydrolysate......................................................................... 10.0g Plant peptone............................................................................... 10.0g L-Lysine hydrochloride ............................................................... 10.0g Yeast extract.................................................................................. 3.0g Agar .............................................................................................. 2.0g Glucose ......................................................................................... 1.0g Ferric ammonium citrate............................................................... 0.5g Bromcresol Purple ...................................................................... 0.02g pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from HiMedia.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC

on their motility, indole production, and ornithine decarboxylase activity.

Motility Indole Ornithine Medium (MIO Medium) (BAM M99) Composition per liter: Tryptone...................................................................................... 10.0g Peptone ....................................................................................... 10.0g L-Ornithine·HCl ............................................................................ 5.0g Yeast extract.................................................................................. 3.0g Agar .............................................................................................. 2.0g Glucose ......................................................................................... 1.0g Bromcresol Purple ...................................................................... 0.02g pH 6.6 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the differentiation of Gram-negative enteric bacteria based on their motility, indole production, and ornithine decarboxylase activity.

Motility Medium Composition per liter: Pancreatic digest of casein.......................................................... 10.0g Glucose ......................................................................................... 5.0g Agar .............................................................................................. 3.0g Na2HPO4 ...................................................................................... 2.5g Yeast extract.................................................................................. 2.5g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring

1224

Motility Medium S

to boiling. Distribute into tubes in 2.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to stand at 25°C for 2 days prior to inoculation.

Source: This medium, without glycerol, is available as a premixed

Use: For the cultivation and observation of motility of Bacillus cereus.

nents to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes in 4.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Motility Medium S Composition per liter: Beef heart, solids from infusion................................................ 500.0g Gelatin......................................................................................... 30.0g Enzymatic hydrolyzate of protein............................................... 10.0g NaCl .............................................................................................. 5.0g K2HPO4 ......................................................................................... 2.0g KNO3 ............................................................................................ 2.0g Agar .............................................................................................. 1.0g 2,3,5-Triphenyltetrazolium chloride solution ..........................10.0mL pH 7.2 ± 0.2 at 25°C

powder from HiMedia.

Preparation of Medium: Add glycerol followed by other compo-

Use: For the cultivation and observation of motility and nitrate reduction in a variety of Gram-negative bacteria.

Motility Nitrate Medium (FDA M101) Composition per liter:

2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL:

Beef heart, solids from infusion................................................ 100.0g Tryptose ...................................................................................... 12.0g Agar .............................................................................................. 3.0g NaCl.............................................................................................. 1.0g KNO3, nitrite free ......................................................................... 1.0g pH 7.4 ± 0.2 at 25°C

2,3,5-Triphenyltetrazolium chloride ............................................. 0.1g

Preparation of Medium: Add components to distilled/deionized

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solution: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into screw-capped tubes in 4.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except 2,3,5-triphenyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 60°C. Aseptically add 10.0mL of the sterile 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Aseptically distribute into sterile tubes. Keep at 4°–8°C until used.

Use: For the determination of bacterial motility.

Motility Nitrate Agar Composition per liter: Beef heart, solids from infusion................................................ 100.0g Tryptose ...................................................................................... 12.0g Agar .............................................................................................. 3.0g NaCl .............................................................................................. 1.0g KNO3 ............................................................................................ 1.0g Glucose ......................................................................................... 0.5g pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes in 4.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.

Use: For the cultivation and observation of motility and nitrate reduction in a variety of Gram-negative bacteria.

Motility Nitrate HiVeg Medium, Buffered Composition per liter: Galactose....................................................................................... 5.0g Plant extract .................................................................................. 3.0g Plant peptone................................................................................. 5.0g KNO3 ............................................................................................ 5.0g Agar .............................................................................................. 3.0g Na2HPO4 ....................................................................................... 2.5g Glycerol .....................................................................................5.0mL pH 7.4 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC

Use: For the cultivation and differentiation of Gram-negative nonfermentative bacteria from cosmetics based on their motility and their ability to reduce nitrate to nitrite.

Motility Nitrate Medium Composition per liter: Beef heart, solids from infusion................................................ 100.0g Tryptose ...................................................................................... 12.0g Agar .............................................................................................. 3.0g NaCl.............................................................................................. 1.0g KNO3 ............................................................................................ 1.0g

Use: For the cultivation and observation of motility and nitrate reduction in a variety of Gram-negative nonfermentative bacteria isolated from cosmetics.

Motility Nitrate Medium (BAM M101) Composition per liter: Tryptose ...................................................................................... 10.0g Agar .............................................................................................. 3.0g Beef heart, infusion from 500g..................................................... 2.0g Tryptose ........................................................................................ 2.0g NaCl.............................................................................................. 1.0g KNO3 ............................................................................................ 1.0g Glucose ......................................................................................... 0.5g pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat with agitation to dissolve agar. Distribute into screw-cap tubes. Autoclave for 15 min at 15 psi pressure–121°C.