Pharma Culture media by Innovation Diagnostics Inc.

Culture Media for the Pharmaceutical Industry

AES CHEMUNEX – Rue Maryse Bastié – Ker Lann – CS17219 – 35172 BRUZ cedex - FRANCE Tel : +33 (0)2 23 50 12 12 – Fax : +33 (0)2 23 50 12 00 http://www.aeschemunex.com – contact@aeschemunex.com TA004B

AES CHEMUNEX is ISO 9001:2000 certified for the design, manufacturing and sales of reagents, instruments, consumables and materials for microbiology laboratories. As to guarantee the quality and the performance of our culture media, we process and control them according to the specifications described in the European Pharmacopoeia (6th edition). The environment process (hygiene, pressurisation, temperature, humidity…) undergoes a harsh and continuous monitoring. Preventive servicing is carried out on the processing machinery. Measurement and test control appliances are calibrated and adjusted to the standard reference. A highly qualified staff and clearly described procedures make the process easily accessible to each employee. Our media go through physical and chemical controls (pH, volume, appearance…) during process and before final validation of batches. Qualitative and quantitative microbiology tests are carried out on each batch before testing their efficiency on stability, fertility and reactivity. Micro-organisms strains used for fertility tests are the ones specified by the European Pharmacopoeia. Through its experience and expertise, AES CHEMUNEX establishes the finest storage and theory shelf life for their media according to the components and the packaging. Once established these theory expiry dates are then validated or amended: •

By quantitative comparison tests between batches of media close to their expiry date and freshly made ones.

•

By fluctuation temperature tests as to characterize the optimum preservation and transport conditions. The “Validation of culture media performances after transport” (5 days) file is available upon request

Our media carry a label or a stamp on which is indicated a preservation temperature. This information is only given as a guide line figure as being the optimum condition for storage established by our laboratory.

More importantly, an isolated derive or a moderate variation in temperature that could occur during transport or incubation does not affect (only in scares exceptions) the quality of our products. Keeping the temperature constant is essential throughout the shelf life, nevertheless, a vast majority of our products have succeeded reactivity tests after suffering moderate temperature changes. This of course not only stands for transport phases but also for their incubation when the media are used. A special file is available, on request, summering up all the stability validation tests after transport. Each one of our packs is sealed with a standard label as a warranty of inviolability (Tamper proof label). On this label figure the following data: •

Name of the product, packaging and quantities

•

Storage temperature,

•

Reference, Batch number, expiry date.

A quality control certificate known as “detachable” and “self-adhesive” is composed of the following information: •

Name of the product, packaging and quantities

•

Reference, Batch number, expiry date and pH.

•

A conformity statement notifying that the quality control has been carried out following a Standard Quality Control Sheet « QCP » whose auditing index is referred to. An up date of the latest QCP version is easily accessible through our web site or just by contacting our microbiology technical department (Phone or mail). To have access to the QCP via the web site you will be given a password when you first login.

Moreover, the label is composed of 12 stickers on which figure the following details: Name of product, batch number, and expiring date (see QCP and control certificate management).

A new irradiating equipment allows to produce perfectly controlled and homogeneous radiosterilised media (for more details please contact us). Finally, as a token of our professionalism and transparency, we would like to stress that our production site can be visited by all our customers upon request. Please, contact us for more details.

Visit our web site: www.chromogenic-media.com

QCP and control certificate management A vast majority of our media have now detachable self-adhesive quality control certificate that you can find on their packaging. This new form of certificate will firstly shorten the time spent on controlling the different statements and secondly minimise the volume of papers filed. The self-adhesive certificate refers to our protocol and control certifications, known as Quality Control Protocols (QCP). These protocols carry an auditing index up dated when the protocol is modified (new packaging, use of new micro-organism strains …).The selfadhesive label gives the variable data belonging to the batch (ie: number, pH, expiring date). The QCP lists all the other details of the control: appearance, sterility, fertility… By sticking the quality control certificate to the QCP it refers to, all the details of the data controlled on the batch are then filed. The self-adhesive certificate guarantees that all the different steps of the procedure listed on the QCP have been validated before releasing the batch. Any modification given to the QCP by AES CHEMUNEX, entails a revision of the auditing index. This modification then automatically appears on the self-adhesive certificate of the newly controlled batches. As to keep your paper work up dated you then just need to obtain the new version by contacting our microbiology technical department (phone, mail, fax) or logging onto our website: www.aeschemunex.com To get access to the QCP on our website you need to register (free service) on our on-line service sheet. This formality not only allows you to get access to the QCP but also to the media’s technical sheets, and most importantly to be informed by mail that a QCP has been revised. This service will help guaranty the follow-up and up date of your media files.

Quality Control Protocol reference and index revision letter (Download Quality Control Protocol (QCP) from our web site

www.aeschemunex.com

pH measured of the batch

Detachable sticker

Self adhesive detachable Q.C. certificate

6 individual self adhesive & detachable stickers.

Product’s abbreviation

N.B : All our culture media follow the QCP system excepting dehydrated versions, packs of 5 tubes and packs of 10 ready poured dishes. Your contact: Claire GARDYN – phone: 00 33(0)2 99 73 36 82 - fax : 00 33 (0)2 23 50 12 00 e-mail : c.gardyn@aeschemunex.com

In parallel of our system QCP/certificates stickers, we offer to you via our on-line service a new tool allowing you to print by batch a quality control certificate including raw data from our quality control laboratory. To get to these certificates, click on Download area on our website www.aeschemunex.com. Enter your Login and Password. To print the certificates, it will be necessary for you to enter the code number of the product as well as its batch number. Example of certificate:

Our advantages Tailor-made culture media Beyond the classic range of media commonly used in the pharmaceutical and cosmetics industries, AES CHEMUNEX proposes to manufacture culture media according to your specifications*. These specifications can be: - the formula of the medium - the origin of the raw materials - the packaging - the validation controls (test strains, sampling, quarantine…)… * subject to technical feasibility and minimum annual agreement (quantities) Our Scientific department is at your disposal to study your requests.

Plasma flasks

Sirup flasks

Twist-off jars and milk jar

Dilubags (pouches of 3 or 5 litres)

Sterility test: our ready to use media used to carry out sterility tests can be available double wrapped. Please, see our price list.

Media fill test: to help you carry out these tests, AES CHEMUNEX proposes ionized dehydrated Tryptic Soy Broth (AEB141502RSTE) and Thioglycollate Resazurin broth (AEB141412RSTE). Depending on your process, ready to use Tryptic Soy Broth in 5 litres Dilubags® (AEB911505/2) is also available.

Biosafe container*: disposable packaging for a safe transport of radio-

cosmetics industries sterilized culture media used for the microbiological control in sterile environment. This system avoids having to use peracetic acid to sterilise reagents. * Complete file upon request

Culture media for the pharmaceutical environment monitoring Our manufacturing process expertise enables us to offer radio sterilized pre-poured agars triple wrapped with a shelf life from manufacturing day extended to 6 months for contact plates and to 9 months for Petri dishes. Available with standing order only Designations Sabouraud Agar : Medium C Trytpic Soy Agar : Medium B (TSA) TSA 4 N IONISED LONG LIFE HYGICOUNT 4N SABOURAUD 4N IONISED LONG LIFE

Code numbers AEB522209LL AEB522879LL AEB530149LL AEB530169CLL AEB522239CLL

Packaging 120 plates 90mm 120 plates 90mm 120 plates 90mm 120 plates 65 mm 120 plates 65 mm

Culture media for the pharmaceutical industry 1. Diluents & sterility tests AOAC Letheen broth ....................................................................................p.13 DNP: NPD (Neutralizing Pharmacopoeia Diluent) ........................................p.14 pH7 Pharmacopoeia diluent .........................................................................p.15 Fluid K ..........................................................................................................p.16 Meat peptone â&#x20AC;&#x201C; Fluid A.................................................................................p.17 Tryptic Soy Broth (TSB)................................................................................p.18 Thioglycollate resazurin ................................................................................p.19

2. Testing Total Viable Aerobic Count (TAMC & TYMC) ..............................................p.21 Tryptic Soy Agar (TSA) ...........................................................................p.24 Sabouraud Agar ......................................................................................p.25 Gram negative bacteria resistant to bile salts ...............................................p.27 EE Broth Mossel .....................................................................................p.28 VRBG Pharma ........................................................................................p.29 E.coli.............................................................................................................p.30 Mac Conkey broth ..................................................................................p.31 Mac Conkey agar ....................................................................................p.32 Pseudomonas...............................................................................................p.33 Cetrimide Pharmacopoeia agar...............................................................p.34 Staphylococci ...............................................................................................p.35 Chapman.................................................................................................p.36 Salmonella ...................................................................................................p.37 Rappaport Vassiliadis Pharma ................................................................p.38 XLD .........................................................................................................p.39 Clostridium....................................................................................................p.41 RCM ........................................................................................................p.42 Columbia 3 ..............................................................................................p.43 Candida ........................................................................................................p.44 Sabouraud Broth .....................................................................................p.45 Sabouraud Agar ......................................................................................p.46

3. Control of the environment R2A .........................................................................................................p.49 TSA 4N IONISED long life.......................................................................p.50 Hygicount and Hygicount 4N...................................................................p.51 Sampl’air .................................................................................................p.53 EP – USP – AES CHEMUNEX ....................................................................p.54 Alphabetical index ........................................................................................p.56 Your contacts................................................................................................p.57

Diluents & sterility tests

AOAC LETHEEN BROTH Broth for the determination of the phenol coefficient in cationic surface-active materials In Vitro use only Store between 2 and 25°C

PRINCIPLE AOAC Letheen is used for determining the phenol coefficient of cationic surface-active materials. Letheen broth was developed as a subculture medium for the neutralization of quaternary ammonium compounds in disinfectant testing. Quisno, Gibby and Foter, found that the addition of lecithin and Polysorbate 80 to F.D.A. broth resulted in a medium that neutralized high concentrations of quaternary ammonium salts. The resulting medium, termed “Letheen” was easy to prepare and clear in appearance which aided in visual inspection for growth. FORMULA In grammes per litre of purified water Peptone 10.0 Beef extract 5.0 Lecithin 0.7 Polysorbate 80 5.0 Sodium chloride 5.0 Final pH : 7,0 + 0,2 at 25°C PROCEDURE AND RESULTS Refer to appropriate references such as the AOAC. BIBLIOGRAPHY 1. Official methods of analysis of the AOAC, 14th Edition (william – 1984) PACKAGING Ready to use medium AEB110059 : Pack of 100 tubes of 9 ml AEB610058M : 6 flasks of 500 ml with septum Special packaging (Please contact us ) AEB610050M :6 flasks of 500 ml with septum AEB610054B : 6 flasks of 90 ml (ISO flasks) Made by AES CHEMUNEX - Combourg – France 110059£: 03/11/08 - F

DNP (Buffer) NPD (Neutralizing Pharmacopoeia Diluent) In Vitro use only To be stored between 2 and 25°C

PRINCIPLE DNP buffer is a general-purpose dilution solution used in pharmaceutical industry. Its levels of polysorbate 80 and lecithin can be increased when stronger emulsifying power is needed. Other surface-active agents or inactivators of antimicrobial agents may be added such as: • Sodium laurylsulfate at 0,4% (phenols neutralization) • saponine 3% + chlorhydrate cystine 0,1% (aldehydes neutralization), • Sodium thioglycollate at 0,5% + L-cystine at 0,1% + sodium thiomalate at 0,075% (organomercurials neutralization) • Sodium thiosulfate at 0,5% ( Halogens neutralization) • egg yolk at 5% ( Ammoniums neutralization) • catalase and peroxydase (Peroxydes neutralisation ). DNP buffer is used when testing the sterility of endoscope. When cleaned with peracetic acid then use DNP + 5 g/l of sodium thiosulfate for testing the sterility .

PACKAGING Ready to use medium AEB111279 : Pack of 100 tubes of 9 ml AEB611274 : Pack of 6 flasks of 90 ml AEB611278 : Pack of 6 flasks of 200 ml AEB611279LAF: Pack of 6 flasks of 900 ml AEB611277AF: Pack of 6 flasks of 300 ml Ready to use medium in flask with septum AEB611274M : Pack of 6 flasks de 90 ml AEB611275M : Pack of 6 flasks of 400 ml Ready to use DNP + thiosulfate 0,5% AEB611324 : Pack of 6 flasks of 90 ml AEB611326M : Pack of 6 flasks with septum of 100 ml AEB611327M : Pack of 6 flasks with septum of 600 ml Ready to use DNP + Lactose AEB611284 : Pack of 6 flasks of 90 ml Made by : AES CHEMUNEX -Combourg - France

FORMULA grammes per litre of purified water. Polysorbate 80 Lecithin Histidin HCl Casein Peptone Sodium Chloride Potassium Phosphate monobasic Sodium Phosphate Dibasic

BIBLIOGRAPHY th 1. European Pharmacopoeia 6 edition.

30,0 3,0 1,0 1,0 4,3 3,6 7,2

111279£: 03/11/08 - N

Final pH : 7,0 + 0,2 at 25°C PROCEDURE Homogenize the sample to be tested or its decimal dilutions in the diluents. Let the diluent works at room temperature for 30 minutes to 1 hour. Then inoculate account agar plates with the neutralized specimen, or proceed according to the laboratory protocol. Endoscopes sterility testing : procedure available when requested.

pH 7 PHARMACOPOEIA DILUENT Buffered sodium chloride peptone solution according to European Pharmacopoeia In Vitro use only

PRINCIPLE pH7 Pharmacopoeia diluent corresponds to the buffer medium recommended by the harmonized method of pharmocopoeias as a general-purpose diluent for microbiological analysis. To this basal medium several compounds may be added to neutralize the activity of disinfectants. Te most communly used neutralizing agents and recommended by EP are : • Tween 80, Lecithin and Histidin HCl added to this medium form the DNP buffer. Other surface-active agents or inactivators of antimicrobial agents may be added such as: • Sodium laurylsulfate at 0,4% (phenols neutralization) • saponine 3% + chlorhydrate cystine 0,1% (aldehydes neutralisation), • Sodium thioglycolate at 0,5% + L-cystine at 0,1% + sodium thiomalate at 0,075% (organomercurials neutralization) • Sodium thiosulfate at 0,5% ( Halogens neutralisation) • egg yolk at 5% ( Ammoniums neutralization) catalase and peroxydase (Peroxydes neutralization ). FORMULA In grammes per litre of purified water Tryptone 1,00 Sodium chloride 4,30 Potassium Dihydogen phosphate 3,56 Disodium hydrogen Phosphate Anhydrous 5,76 (corresponding to 7,23 g of disodium phosphate dihydrated) Final pH : 7,0 + 0,2 at 25°C METHOD Suspend 14.6 g of powder into 1 litre of purified water. Bring slowly to the boil, stirring to obtain complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C. PROCEDURE Homogenize the sample and /or prepared decimal dilutions of the sample using this diluent. Leave at room temperature for 30-60 minutes then inoculate the appropriate culture media.

BIBLIOGRAPHY ème 1. Pharmacopée Européenne. 6 édition.. PACKAGING - Dehydrated medium (Store between 1 and 30°C) AEB141292 : 500g - Ready to use medium (Store between 2 and 25°C) AEB611294 : Pack of 6 flasks of 90 ml AEB611294NO: Pack of 6 flasks of 90ml AEB611297 : Pack of 6 flasks of 200 ml AEB611313 : Pack of 6 flasks of 300 ml AEB111299 : Pack of 100 tubes of 9 ml AEB611301LAF: Pack of 6 flasks of 1000 ml - Ready to use medium with septum (Store between 2 and 25°C) AEB611294M : Pack of 6 flasks of 90 ml AEB611298M : Pack of 6 flasks of 100 ml AEB112273M : Pack of 6 flasks of 990 ml AEB612273MDE : Pack of 6 flasks of 990 ml (Double sealed) AEB611294MDE : Pack of 6 flasks of 90 ml (Double sealed) - Ready to use medium with 0.1% Tween (Store between 2 and 25°C) AEB611311 : Pack of 6 flasks of 100 ml - Ready to use medium with 1% Tween (Store between 2 and 25°C) AEB611314 : Pack of 6 flasks of 300 ml - Ready to use medium with 3% Tween (Store between 2 and 25°C) AEB611358 : Pack of 6 flasks of 90 ml AEB611309L : Pack of 6 flasks of 900 ml - Ready to use medium with 5% Tween (Store between 2 and 25°C) AEB611310 : Pack of 6 flasks of 90 ml AEB811311GAF: Pack of 8 flasks of 990ml - Ready to use medium with 0.5% Thiosulfate (Store between 2 and 25°C) AEB611316MAF: Pack of 6 septumed flasks of 90 ml Made by : AES CHEMUNEX - Combourg – France 141292£: 03/11/08 - K

Fluid K Rinsing solution (USP) For in vitro use To be stored between 2 and 25°C

PRINCIPLE Fluid K solution is used in the frame work of the sterility tests of pharmaceutical products according to the USP. It allows the humidification and rinse of the filtration membranes used for the analysis of samples containing vaseline (petrolatum). FORMULA In grammes per litre of purified water Peptic digest of animal tissues Beef extract Polysorbate 80

5,0 3,0 10,0

pH at 25°C : 6,9 ± 0,2 at 25°C PROCEDURE Humidify the membranes with 200 µl of K rinse solution. After the sample filtration, rinse the membrane 3 times with 100 ml of solution and continue the analysis according to the USP instructions. BIBLIOGRAPHY 1. USP 26 – Sterility tests (71) – Diluting and rinsing fluids for membrane filtration. PACKAGING Ready to use culture medium – double wrapped : AEB510884MDE : Pack of 12 flasks (with septum) of 400 ml AEB610887MDE : Pack of 6 flasks (with septum) of 200 ml AEB610085MDE : Pack of 6 flasks (with septum) of 650 ml Made by AES CHEMUNEX - Combourg - France 510884£: 22/12/06 - B

MEAT PEPTONE Peptic digest of meat In Vitro use only

PRINCIPLE Peptic digest of meat is obtained by the enzymatic digestion of selected animal tissues. It is characterized by a predominance of high molecular weight polypeptides. Peptic digest of meat is used for the growth of numerous microorganisms, as yeasts and moulds, enterobacteria or staphylococci. 0,1 % solution of peptic digest of meat (Fluid A) is conform to the requirements of the harmonized method of pharmacopoeias as rinsing solution for sterility controls.

MICROBIOLOGICAL CHARACTERISTICS Detection of fermentable sugars slightly positive Reducing sugars absent Indole absent Indole production positive Acetylmethylcarbinol production positive Hydrogen sulfide production positive

PHYSICAL CHARACTERISTICS Appearance : light beige powder Residual humidity : < 5% Solubility in water at 5 % is total. pH : 7,01+/- 0,2 Stable after autoclaving for 15 minutes at 121°C.

CHEMICAL CHARACTERISTICS Sulfuric ash Total nitrogen (Kjeldahl) α-amino nitrogen (Sörensen) α-amino/total nitrogen ratio Not digested proteins Proteoses Nitrites Potassium Chlorides (expressed as NaCl) Iron Sodium Calcium

< 15,0 % 12,2 % 3,5 % 0,28 % absent presence absent 3,5 % 5,0 % 0,004 % 1,7 % 0,05 %

TOTAL AMINO ACIDS (expressed as g per 100 g of product) Aspartic acid 7,9 Threonine 2,8 Serine 2,6 Glutamic acid 9,3 Proline 5,6 Glycine 6,8 Alanine 6,5 Valine 5,1 Tryptophan 0,8 Methionine 1,5 Isoleucine 3,2 Leucine 6,6 Tyrosine 2,0 Phenylalanine 2,5 Lysine 4,8 Histidine 2,9 Arginine 3,5

FORMULA OF THE READY TO USE SOLUTIONS

Meat peptone or fluid A : Peptic digest of meat 1g Water 1000 ml Peptone pepsique de viande et 0,1% tween : Peptic digest of meat 1g Polysorbate 80 1g Water qs 1000 ml of meat peptone solution Peptone pepsique de viande et 1% tween : Peptic digest of meat 1g Polysorbate 80 10 g Eau qs 1000 ml of meat peptone solution

PACKAGING Dehydrated base (Store between 1 and 30°C) AEB170456 : 500 g Ready to use 0,1 % solution (Fluid A) (Store between 2 and 25°C) AEB627456 : Pack of 6 flasks of 100ml AEB627457: Pack of 6 flasks of 200ml Ready to use 0,1 % solution (Fluid A) in flasks with septum (Store between 2 and 25°C) AEB627457M : Pack of 6 flasks of 200ml AEB627453M : Pack of 6 flasks of 300ml AEB627454M : Pack of 6 flasks of 400ml AEB627459M : Pack of 6 flasks of 800ml AEB627460M : Pack of 6 flasks of 990 ml Ready to use 0,1 % solution + Tween 0. 1 % (Store between 2 and 25°C) AEB627487M : Pack of 6 flasks with septum of 200 ml

Specific preparation and packaging also exist. Please contact us: Ready to use 0,1 % solution + Tween 1 % (Store between 2 and 25°C) AEB627488M : Pack of 6 flasks with septum of 300 ml Ready to use 0.1% solution in double sealed packaging (Store between 2 and 25°C) AEB527424MDE: Pack of 12 flasks with septum 400 ml AEB627455MDE: pack of 6 flasks with septum 650 ml AEB527453MDE: Pack of 12 flasks with septum of 250 ml

Made by : AES CHEMUNEX - Combourg - France 170456£: 20/02/09 - O

TRYPTIC SOY BROTH Tryptic Soy broth (TSB) In Vitro use only To be store between 2 to 25°C PRINCIPLE Tryptic soy broth is a general purpose medium used for the cultivation of a wide variety of microorganisms, including fungi, aerobic, aero/anaero facultative and anaerobic bacteria. It is recommended by US and European Pharmacopoeia as a sterility test medium. FORMULA In grammes per litre of purified water Pancreatic digest of casein Papaic digest of soybean meal Sodium chloride Dipotassium phosphate Dextrose

17,00 3,00 5,00 2,50 2,50

Final pH : 7,3 + 0,2 at 25°C PREPARATION Suspend 30,0 grammes of powder in one litre of purified water. Mix thoroughly and heat gently until dissolution is complete. Dispense and sterilize by autoclaving at 121°C for 15 minutes. PROCEDURE Aerobic cultivation This broth will support the growth of aerobic and aero/anaero facultative bacteria, yeasts and molds (Candida albicans). Anaerobic cultivation For the growth of strict anaerobes, such Clostridium, add 0,5 to 1 g/l of Pastagar B. Broth must be used immediately after having been regenerated in a boiling water bath with the caps loosened and cooled with caps tightened. Blood culture Add an anticoagulant in the broth before inoculation. Inoculate aseptically with 5 to 10 ml of blood for 50 ml of broth. Incubate in an aerobic atmosphere enriched with CO2 if presence of Brucella, Streptococcus pneumoniae, is suspected. Sensitivity to antibiotics The tryptic soy broth is recommended for liquid sensitivity test and disk diffusion sensitivity test. Test for specified microorganisms (TSB with neutralizing agents) The addition of egg lecithin (3 g/l), polysorbate 80 (30 g/l), histidine hydrochloride (1 g/l) allows to neutralize the activity of antimicrobial agents used in the pharmaceutical industry.

Test for sterility Tryptic soy broth conforms with the formula specified in US and European Pharmacopoeia for sterility testing of pharmaceutical products, biologics and devices. TSB is also recommended for testing bacterial contaminants in cosmetics. REFERENCES 1. United States Pharmacopeia. Tests, Sterility tests. 1152-1156. 2. European Pharmacopeia . 6th edition. Broth medium A. PACKAGING Dehydrated medium AEB141502 : 500 g Radiosterilized dehydrated medium AEB141502STE : 500g * Ready to use AEB611503 : Pack of 6 flasks of 55 ml* AEB611504 : Pack of 6 flasks of 90 ml AEB611506 : Pack of 6 flasks of 100 ml* AEB611507 : Pack of 6 flasks of 200 ml AEB111510 : 1 Pack of 100 tubes of 9 ml AEB111502 : Pack of 20 tubes of 10 ml AEB111509 : Pack of 100 tubes of 10 ml* AEB111511 : Pack of 100 tubes of 20 ml* AEB111512T : Pack of 100 tubes of 2 ml AEB611511L: Pack of 6 flasks of 900 ml AEB611506GSE : Pack of 6 flasks 100ml with large opening* AEB911505/2 : 2 DilubagS™ of 5 Litres* Ready to use medium in flask with septum AEB611506M : Pack of 6 flasks of 100 ml* AEB111508M : Flask of 500 ml* AEB611506MDE : Pack of 6 flasks of 100 double sealed* Ready to us medium with neutralizing agents AEB611518 : Pack of 6 flasks of 90 ml* Ready to use TSB + Polosorbate 80 AEB611526MDE: Pack of 6 flasks with septum 100 ml double sealed* Made by AES CHEMUNEX - Combourg - France 141502£: 20/11/08 - O * : Product not

stamped.

THIOGLYCOLLATE RESAZURIN thioglycollate regenerated in a Fluid boiling water bath. Incubatemedium 21h+/For in vitro use 3h at 37°C.

PRINCIPLE The fluid thioglycollate medium is used for detecting microorganisms in normally sterile materials. It permits the growth of aerobic and anaerobic microorganisms. It’s prepared according to the formula of the ISO 7937 standard and the harmonized method of Pharmacopoeias. Sodium thioglycollate and L-cystine lower the oxidation-reduction potential of the medium by removing oxygen and preventing the accumulation of peroxides which can be toxic for some organisms. These compounds also neutralize the antibacterial effect of mercurial preservatives, making thioglycollate resazurin broth useful in testing material which contains heavy metals. FORMULA In grams per litre of purified water Pastone Yeast extract Dextrose Sodium chloride L-Cystine Sodium Thioglycollate Resazurin Agar pH final: 7,1 + 0,2 à 25°C

15,00 5,00 5,50 2,50 0,50 0,50 0,001 0,75

PREPARATION Suspend 29.7 grammes of powder in one litre of purified water. Mix thoroughly and warm gently until dissolution is complete. Dispense and sterilize by autoclaving at 121°C for 15 min. PROCEDURE Sterility test: Inoculate the sample prepared according to the recommendation of the harmonized method of pharmacopoeia in appropriate tubes or flasks. Incubate for 14 days at 32,5°C+/-2,5°C. In parallel, carry out the same procedure but this time using tryptic soy broth. Incubate 14 days at 22,5°C+/2,5°C . Validation tests must be carried out in order to validate the essay. Confirmation test for Clostridium perfringens in food industry: When in presence of characteristic colonies on TSC (tryptose sulphite D-cycloserine) plate, subculture 5 colonies in thioglycollate resazurine tube previously

RESULTS: Sterility test: The product complies with the test for sterility if no evidence of microbial growth is found in presence of both culture media (thioglycollate resazurin and tryptic soy broth) and that the controls have passed validated. Confirmation test for Clostridium perfringens in food industry: From the incubated broths: Subculture onto a tryptose sulphite agar base (TSC base). Or transfer 5 drops into a lactose sulphite broth. Carry on confirmation test according to the ISO 7037 standard. LIMITS If more than 20% of the broth shows a pink coloration (due to oxidation), then regenerate the medium by putting the container in boiling water bath for 10 minutes. This treatment must be done only once. Store this medium in the dark. REFERENCES 1. United States Pharmacopeia. Sterility tests. 2. J.O. du 25 Octobre 1978. Essai de stérilité. 82338237. 3. Brewer J.M. 1940. J.A.M.A. 115:598-600. 4. Pittman M. 1946. J. Bacterio. 51:19-32. 5. Pharmacopée Européenne. Essai de Stérilité. 6. NF EN ISO 7937. Méthode horizontale pour le dénombrement de Clostridium perfringens.

PACKAGING

Dehydrated medium (Store between 1 and 30°C) AEB141402 : 500g AEB141412RSTE : 500g* (ionised) Prepared medium (Store between 2 and 25°C) AEB111404: Pack of 100 tubes of 10 ml AEB111402: Pack of 20 tubes of 10ml AEB111409: Pack of 100 tubes of 20 ml AEB611404 : Pack of 6 flasks of 90 ml* AEB611426GSE : Pack of 6 flasks with large opening 100ml Prepared medium in flask with septum (Store between 2 and 25°C) AEB611406M: Pack of 6 flask of 100 ml* AEB611403MAF: Pack of 6 flasks of 50 ml* Ready to use medium double sealed (Store between 2 and 25°C) AEB611406MDE: Pack of 6 flasks with septum of 100 ml AEB611416MDE: Pack of 6 flasks of 100 ml with 1% Polysorbate 80.

Made by : AES CHEMUNEX - Combourg – France 141402£ : 30/10/08 – S * : Product no

stamped.

Testing

Culture media for the pharmaceutical industry

Or less if

Total Viable Aerobic Count Monograph: 2.6.12 European Pharmacopoeia 6th ed. (Mesophilic bacteria and fungi) Harmonized method of Pharmacopoeias Preparation of the sample 10 g (or 10 ml) of the test product in 90 ml of Pharmacopoeia Diluent pH7 (AEB611294 – Pack of 6 flasks - 90ml) or NPD (AEB611274 - Pack of 6 flasks- 90ml) if presence of preservatives or Phosphate buffer pH 7.2 or Tryptic Soy Broth : TSB (AEB611504 - Pack of 6 flasks- 90ml)

Sample Filtration

Filter on the surface of 2 appropriate membrane filters the equivalent of 1 gramme (or 1 millilitre) of product or less if the presumed number of microorganisms is high. Rinse each membrane filter with 3 times 100 ml of Pharmacopoeia Diluent (AEB611313 – Pack of 6 flasks - 300ml) or NPD.

Determination of Total Aerobic Microbial Count (TAMC) : Transfer one membrane filter onto TSA (AEB522860 – Pack of 20 plates ∅ 90 mm) then incubate at (32,5+/2,5)°C for 3 - 5 days. Determination of Total Combined Yeast and Moulds count (TYMC): Transfer the second membrane filter onto Sabouraud Agar (AEB522210 – Pack of 20 plates ∅ 90 mm) then incubate at 22,5+/-2,5°C for 5-7 days.

Count : Select plates with highest number bellow 100 colonies (CFU) on each medium. Total Viable Aerobic Count : Σ (CFUTSA + CFUSAB)

Culture media for the pharmaceutical industry

Total Viable Aerobic Count Monograph: 2.6.12 European Pharmacopoeia 6th ed. (Mesophilic bacteria and fungi) Harmonized method of Pharmacopoeias Plate–count methods Preparation of the plates Pour-plate method Add 1 ml of the prepared sample or its subsequent tenfold dilutions into 2 sterile Petri plates (∅ 90 mm). Pour 15 to 20 ml of liquefied medium (45°C). TAMC: TSA (AEB622857 – Pack 6 of flasks - 200ml) TYMC: Sabouraud (AEB622207 – Pack 6 of flasks - 200ml)

Surface-spread method Spread 0,1 ml of the prepared sample or its subsequent tenfold dilutions onto 2 sterile Petri plates (∅ 90 mm) containing the appropriate medium depending on the targeted germs. TAMC : TSA (AEB522860 – Pack of 20 plates ∅ 90 mm) TYMC : Sabouraud Agar (AEB522210 – Pack of 20 plates ∅ 90 mm)

Incubation Incubate the prepared plates at : TAMC : (TSA) 3-5 days at (32,5+/-2,5)°C TYMC : (Sabouraud) 5-7 days at (22,5+/-2,5°C) Results : Select the plates corresponding to one dilution and showing the highest number of colonies less than 300 (100 for fungi). Take the arithmetic average of the counts and calculate the number of CFU per gramme or millilitre

Culture media for the pharmaceutical industry

Total Viable Aerobic Count Monograph: 2.6.12 European Pharmacopoeia 6th ed. (Mesophilic bacteria & fungi) Harmonized method of Pharmacopoeias Most Probable Number method Preparation of the test Prepare a series of at least 3 subsequent tenfold dilutions of the prepared product (ex : 10-1 ; 10-2 ; 10-3â&#x20AC;Ś). From each level of dilution, 3 aliquots (1 g or 1 ml) are used to inoculate TSB tubes (9 10 ml added if necessary with antimicrobial inactivator or surface active agent). Incubate the tubes 5 days at (32,5+/-2,5)Â°C. For each dilution note the number of tubes showing a microbial growth. Calculate the number of microorganisms using the MPN table (ex: Table 2.6.12-1)

Note : The precision and accuracy of the MPN method is less than that of the membrane filtration method or of plate count methods. This method should be used only if the product can be tested with the two first ones.

TRYPTIC SOY AGAR Tryptic Soy agar (TSA) In vitro use only Store between 2 and 25°C

PRINCIPLE The Tryptic Soy Agar is a multi-purpose medium that contains two peptones to grow a variety of germs either fastidious or non-fastidious. It is used for aerobes as well as anaerobes. The medium can also be used as a base for a blood agar : if so, add 5% of sterile defibrinated horse or sheep blood to agar that has been previously melted and cooled down to 42°C +/-1°C. The Tryptic Soy Agar can also be a base for the preparation of the “chocolate agar”.

PACKAGING Dehydrated medium (Store between 1 & 30°C) AEB152852 : 500 g

Our control of the manufacturing process enables us to provide you with triple wrapped ionised ready to use products whose shelf life, from manufacturing day, extends to 9 months for Petri plates.

Ready poured medium AEB522860 : Pack of 20 plates 90mm∅ AEB522859 : Pack of 120 plates 90mm∅ AEB522879 : Pack of 120 radiosterilised plates 90mm ∅* AEB122861: Pack of 10 plates 55 mm ∅*

FORMULA In grammes per litre of purified water. Pancreatic digest of casein Papaic digest of soya bean Sodium Chloride Agar

15,00 5,00 5,00 15,00

Final pH: 7,3 +/- 0,2 to 25°C PROCEDURE Pour 40 g of powder into 1 litre of purified water. While bringing to the boil stir to achieve complete dissolution. Autoclave for 15 minutes at 121°C. When adding 0,5ml of a 1% tellurite potassium solution, the Trypticase Soy Agar can be used for the growth and the selective isolation of Corynebacterium diphteriae, Candida albicans, Listeria and Streptococcus. BIBLIOGRAPHY 1. Abbot J.D.and Graham J.M. 1961. Mon Bull. Min Hlth. Pub. Hlth. Lab. Serv. 20 : 51-58. 2. Mitchell T.G. 1964. J. Appl. Bact. 27(1) :45-52. 3. Barnes E.M. and Shrimpton D.H. 1958. J.Appl. Bact. 2(2) :313-329. 4. Facklam R.R. and Carey R.B. 1985. Manual of th Clinical Microbiology 4 Ed.-ASM – 154-175 5. European Pharmacopea. Medium B

Ready to use medium AEB122854 : 5 slanted tubes* AEB122859 : 100 slanted tubes* AEB122858 : 100 tubes of 15ml* AEB122869 : 100 tubes of 20ml* AEB622856 : 6 Flasks of 100ml AEB622857 : 6 Flasks of 200ml

Ready to use triple wrapped ionised plates with extended shelf life AEB522879LL : Pack of 120 plates 90mm ∅* T.S.A. + 5% horse blood AEB522871 : Pack of 20 plates 90mm ∅* AEB522861 : Pack of 120 plates 90mm ∅* T.S.A. + 5% sheep blood AEB522870 : Pack of 20 plates 90mm ∅* AEB522869 : Pack of 120 plates 90mm ∅* Made by AES CHEMUNEX – Combourg – France 152852£ : 09/07/07 - M

* : Product not

stamped.

SABOURAUD Dextrose Agar Isolation dextrose agar In Vitro use only

DESCRIPTION Sabouraud agar is used for : * molds isolation from pathological samples without germs or saprophytes molds. * molds culture for their identification. * sterility tests of pharmaceutic or cosmetic products . In this case, incubation is necessary for 15 days. * Candida differentiation, when adding 100mg/l of triphenyl-2,3,5-tetrazolium (T.T.C.) . * special media preparation when adding antibiotics (chloramphericol 0,05 g/l, gentamicin 0,1 g/l). It is recommended by European and American Pharmacopoeia for the microbiology monitoring. Our control of the manufacturing process enables us to provide you ionised ready to use products with triple wrapped whose shelf life, from manufacturing day, extends to 6 months for the contact plates and to 9 months for Petri plates. FORMULA In grammes per litre of purified water Sabouraud Dextrose Peptic digest meat and casein Monohydrated dextrose Agar final pH : 5,6 + 0,2 at 25°C

10,00 40,00 15,00

Sabouraud Dextrose Chloramphenicol Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Chloramphenicol 0.05 Agar 15,00 final pH : 5,6 + 0,2 at 25°C Sabouraud Dextrose Chloramphenicol - Gentamicin Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Chloramphenicol 0.05 Gentamicin 0.10 Agar 15,00 final pH : 5,6 + 0,2 at 25°C Sabouraud Dextrose with neutralizing chemicals Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Lecithin 0,70 Polysorbate 80 5,00 Histidine 1,00 Sodium thiosulfate , 5 H2O 0,50 Agar 15,00 final pH : 5,6 + 0,2 at 25°C

METHOD Pour 65,0 gr (of Sabouraud dextrose agar or Sabouraud dextrose Agar with chloramphenicol) of powder into 1 litre o Purified water. Bring slowly to the boil, under continuous homogenization as to obtain complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C. PROCEDURE Please refer to different Pharmacopoeias laboratory procedure. Generally, incubate for 3 to 5 days at 20-25°C.

RESULTS Separate or combined use of gentamicin and chloramphenicol gives a very large antibacterial spectrum. Consequently, this medium prevents from most germs growth (Gram positive or negative). It also prevents from the growth of other germs resistant to other antibiotics with no influence on yeasts development. TTC use gives the following pigmentations : C. albicans : white (creamy) C. guilliermondii : pink-red C. krusei : white (mat ) C. parapsilosis, pseudotropicalis, pulcherrima and stellatoïdea : pink C. tropicalis : red-violet C. zeylanoïdes : pink-white PACKAGING Dehydrated medium (Store between 1 and 30°C) Sabouraud AEB152202 : Flask of 500 g Sabouraud chloramphenicol AEB152352 : Flask of 500 g Ready to use media (Store between 2 and 25°C) Sabouraud AEB122202 : 20 tubes with slant AEB122208 : Pack of 100 tubes of 15 ml* AEB122209 : Pack of 100 tubes with slant AEB122219: Pack of 100 tubes 20ml AEB622207 : Pack of 6 flasks of 200 ml Sabouraud chloramphenicol AEB122359 : Pack of 100 tubes AEB622356 : Pack of 6 flasks of 100 ml AEB622357 : Pack of 6 flasks of 200 ml AEB122350 : Pack of 100 tubes of 15 ml* AEB122358 : Pack of 100 tubes of 20 ml*

SABOURAUD Dextrose Agar Isolation dextrose agar In Vitro use only

Ready to use media (Store between 2 and 25°C) Sabouraud chloramphenicol gentamicin* AEB122399 : Pack of 100 tubes of 15 ml* AEB122369 : Pack of 100 tubes with slant Pre-poured media (Store betwen 2 and 25°C) Sabouraud AEB522210 : Pack of 20 plates of 90 mm* AEB522209 : Pack of 120 plates of 90 mm* Sabouraud chloramphenicol AEB522360 : Pack of 20 plates of 90 mm AEB522359 : Pack of 120 plates of 90 mm AEB522379 : Pack of 120 plates of 90 mm ionised* AEB122360C : Pack of 10 contact plates * Sabouraud chloramphenicol gentamicin AEB122371 : Pack of 10 plates ( 55 mm)* AEB522370 : Pack of 20 plates (90 mm) AEB522369 : Pack of 120 plates ( 90 mm)* Sabouraud/trypto-casein-soja AEB526750 : Pack of 20 biplates (90 mm) Ready to use triple wrapped ionised plates with extended shelf life AEB522209LL : Pack of 120 Petri plates ∅ 90 mm ionised Sabouraud 4N ionised long life (4 neutralizing chemicals) AEB522239CLL: Pack of 120 contact plates ionised* Made by AES CHEMUNEX - Combourg – France 152202£ : 30/10/08 - O * : Product not

stamped

Culture media for the pharmaceutical industry

Gram negative bacteria resistant to Bile Salts Monograph : 2.6.13. European Pharmacopoeia 6th ed. Harmonized method of Pharmacopoeias Prepare the sample : tenfold dilution of at least 1 gramme of product to analyzed in a broth of Tryptic Soy Broth (Pack of 6 flasks of 90ml – AEB611504)

After homogenization, incubate at 22.5-/+2.5°C for 2 to 5 hours to ensure a good revive of the bacteria.

Presence/Absence Protocol

Place the equivalent of 1 gramme of product revived in an appropriate volume of EE Broth Mossel (Pack of 6 flasks of 100 ml – AEB611376). Incubate at 32.5°C +/-2.5°C For 24 to 48 hours

Subculture on a VRBG Pharma agar (Pack of 20 plates 90 mm AEB523230) Incubate at 32.5°C +/-2.5°C for 18 to 24 hours

The product complies with the test if no growth of colonies is detected.

Enumeration Protocol

From the sample revived inoculate the equivalent of 0.1grs, 0.01grs, 0.001grs or (0.1ml, 0.01ml, 0.001ml) in an adequate volume of EE Broth Mossel (Pack of 100 tubes of 9 ml : AEB111379) Incubate at 32.5°C +/-2.5°C for 24 to 48 hours

Subculture on a VRBG Pharma agar (Pack of 20 plates 90 mm AEB523230) Incubate at 32.5°C +/-2.5°C for 18 to 24 hours

Calculate the most probable number from the results given from VRBG Pharma agar.

EE BROTH MOSSEL Enrichment broth for Enterobacteria according to Mossel In Vitro use only

DESCRIPTION This liquid medium whose formulation fits with Mossel recommendations, is used for the selective enrichment of Enterobacteriaceae for the bacteriological control of pharmaceutical products that may not be sterile, human and animal food. This broth is the result of a modification of Brilliant Green broth, in which lactose was replaced by glucose and whose buffer effect is reinforced to obtain an early growth and avoid microorganisms destruction by acidification. Using glucose instead of lactose makes the medium adequate for all Enterobacteriaceae. The medium also contains brilliant green and bile salts, necessary for the secondary flora inhibition. FORMULA In grammes per litre of purified water Peptone Ox Bile Glucose Disodium phosphate anhydrous Monopotassium phosphate Brilliant green (1)

10,00 20,00 5,00 (1) 6,45 2,00 0,015

Corresponds to 8 g of disodium phosphate 2H2O

Final pH : 7,2 + 0,2 at 25°C PREPARATION Pour 43,5 g of powder into 1 litre of purified water. Stir slowly until complete dissolution. Dispense into tubes or flasks. Heat up to 100°C for 30 minutes or 110°C for 20 minutes. DO NOT OVERHEAT. Once the broth has cooled down, a slightly white precipitate may appear at the bottom of the tubes or flasks. PROCEDURE 1. Perform a revivification by incubating dilutions of the product to be analyzed (1/10th in Tryptic Soy Broth), at 20-25°C for 2 to 8 hours 2. After revivification mix Mossel broth with the sample (10 times the initial volume). Homogenize carefully. 3. Incubate at 30-35°C for 18-48 hours.

RESULTS When the medium becomes cloudy or change colour (yellow / green), it reveals the presumptive presence of Enterobacteriaceae. A final diagnosis should be obtained with isolation on a selective medium such as VRBG. NOTES For an enumeration (between 1 and 100 for 1 ml or gramme of the sample), the “most probable number” method should be used (after a 37°C incubation for 24 hours). LIMITS AND PRECAUTIONS Autoclaving at 110°C for 20 minutes is ideal if the medium can be cooled down quickly. In case of a preparation with double concentration, it is advised to heat up to 100°C for 30 minutes. In any case, the medium should be green at the end of sterilization. BIBLIOGRAPHY 1. Mossel D.A.A., Visser M. and Cornelissen A.M.R. 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of all Enterobacteriaceae. J. Bact., 84:381. 2. Mossel D.A.A., Visser M. and Cornelissen A.M.R. 1963. The examination of foods for Enterobacteriaceae using a test of the type generally adopted for the detection of Salmonella. J. Appl. Bacteriol. 26:444-452. 3. Pharmacopée Européenne. Milieu E. ème 4. Pharmacopée Européenne 6 ed. Harmonisation PACKAGING Dehydrated medium (Store between 1 & 30°C) AEB140372 : 500 g flask Ready to use medium (Store between 2 & 25°C) AEB611376 : Pack of 6 flasks of 100 ml AEB111379 : Pack of 100 Tubes of 9 ml Made by AES CHEMUNEX - Combourg – France 140372£: 25/04/08 - L

V.R.B.G. PHARMA Violet crystal, neutral Red, Bile Glucose (dextrose) Pharma agar In Vitro use only To be stored between 2 & 25°C

PRINCIPLE Violet crystal, neutral red bile glucose Pharma agar (VRBG Pharma) is used for screening and enumeration of Bile tolerant Gran-negative bacteria. Crystal violet and bile salts inhibit the growth of Gram positive bacteria.

PACKAGING Ready to use medium AEB623236 : Pack of 6 flasks of 100 ml

FORMULA En grammes par litre d'eau distillée

Made by : AES CHEMUNEX - Combourg - France

Yeast extract Pancreatic digest of gelatin Sodium chloride Bile salts Dextrose Neutral red Violet crystal Agar

3,00 7,00 5,00 1,50 10,00 0,03 0,002 13,00

Ready poured media AEB523230 : Pack of 20 dishes 90 mm

623236 : 17/01/07 - A

Final pH : 7,4 + 0,2 at 25°C PROCEDURE Liquefy the medium in a boiling water bath then cool to 45°C before dispatching in Petri plates. Inoculate the plates previously dried with the enriched selective broth. Incubate at 32,5°C+/-2,5°C for 18 to 24 hours.

RESULTS In the frame work of the test for absence, the product complies with the test if there is no growth of colonies. In the frame work of the quantitative test, growth of colonies constitutes a positive result. Estimate the number of bacteria using the MPN chart used given in the European Pharmacopoeia.

BIBLIOGRAPHY th European Pharmacopoeia 5 edition.

Culture media for the pharmaceutical industry

Detection of Escherichia coli Monograph : 2.6.13. European Pharmacopoeia 6thed. Harmonized method of Pharmacopoeias

Preparation of the sample

or or or

10 g or 10 ml of sample in 90 ml of Pharmacopoeia diluent pH 7 (AEB611294 – Pack of 6 flasks of 90ml) of DNP (AEB611274 - Pack of 6 flasks of 90ml) if presence of preservatives of Phosphate Buffer pH 7,2 of TSB broth (AEB611504 – Pack of 6 flasks of 90ml)

Enrichment Inoculate 10 ml (= 1 g or 1 ml of product) in 100 ml of broth TSB ( (AEB611506 – Pack of 6 flasks of 100ml) Incubate at 32,5 + 2,5°C for 18 to 24H

Inoculate 1 ml in 100 ml of Mac Conkey broth (AEB610606 – Pack of 6 flasks of 100ml) Incubate at 43 + 1°C for 24 to 48H Isolation Carry out subcultures on Mac Conkey agar (AEB521610 – Pack of 6 flasks of 100ml) Incubate at 32,5 + 2,5°C for 18 to 72H

Reading and confirmation Probable presence of Escherichia coli if growth of red non-mucoid colonies of Gram – rods. Carry out confirmation tests

MAC CONKEY BROTH Pharmacopoeia Mac Conkey Broth In Vitro use only To be stored between 2 and 25°C

PRINCIPLE Pharmacopoeia Mac Conkey broth is used to screen d'Escherichia coli in none obligatory sterile products. It is also used when analysing milk or water samples using a similar protocol to the one use in pharmacopoeia. This medium contains Lactose that when it is metabolised results in an acidification shown by a change of colour of the pH indicator from purpil to yellow and a production of gas captured in the Durham enclosure. These two criteria revel the presence of Coliforms. FORMULA In grammes per litre of purified water Peptone Monohydrated Lactose Dehydrated Oxgall Brom Cresol Purple

20,00 10,00 5,00 0,01

Final pH : 7,3 + 0,2 at 25°C

BIBLIOGRAPHY 1.Mac Conkey A. 1908. Bile salt media and their advantage in some bacteriological examination. J. Hyg., 8:322-334. 2. Pharmacopée Européenne. Contamination microbienne, milieux de culture. Milieu G. PACKAGING Milieu déshydraté (Mac Conkey Broth) BBL20100 : 500 g Ready to use medium Mac Conkey single concentration AEB610606 : Pack of 6 flasks of 100 ml AEB110609 : Pack of 100 tubes of 10 ml

Made by AES CHEMUNEX - Combourg - France

20100£: 06/03/09 - H

METHOD Suspend 35,0 g of powder in 1 litre of purified water. Homogenise until completely dissolved. Dispense in tubes with Durham closures. Autoclave 15 minutes at 121°C. PROCEDURE PHARMACEUTICAL PRODUCTS : Inoculate 100 ml of Mac Conkey Pharmacopoeia broth with 1 ml the tested sample previously enriched in TSB. Incubate at 43 + 1°C for 24 to 48 hours and then subculture on Mac Conkey Crystal violet agar. OTHER PROTUCTS : According to the level of contamination two protocols are possible: - 10 or 100 ml of the sample or its decimal dilutions are added to equal amount of double concentrated Mac Conkey broth. - 1 ml of the sample or its decimal dilutions are added to 9 ml of single concentrated Mac Conkey broth. Incubate at 37°C for 48 hours.

MAC CONKEY AGAR Mac Conkey with violet cristal (n°3) In Vitro use only

PRINCIPLE Mac Conkey agar (Mac Conkey N°3) is a selective and differential plating medium mainly used for the detection and isolation of gram-negative organism from dairy, foodstuff and waters samples. In clinical specimen this medium is adapted for the screening of pathogen enterobacteria especially in babies stools. pharmaceutical and cosmetic industries this medium is used to detect the presence or the absence of Escherichia coli. Bile salts and Crystal Violet inhibits Gram positive germs growth. The pH indicator, neutral red, helps to differentiate lactose-fermenting from lactose none fermenting gram negative enteric bacilli. FORMULA In grammes of purified water Peptone Proteose Peptone Monohydrated Lactose Bile salts Sodium chloride Neutral red Crystal violet Agar

17,00 3,00 10,00 1,50 5,00 0,03 0,001 13,50

Final pH : 7,1 + 0,2 at 25°C METHOD Pour 50,0 g of powder into 1 litre purified water. Bring slowly to the boil, homogenise until complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C. Liquefy the medium then cool to 45-50°C and dispense into sterile Petri plates. Inoculate the surface in order to obtain isolated colonies. PROCEDURE Application n°1: When analysing, water, dairy, foodstuff, dispense 1 ml of the sample or its decimal dilutions into a sterile Petri plate then pour about 15 ml of medium (4557°C), homogenise and let the medium set. Incubate 18 to 24 hours at 37°C Application n°2: When screening for pathogen Enterobacteria in babies stools or urine swab the specimen (or the enriched specimen) onto the surface of a Mac Conkey plate. Repeat the procedure with a second selective medium such as D.C.L.S for Salmonella and Shigella. Incubate 18 to 24 hours at 37°C.

Application n°3: When screening for Escherichia coli in pharmaceutical and cosmetic products subculture the enriched sample Mac Conkey broth for pharmaceutical preparations and LT100 for cosmetic products) onto the surface of a Mac Conkey plate. Incubate at 32,5+/-2,5°C for 18 to 72 hours. RESULTS Lactose – germs grow as colourless colonies as oppose to lactose + germs that grow as pink to brickred colonies with or with out a precipitation zone. BIBLIOGRAPHY 1. Mac Conkey A. 1905. Lactose-fermenting bacteria in faeces. J. Hyg. 8:333-379. 2. Mac Conkey A. 1908. Bile salt media and their advantage in some bacteriological examination. J. Hyg., 8:322-334. 3. Pharmacopée Européenne. Milieu H. PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB151602 : Flacon de 500 g* Ready to use medium(Store between 2 and 25°C) AEB621606 : Pack of 6 flasks of 100 ml* AEB621607 : Pack of 6 flasks of 200 ml* Ready poured medium (Store between 2 and 25°C) AEB521610 : Pack of 20 plates 90 mm ∅ Made by : AES CHEMUNEX - Combourg - France 151602: 06/03/09-I * : Products not

stamped.

Culture Culturemedia mediafor forthe thepharmaceutical pharmaceuticalindustry industry

Detection of Pseudomonas aeruginosa : Monograph: 2.6.13 European Pharmacopoeia 6th ed. Harmonized method of Pharmacopoeias

Preparation of the sample 10 g (or 10 ml) of the test product in 90 ml of : Pharmacopoeia Diluent pH7 (AEB611294 – Pack of 6 flasks – 90 ml) or NDP (AEB611274 – Pack of 6 flasks – 90 ml if presence of preservatives) or Phosphate buffer pH 7.2 or Tryptic Soy Broth : TSB (AEB611504 - Pack of 6 flasks- 90ml)

Inoculate 10 ml (= 1 g or 1 ml of product) in 100 ml of TSB (AEB611506 – Pack 6 flasks – 100 ml) Incubate at 32.5 + 2.5°C for 18 to 24H

Subculture on Cetrimide (AEB520260 – Pack 20 plates ∅90mm) Incubate at 32.5 + 2.5°C for 18 to 72H

If no growth of microorganisms is detected, the product passes the test. If growth: Carry out confirmation tests and identification.

CETRIMIDE PHARMACOPOEIA AGAR Selective medium for the isolation of Pseudomonas aeruginosa In vitro use only

PRINCIPLE Cetrimide is a selective medium used for the isolation and the numeration of Pseudomonas aeruginosa in pharmaceutical compounds, water, foodstuffs and biological specimen. Its formula is similar to Pseudomonas Agar P (King A), to which is added Cetrimide a potent broad spectrum antimicrobial that is not active against Pseudomonas. FORMULA In grammes per litre of purified water Peptone 20,00 Cetrimide 0,30 Magnesium Chloride 1,40 Potassium Sulfate (K2SO4) 10,00 Agar 15,00 The ready poured and ready prepared media already contain 10% glycerol. Final pH : 7,2 + 0,2 at 25°C METHOD Suspend 46,7 g of the powder in 1 litre of distilled water. Add 10 ml of glycerol. Heat with frequent agitation up to boiling point to completely dissolve the powder. Dispense and sterilize by autoclaving at 121°C for 15 minutes. PROCEDURE Liquefy the medium and cool to 45-50°C then pour in sterile plates. Let the medium set and dry the plates in the incubator with the lid slightly open. General purpose procedure: Spread 0.1 ml of the sample or its decimal dilution on the surface of a prepared plate. Incubate at 37°C for 48 hours. Harmonized method of pharmacopoeias: Incoculate a plate of cetrimide with the enriched sample. Turn the plate upside-down then incubate it at 32.5+/-2.5°C for 18 to 72 hours. RESULTS General purpose procedure: Colonies that turn out of a greenish colour, flat, rough with a metallic shine, pigmented or mucus colonies of a greyish colour with or without pigment are considered as suspect. Suspected isolated colonies undergo three confirmation tests which are:

Oxydase cytochrom test. Production of Pyocyanin pigment on Pseudomonas Agar P (King A) • Growth at 42°C in TSB broth . A Gram negative bacilli that grows on Cetrimide agar must be considered as being a Pseudomonas aeroginosa if it turns out to have an Oxydase cytochrom (oxydase +) and to be able to produce pyocyanin and to grow at 42°C in TSB. These three conditions must be observed simultaneously. Harmonized method of pharmacopoeias: The product passes the test if no growth of microorganisms is detected. If growth is detected the confirmation and identification test must be carried out (see general purpose procedure of this paragraph for example). • •

LIMITATIONS AND PRECAUTIONS Cetrimide agar can also be used when controlling hygiene in hospitals or in industries: surfaces and instruments. BIBLIOGRAPHY 1. Pharmacopée Européenne. Milieu N. 2. Ministère de l'Agriculture. Commission XXX. Cosmétologie. Recherche de Pseudomonas aeruginosa dans les produits cosmétologiques. 3. King B.S., Ward M.K.W. and Roney D.E. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44:301. PACKAGING Dehydrated medium (Storage between 1 and 30°C) AEB150252 : 500 g Flask Ready prepared medium (Storage between 2 and 25°C)

AEB620256 : Pack of 6 flasks of 100 ml AEB620257 : Pack of 6 flasks of 200 ml Ready poured (Storage between 2 and 25°C)

AEB520260 : Pack of 20 plates (90 mm) AEB120260C : Pack of 10 contact plates * Made by : AES CHEMUNEX – Combourg – France 150252£: 05/03/09 - L * : Product not

stamped.

Culture media for the pharmaceutical industry

Detection of Staphylococcus aureus Monograph: 2.6.13 European Pharmacopoeia 6th ed. Harmonized method of Pharmacopoeias

Preparation of sample 10 g (or 10 ml) of the test product in 90 ml of : Pharmacopoeia Diluent pH7 (AEB611294 – Pack of 6 flasks - 90ml) or NDP (AEB611274 – Pack of 6 flasks – 90 ml if presence of preservatives) or Phosphate buffer pH 7.2 or Tryptic Soy Broth : TSB (AEB611504 - Pack of 6 flasks- 90ml)

Inoculate 10 ml (= 1 g or 1 ml of product) in 100 ml of TSB (AEB611506 – Pack 6 flasks - 100ml) Incubate at (32.5 + 2.5)°C for 18 to 24H

Subculture onto a Mannitol Salt Chapman Agar (AEB520510 – Pack of 20 plates – ∅ 90 mm) Incubate at (32.5 + 2.5)°C for 18 to 72H

Probable presence of Staphylococcus aureus if growth of yellow / white colonies surrounded by a yellow zone. Carry out confirmation test The product passes the test if colonies of the types described do not appear or if the confirmatory biochemical tests are negative

CHAPMAN Mannitol salt Agar In Vitro use only

PRINCIPLE Chapman Agar is used for the selective isolation and identification of Staphylococci in all types of samples. This medium is compliant with the formula described by Chapman, it contains a high level of Sodium Chloride (75 g/l) which inhibits the growth of most interfering flora (except a few Bacillus, Corynebacterium & Streptococcus faecalis). Acid productions resulting from the mannitol fermentation cause a colour change of the pH indicator, phenol red, from red to yellow. Pathogen Staphylococci, coagulase positive, grow as larges colonies surrounded by a yellow halo. Staphylococci coagulase negative colonies are small and do not cause a change of colour. FORMULA In grammes per litre of purified water Tryptone Peptic digest of animal tissue Meat extract Mannitol Sodium chloride Phenol red Agar Final pH : 7,4 + 0,2 at 25°C

5,00 5,00 1,00 10,00 75,00 0,025 15,00

METHOD Suspend 111 grammes of powder in one litre of purified water. Bring to the boil under constant homogenisation until completely dissolved. Dispatch in tubes or flasks. Autoclave 15 minutes at 121°C. PROCEDURE Liquefy the medium in boiling water then cool it to 45 50°C (according to standard specifications) in a second water bath. Pour the medium into sterile Petri plates, let the plates set on an horizontal surface. Medical bacteriology: Inoculate the surface of the plate with the specimen. Turn the plates upside-down before incubating them at (36+/2)°C. Cultures are examined generally after 24 to 48 hours incubation. Harmonized method of pharmacopoeias: Incoculate a plate of Chapman with the enriched sample. Turn the plate upside-down then incubate it at 32.5+/2.5°C for 18 to 72 hours. Water analyses: (According to a French standard) Filter the appropriate volume of sample onto a membrane with 0,45µm pores. Transfer the membrane onto the Chapman plate. Take care not to capture air between the membrane and the agar. Turn the plate upside-down before incubating at 36+/-2°C for 44+/-4 hours.

RESULTS Pathogen Staphylococci grow as luxuriant colonies that produce their own pigment. These colonies are surrounded by a yellow halo due to mannitol’s fermentation. Non pathogen Staphylococci grow mainly as small red colonies. REMARKS The temperature of incubation of the agar is chosen according to procedure and current regulations. Staphylococcus epidermidis strains are able to ferment mannitol. Under those circumstances it is essential to carry out further investigation by look for example for the production of free coagulase and/or the A protein, clumping factor or even Dnase test. PRECAUTIONS It is important not to dry the medium in an incubator since this might increase the sodium chloride concentration making the medium to selective. BIBLIOGRAPHY 1. Chapman G.H. 1945. The significance of sodium chloride in studies of staphylococci. J. Bact. 50:201-203. 2. Chapman G.H. 1948. An improved Stone medium for the isolation and testing of food poisonning staphylococci. Food Research, 13:100-105. 3. The United States Pharmacopeia. 4. European Pharmacopoeia 6th edition. 5. Norme XF T90-412 : juin 2006 - Qualité de l’eau : Recherche et dénombrement des staphylocoques pathogènes- Méthode par filtration sur membrane. PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB150502 : 500 grammes Ready to use medium controlled according to the harmonized method of pharmacopoeias (Store between 2 and 25°C) AEB620507 : Pack of 6 flasks of 200 ml AEB520510 : Pack of 20 plates 90 mm AEB520509 : Pack of 120 plates 90 mm* AEB120510C : Pack of 10 contact plates* AEB120504: Pack of 5 tubes of 20 ml Ready to use medium used in water analysis (Store between 2 and 25°C) AEB120511 : Pack of 10 plates 55 mm* AEB520511 : Pack of 120 plates 55 mm* Made by AES CHEMUNEX - Combourg – France 150502: 06/02/09 – J * : Product none

stamped

Culture media for the pharmaceutical industry

Detection of Salmonella spp : Monograph: 2.6.13 European Pharmacopoeia 6th ed. Harmonized method of Pharmacopoeias

10 g or 10 ml of the product in 90 ml of TSB (AEB611504 – pack of 6 flasks - 90ml) Incubate at 32.5 + 2.5°C for 18 to 24H

Inoculate 0.1 ml in 10 ml Rappaport Vassilidis Pharma (AEB110985 – 5 tubes - 10ml) Incubate at 32.5± ±2.5 for 18 to 24H

Subculture onto XLD Agar plates (AEB523410 – Pack 20 plates ∅ 90mm) Incubate at 32.5 + 2.5°C for 18 to 48H

Confirm suspect colonies: Red well developed colonies with or without a black centre.

The product passes the test if colonies of the types described do not appear or if the confirmatory biochemical serological tests are negative

RAPPAPORT-VASSILIADIS PHARMA Rappaport-Vassiliadis enrichment broth In Vitro use only To be stored between 2 and 25째C

PRINCIPLE Rappaport-Vassiliadis PHARMA broth is used as a selective enrichment broth when screening for Salmonella (except S. typhi and paratyphi) in pharmaceutical products. The selectivity of this medium towards Salmonella lies on: * The ability of Salmonella to survive to high osmotic pressures (high concentration of MgCl2), * The ability of salmonella to grow when pH levels are acide (pH = 5,2), * Resistance of Salmonella towards Malachite Green Oxalate (but inhibition of S. typhi and paratyphi and Shigella), * Salmonella low needs of nutrients. FORMULA In grammes for 1 L of purified water Soy peptone Sodium chloride Dipotassic phosphate Monopotassic phosphate Magnesium chloride 6H2O Malachite Green Oxalate

4,50 8,00 0,40 0,60 29,00 0,036

Final pH : 5,2 + 0,2 at 25째C PROCEDURE Inoculate the tubes with 0,1 ml of enrichment broth. Homogenize well and incubate at 32,5+/-2,5째C for 18 to 24 hours. RESULTS After incubation subculture onto XLD plates and incubate 18 to 24 hours at 32,5+/-2,5째 . BIBLIOGRAPHY th European Pharmacopoeia 5 edition. PACKAGING Ready to use medium AEB110985 : 5 tubes of 10 ml AEB110879N : Pack of 100 tubes of 10 ml Made by AES CHEMUNEX - Combourg - France 140989:17/01/07 A

X.L.D Xylose Lysine Desoxycholate Agar In Vitro use only

PRINCIPLE X.L.D. (Xylose Lysine Desoxycholate), as described by Taylor, was developed principally for isolating Shigella and Providencia from stools. It has been shown to be more effective than other enteric differential media. The principal assets of this medium are : • Acid production : due to Lactose and/or saccharose and/or xylose fermentation (Medium colour change from red to yellow) • Alkaline reversion : due to Lysine decarboxylation into cadaverin, (LDC+ colonies turn out red). • Hydrogen sulfide (H2S): Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions. This medium inhibits the growth of gram+ microorganisms and most unwanted coliforms. FORMULA In grammes per litre of purified water Yeast Extract L-Lysine Xylose Saccharose Lactose (monohydrated) Sodium desoxycholate Sodium Chloride Sodium thiosulfate Ferric Ammonium Citrate Phenol Red Agar Final pH : 7,4 + 0,2 at 25°C

3,00 5,00 3,50 7,50 7,50 2,50 5,00 6,80 0,80 0,08 13,50

METHOD Suspend 55,0 grammes of powder in 1 litre of purified water. Heat slowly under constant agitation up to 90°C, until the agar is completely dissolved). DO NOT AUTOCLAVE. It is important not to boil the medium, as soon as the medium is dissolved to 50°C then pour into sterile Petri plates. The medium must be translucide and of orangey-red colour. An excess heating or prolongated periode at 50°C can cause an unwanted precipitation making the reading difficult. A filtration, of the liquefied medium allows to regain the classic appearance, nevertheless the efficiency of the medium is lowered. Home made medium have to be stored at 4°C in the dark and up to 15 days. Manufactured medium can de stored at room temperature for several months.

PROCEDURE Inoculate directly the plates with the sample and/or the selective enriched sample Harmonized method of pharmacopoeias: Incoculate a plate of XLD with the enriched sample. Turn the plate upside-down then incubate it at 32.5+/2.5°C for 18 to 48 hours. RESULTS Enterobacteria ferment very quickly the xylose (except Edwardsellia, Proteus morganii, Proteus rettgeri, Providencia, and Salmonella paratyphi A that are xylose -) This allows to defferentiate from Shigella (Red colonies). As xylose is exhausted Salmonella then decarboxylate lysine causing reversion to alkaline conditions. Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions. Alkaline conditions reversion by other lysine-positive organisms is prevented by excess acid production from fermentation of lactose and saccharose. Moreoer these acid conditions also inhibits the H2S production. Yellow opaque colonies : Citrobacter, Enterobacter, Escherichia coli, Klebsiella, Proteus et Serratia. Red colonies : Providencia, Salmonella H2S - and Shigella. Red colonies with a black center: Arizona, Edwardsiella et Salmonella LIMITS AND PRECAUTIONS An excess incubation ends by diluting the produced acids and therefore a colour variation of the pH indicator occurs making the reading difficult. Proteus mirabilis have similar colonies to Salmonella since they ferment saccharose very slowly. Ready to use XLD Agar can contain white particles that cause no prejudice to its performances and

therefore not affect the reading. BIBLIOGRAPHY 1. Taylor W.I. 1965. Isolation of Shigellae. I. Xylose Lysine Agars; New media for the isolation of enteric pathogens. Am. J. Clin. Path. 44:471-475. 2. Taylor W.I. and Harris B. 1965. Isolation of Shigellae. II. Comparison of plating media and enrichment broths. Am. J. Clin. Path. 44(4):476-479. 3. Taylor W.I. and Harris B. 1967. Isolation of Shigellae. III. Comparison of new and traditional media with stool specimens. Am. J. Clin. Path. 48:350-355 4. Pharmacopée Européenne (Milieu K).

X.L.D Xylose Lysine Desoxycholate Agar In Vitro use only

PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB153402 : 500 g Ready to use medium To be stored between 2 and 25°C AEB623406 : Pack of 6 flasks of 100 ml Ready poured-plate medium To be stored between 2 and 25°C AEB523410 : Pack of 20 dishes 90 mm ∅ AEB523409 : Pack of 120 dishes 90 mm ∅ Made by : AES CHEMUNEX - Combourg - France

153402£: 02/04/09 - N

Culture media for the pharmaceutical industry

Detection of Clostridies Monograph : 2.6.13. European Pharmacopoeia 6th ed. Harmonized method of Pharmacopoeias Preparation of the sample

or or or

10 g or 10 ml of sample in 90 ml of Pharmacopoeia Diluent pH 7 (AEB611294 – Pack 6 flasks of 90ml) of DNP (AEB611274 - Pack 6 flasks of 90ml) if presence of preservatives of Phosphate Buffer pH 7,2 of TSB broth (AEB611504 – Pack 6 flasks of 90ml)

Pre-treatment and enrichment Prepare 2 aliquots of 10 ml of the sample. Treat one at 80°C for 10 min then inoculate each sample in 100 ml of RCM broth (AEB152132 – Dehydrated medium Flask of 500g) Incubate at 32,5 + 2,5°C for 48H in anaerobic atmosphere

Subculture on Columbia Agar (Medium Q : AEB620657 – Pack 6 flasks of 200ml) + 20 mg/L of Gentamicine (AEB184004 ) Incubate at 32,5 + 2,5°C for 48H in anaerobic atmosphere

Presence of Clostridies if growth of rods with or without endospore Gram + and Catalase -

R.C.M. Reinforced Clostridium Medium (broth R.C.M.) In Vitro use only To be stored between 2 and 25°C

PRINCIPLE Semi-solid R.C.M. (Reinforced Clostridium Medium), according to Hirsch et Grinstead formula, is used for cultivating and enumerating Clostridia and other anaerobes species of bacteria from foods and clinical specimen. FORMULA In grammes per litre purified water Yeast extract Beef extract Peptone Monohydrated Dextrose Sodium Chloride Sodium Acetate Cysteine Hydrocloride Starch Agar

3,00 10,00 10,00 5,00 5,00 3,00 0,50 1,00 0,50

Fianl pH : 6,8 + 0,2 at 25°C

BIBLIOGRAPHY 1. Hirsch A. and Grinstead E. 1954. Methods for the growth and enumeration of anaerobic sporeformers from cheese, with observations on the effects of nisin. J. Dairy Res. 21:101-110. 2. Pharmacopée Européenne – Milieu P. PACKAGING Dehydrated medium AEB152132 : 500 g Ready to use medium AEB112137 : Pack of 100 tubes of 10 ml AEB612136 : Pack of 6 flasks of 100 ml Made by : AES CHEMUNEX - Combourg - France 152132£: 19/01/07 - D

METHOD Suspend 38.00 g of the powder in 1 litre of purified water. Heat with frequent agitation to boiling until the powder is completely dissolved. Dispense 10 ml in 16x160 mm tubes and sterilize by autoclaving at 121°C for 15 minutes. PROCEDURE When the medium has been prepared in advance, regenerate the tubes 20 minutes at 100°C, then cool to room temperature. Heat the sample to destroy any vegetative forms and activate spores germination. Inoculate each tube with 1 ml of the treated product or its decimal dilutions. Add on top 2 ml of sterile liquid paraffin. Incubate at 37°C. RESULTS Read after 48 hours then daily up to 5 days. Positive tubes (growth + gas production) undergo the numeration by the M.PN. technique ( Most Probable Number).

COLUMBIA 3 Columbia 3 Agar base In vitro use only

PRINCIPLE

RESULTS

Columbia 3 agar is a rich peptone medium used for the growth of fastidious microorganisms with or without the addition of blood, for example Streptococcus or Pneumococcus. Columbia 3 agar has a similar formula to the previous one, but a more accurate choice of peptone helps to improve the haemolysis reading.

Fresh blood agar is recommended for the growth of Streptococcus, Staphylococcus, Listeria and Erysipelothrix. Chocolate agar is used to grow Haemophilus and Neisseria.

FORMULA In grammes per litre of purified water Special mix of peptone Starch Sodium Chloride Agar

23,00 1,00 5,00 12,00

Final pH : 7,3 + 0,2 at 25°C

METHOD Suspend 41,0 grammes of powder in 1 litre of purified water. Heat with frequent agitation up to boiling point to completely dissolve the agar. Dispense and sterilize by autoclaving at 121°C for 15 minutes.

BIBLIOGRAPHY 1. Elek S.D. 1949. The plate virulence test for diphteria. J. Clin. Pathol. 3:250-258. 2. Hermann G.L., Moore M.S. and Parsons E.J. 1958. A substitute for serum in the diphteria in vitro toxigenicity test. Amer. J. Clin. Pathol. 29:181-182. 3. Ellner P.D., Stoessel C.I., Drakeford E. and Vasi F. 1966. A new culture medium for medical bacteriology. Amer. J. Clin. Pathol. 45:502-504. 4. Goldberg R.L. and Washington J.A. 1976. Comparison of isolation of Haemophilus vaginalis (Corynebacterium vaginale) from Peptone-Starch-Dextrose Agar and Columbia Colistin-Nalidixic Acid Agar. J. Clin. Microbiol. 4:245-247.

PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB151002N : 500 g Flask *

PROCEDURE

Ready to use medium base To be stored between 2 and 25°C AEB620657 : Pack of 6 flasks of 200 ml

Columbia 3 agar can be used to grow Enterobacteriaceae Brucella abortus, Yersinia pestis and Clostridium perfringens, without having to undergo an enrichment step.

Ready poured medium with Sheep’s blood To be stored between 2 and 8°C AEB520680 : Pack of 20 dishes 90 mm ∅

By adding 5% of serum and antitoxin, Columbia 3 agar can be used for the identification of diphtheria bacilli when using an immuno-precipitation method such as Elek test. The reading of the precipitation lines take place after a 48 hours incubation. In order to prepare fresh blood agar, add aseptically 5% of defibrinated blood (horse or sheep) and eventually CNA supplement to liquefied Columbia 3 base that was cooled to 42°C + 1°C. Mix well and dispense into sterile Petri plates. The addition of Colistin sulfate and Nalidixic Acid (CAN) contributes to inhibit the growth of gram negative bacilli and a majority of Bacillus. To prepare chocolate agar or « Cooked blood agar », add 10% of defibrinated sterile horse blood to liquefied Columbia 3 agar at 42° C + 1°C. Under frequent agitation raise the temperature to 80°C and cook until the medium takes a chocolate colour, then dispense in sterile plates.

Dehydrated medium + CNA To be stored between 1 and 30°C AEB150662 : 500g Flask* Ready to use medium base with CNA To be stored between 2 and 25°C AEB620666 : Pack of 6 flasks of 100 ml Ready poured medium with Sheep’s blood and CNA To be stored between 2 and 8°C AEB520690 : Pack of 20 dishes 90 mm ∅ AEB125970 : 10 half dishes (Drigalsky/Columb. CNA SB) Made by AES CHEMUNEX - Combourg – France Réactif enregistré à l'Agence du Médicament 151002N£: 28/03/08 -G * : Product not

stamped.

Culture media for the pharmaceutical industry

DETECTION OF Candida albicans : MONOGRAPH : 2.6.13. EUROPEAN PHARMACOPOEIA 6th ed. Harmonized method of Pharmacopoeias

Sample preparation

or or or

10 g or 10 ml of sample in 90 ml of Pharmacopoeia Diluent pH 7 (AEB611294 – Pack of 6 flasks of 90ml) of NPD(AEB611274- Pack of 6 flasks of 90ml) if presence of preservatives of Phosphate Buffer pH 7,2 of TBS broth (AEB611504 – Pack of 6 flasks of 90ml)

Enrichment

Inoculate 10 ml in 100 ml of Dextrose Sabouraud Broth (AEB611106 – 6 flasks of 100ml) Incubate at 32,5 + 2,5°C for 3 to 5 days

Isolation

Subculture on Sabouraud Agar (AEB522210 – Pack of 20 plates ∅90mm) Incubate at 32,5 + 2,5°C for 24 to 48H

Confirmation

Confirm the suspected colonies: white colonies

SABOURAUD BROTH Sabouraud Broth In Vitro use only

PRINCIPLE Sabouraud broth can be used for: • Blood culture when a fungi septicaemia is incriminated. In that case and before sterilizing add 5g/L of Sodium Citrate and 0.5 g/l of chloramphenicol. • For detection of Candida albicans in pharmaceutical products. • Subculture of yeast and moulds.

PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB141102 : 500 g Ready to use medium (Store between 2 and 25°C) AEB111102 : 20 tubes of 10 ml AEB611106 : Pack of 6 Flasks of 100 ml*

FORMULA In grammes per litre of purified water

Ready to use Flasks with septum (Store between 2 and 25°C) AEB611106M : Pack 6 Flasks of 100 ml*

Tryptone Peptic digest meat Dextrose

Medium with chloramphenicol (Store between 2 and 25°C) AEB611110: Pack of 6 flasks of 100 ml*

5,00 5,00 20,00

Final pH : 5,6 + 0,2 at 25°C PREPARATION Suspend 30.0 g of powder in 1 litre of purified water. Heat to boiling point until completely dissolved. Dispense in tubes or flasks. Autoclave 15 minutes at 121°C.

Made by : AES CHEMUNEX- Combourg - France

PROCEDURE General use: For the growth of yeast and moulds, incubate the Sabouraud broth 3 to 14 days at 25°C. The choice of the temperature of incubation is of the responsibility of the user and must be compliant with the application and the current standards.

* : Produit non marqué CE

141102£: 30/10/08 - J

In the frame work of the detection of Candida albicans in pharmaceutical products: Inoculate the broth with the prepared sample then incubate for 3 to 5 days at 30-35°C before carrying out an isolation on Sabouraud dextrose agar. LIMITS AND PRECAUTIONS Due to a large concentration of dextrose it is recommended not to overheat the medium when preparing in order to prevent an unwanted tanning. It is common to test in parallel thioglycolate medium when screening for contaminating germs. BIBLIOGRAPHY 1. European Pharmacopoeia 2. The United States Pharmacooeia

SABOURAUD Dextrose Agar Isolation dextrose agar In Vitro use only

10,00 40,00 15,00

Bring slowly to the boil, under continuous homogenization as to obtain complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C. PROCEDURE Please refer to different Pharmacopoeias laboratory procedure. Generally, incubate for 3 to 5 days at 20-25°C.

Sabouraud Dextrose Chloramphenicol Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Chloramphenicol 0.05 Agar 15,00 final pH : 5,6 + 0,2 at 25°C

PACKAGING Dehydrated medium (Store between 1 and 30°C) Sabouraud AEB152202 : Flask of 500 g Sabouraud chloramphenicol AEB152352 : Flask of 500 g

Sabouraud Dextrose Chloramphenicol - Gentamicin Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Chloramphenicol 0.05 Gentamicin 0.10 Agar 15,00 final pH : 5,6 + 0,2 at 25°C

Ready to use media (Store between 2 and 25°C) Sabouraud AEB122202 : 20 tubes with slant AEB122208 : Pack of 100 tubes of 15 ml* AEB122209 : Pack of 100 tubes with slant AEB122219: Pack of 100 tubes 20ml AEB622207 : Pack of 6 flasks of 200 ml Sabouraud chloramphenicol AEB122359 : Pack of 100 tubes AEB622356 : Pack of 6 flasks of 100 ml AEB622357 : Pack of 6 flasks of 200 ml AEB122350 : Pack of 100 tubes of 15 ml* AEB122358 : Pack of 100 tubes of 20 ml*

Sabouraud Dextrose with neutralizing chemicals Peptic digest meat and casein 10,00 Monohydrated dextrose 40,00 Lecithin 0,70 Polysorbate 80 5,00 Histidine 1,00 Sodium thiosulfate , 5 H2O 0,50 Agar 15,00 final pH : 5,6 + 0,2 at 25°C PREPARATION Pour 65,0 gr (of Sabouraud dextrose agar or Sabouraud dextrose Agar with chloramphenicol) of powder into 1 litre o Purified water.

Ready to use media (Store between 2 and 25°C) Sabouraud chloramphenicol gentamicin* AEB122399 : Pack of 100 tubes of 15 ml* AEB122369 : Pack of 100 tubes with slant

SABOURAUD Dextrose Agar Isolation dextrose agar In Vitro use only

Pre-poured media (Store betwen 2 and 25°C) Sabouraud AEB522210 : Pack of 20 plates of 90 mm* AEB522209 : Pack of 120 plates of 90 mm* Sabouraud chloramphenicol AEB522360 : Pack of 20 plates of 90 mm AEB522359 : Pack of 120 plates of 90 mm AEB522379 : Pack of 120 plates of 90 mm ionised* AEB122360C : Pack of 10 contact plates * Sabouraud chloramphenicol gentamicin AEB122371 : Pack of 10 plates ( 55 mm)* AEB522370 : Pack of 20 plates (90 mm) AEB522369 : Pack of 120 plates ( 90 mm)* Sabouraud/trypto-casein-soja AEB526750 : Pack of 20 biplates (90 mm) Ready to use triple wrapped ionised plates with extended shelf life AEB522209LL : Pack of 120 Petri plates ∅ 90 mm ionised Sabouraud 4N ionised long life (4 neutralizing chemicals) AEB522239CLL: Pack of 120 contact plates ionised* Made by AES CHEMUNEX - Combourg – France 152202£ : 30/10/08 - O * : Product not

stamped

Control of the environment

R2A Enumeration of aerobic mesophilic micro-organisms in water For In Vitro diagnosis

PRINCIPLE R2A agar is a medium for the bacterial examination of treated water. This medium is less nutrient than the ones used classically for the enumeration of cultivable flora in water samples; Incubated at 20°C for a period superior to 48 hours, this medium favours the growth of stressed bacteria and chlorine-tolerant. The estimation of the culturable flora is therefore more accurate than the use of classic methods using a richer medium and incubation at 37°C. This medium is recommended for the microbiological monitoring of purified water and water for injections with in the frame work of the European Pharmacopoeia. It is also used for the control of haemodialysis waters in hospitals. FORMULA In grammes per litre of distilled water Yeast extract Proteose peptone Casamino Acids Dextrose Soluble starch Potassium phosphate Magnesium sulfate Sodium pyruvate Agar

0,50 0,50 0,50 0,50 0,50 0,3 0,024 0,3 15,00

Final pH : 7,2 + 0,2 at 25°C PREPARATION Suspend 18,2 grammes of powder in 1 litre of purified water. Bring to the boil under continuous homogenisation until the powder is completely dissolved. Dispatch in tubes or flasks before autoclaving 15 minutes at 121°C. PROCEDURE Filter on a membrane the appropriate quantity of water. Within the frame work of the control of water for injections according to the European Pharmacopoeia, incubate the plates at 32,5+/-2,5 °C for 5 days.

For the control of microbiological managed waters in hospitals, incubate at 20-22°C for 7 to 14 days. RESULTS The number of colonies on plate or membrane are counted and reported as CFU (colony-forming units) per volume of sample (multiplied by the sample dilution if any). LIMITS & PRECAUTIONS After regenerating ready to use flasks or tubes, the medium can show presence of precipitates without any incidence of its performances. BIBLIOGRAPHY 1. Greenberg, Trussel and Clesceri (ed.). 1985. Standard methods for the examination of water th and wastewater, 16 edition APHA, Washington, D.C. 2. European Pharmacopoeia – medium S 3. Circulaire DGS/DH/AFSSAPS n°311, 07/06/00, Contrôles des eaux de dialyse. PRESENTATION Dehydrated medium (Store between 1 and 30°C) AEB153482: 500g Ready to use medium (Store between 2 and 25°C) AEB623486 : Pack of 6 flasks of 100ml AEB623487 : Pack of 6 flasks of 200ml AEB123488: Pack of 100 tubes of 15 ml Ready to use plates (Store between 2 and 25°C) AEB523480 : Pack of 20 dishes 90 mm ∅ AEB123487 : Pack of 10 dishes 55 mm ∅ AEB523487 : Pack of 120 dishes 55 mm ∅ Made by : AES CHEMUNEX - Combourg – France 523480£ : 29/07/09 – K

TSA 4N IONISED long life Tryptic soy agar ionised with 4 neutralising chemicals Use In Vitro

DESCRIPTION This medium intended for the environment monitoring of isolators and other controlled areas. It is used for the dynamic or static microbiological control of air and the microbiological control of gloves This medium uses as nutrient base Tryptic soy agar and is added with 4 neutralising chemicals. Lecithin neutralizes quaternary ammonium compounds and chlorexidine, Polysorbate 80 neutralises phenolic disinfectants, hexachlorophene, formalin and mercurial products. The combined action of lecithin and Polysorbate 80 neutralizes ethyl alcohol. Sodium Thiosulfate neutralises halogen residuals L-histidine allows the neutralization of aldehyde residuals. Our control of the manufacturing process enables us to provide you ready to use ready to use ionised products with triple wrapped whose shelf life, from manufacturing day, extends to 6 months for the contact plates and to 9 months for Petri plates.

PACKAGING Ready to use triple wrapped ionised plates with extended shelf life (to be stored between 2 and 25°C) AEB530149LL : Pack of 120 Petri plates ∅ 90 mm Made by : AES CHEMUNEX - Combourg – France 530149LL : 03/10/08-A

FORMULA In grammes per litre of purified water. Casein Peptone Soybean Peptone Sodium Chloride Lecithin Polysorbate 80 Sodium Thiosulfate 5H2O L-histidine Agar

15,00 5,00 5,00 0,70 5,00 0,50 1,00 15,00

Final pH : 7,3 + 0,2 at 25°C PROCEDURE 1. Open the cardboard box and take off the exterior wrapping in the air-lock accessing to the controlled zone. 2. Suspend the stack of plates using the eyelet of the second wrapping to optimize decontamination. 3. Discard the two remaining wrapping before once the decontamination procedure is over. 4. Use the plates for your tests. RESULTS Count the visible colonies. The results are interpreted according to the type of surface, the treatment, and the acceptable risk level.

HYGICOUNT and HYGICOUNT 4N Surfaces control medium Use In Vitro

DESCRIPTION Hygicount medium is used for the detection and enumeration of micro-organisms present on surfaces (walls, floors, equipment…). It contains Lecithin and Polysorbate 80, according to the recommendations from the A.O.A.C. (Association of the Official Analytical Chemists). These agents inactivate residual disinfectants: Lecithin neutralizes quaternary ammonium compounds and chlorexidine, and Polysorbate 80 neutralises phenolic disinfectants, hexachlorophene, formalin and mercurial products. The combined action of Lecithin and Polysorbate 80 neutralizes ethyl alcohol. The addition of Sodium Thiosulfate and L-histidine in the Hygicount 4 N allows the neutralization of halogened and aldehyde products. Poured in plates with a grid, his medium is recommended for bacteriological surface sampling that is performed in hospitals, industry or any place with strict hygiene regulation Our control of the manufacturing process enables us to provide you ready to use ready to use ionised products with triple wrapped whose shelf life, from manufacturing day, extends to 6 months for the contact plates and to 9 months for Petri plates. FORMULA In grammes per litre of purified water. Hygicount Casein Peptone Soybean Peptone Sodium Chloride Lecithin Polysorbate 80 Agar Hygicount 4N Casein Peptone Soybean Peptone Sodium Chloride Lecithin Polysorbate 80 Sodium Thiosulfate 5H2O L-histidine Agar

15,00 5,00 5,00 0,70 5,00 15,00 15,00 5,00 5,00 0,70 5,00 0,50 1,00 15,00

For the Hygicount 4N medium, add the extra components to the basic medium. Dispense into 17ml flasks or tubes. Autoclave for 15 minutes at 121°C. PROCEDURE Frequency and sampling spots should be determined according to the quality insurance system of the tested site. You may also refer to the normalised sampling scheme as defined in NF 06-22 and NF 0623 standards. It is important to take samples according to the same procedure, in order to compare the various results. Apply the agar onto a dry area, giving a 500 grammes pressure for 10 seconds. (future NF EN 1632-3 standard). Clean the sampled area once the agar has been applied. The incubation may be for 3 days at 30°C or 25°C for the detection of aerobe mesophilic flora. RÉSULTS Count the visible colonies. The results are interpreted according to the type of surface, the treatment, and the acceptable risk level. Below are some examples of interpretations: Risk 4 (very high risk) 3 (high risk) 2 (medium risk) 1 (low or negligible risk)

Colonies per 25 cm² (1) <5 <5 < 20 < 125

Colonies per 100 cm² (2) < 10 < 100 < 1000 > 1000

(1) Guide du « bionettoyage » - J.O. Recommandations N° E-190 (1991). (2) Futur standard NF EN 1632-3

Classes A B C D

UFC/ 55 mm dishes <1 5 25 50

Interpretation according to the « Manufacturing of sterile medicines ».

European

GMP

Final pH : 7,3 + 0,2 at 25°C The radiosterilised Hygicount agar is enriched with growth factors to ensure optimal fertility. PRÉPARATION Pour 45,7 grams of powder into 1 litre of purified water. Bring slowly to the boil, while stirring until complete dissolution.

HYGICOUNT and HYGICOUNT 4N Surfaces control medium Use in Vitro

BIBLIOGRAPHY 1. « Guide du bionettoyage ». Journal officiel de la République Française. Recommandations n° E-190 (1991) – Chapitres 5 et 6. 2. Projet de norme européenne NF EN 16323. Méthode d’analyse et de mesurage de la biocontamination des surfaces dans les zones à risques. 3. GMP for pharmaceutical products / BPF Européennes « Fabrication des médicaments stériles ». PACKAGING Dehydrated medium (to be stored between 1 and 30°C) AEB160132 : Flask of 500 g Prepoured medium ( 65 mm contact plates) (to be stored between 2 and 8°C) AEB130140C : Pack of 10 plates Hygicount AEB630140C : Pack of 60 plates Hygicount AEB130160C : Pack of 10 plates Hygicount 4N AEB630160C : Pack of 60 plates Hygicount 4N Prepoured medium (65 mm radio-sterilised contact dishes, with triple wrapping) (to be stored between 2 and 25°C) AEB530169C: Pack of 120 plates Hygicount 4N Prepoured medium (65 mm radiosterilised contact dishes packed in Biosafe® container) (to be stored between 2 and 25°C) AEB530170CB : Container of 100 plates Hygicount 4N AEB130160B : Container of 10 plates Hygicount 4N AEB130163B : Container of 30 plates Hygicount 4N Ready to use triple wrapped ionised plates with extended shelf life (to be stored between 2 and 25°C) AEB530169CLL : Pack of 120 contact plates Hygicount 4N Made by : AES CHEMUNEX - Combourg – France 160132£: 29/07/09 - Q

Sampl'air for accuracy and traceability in microbial air sampling

The Sampl’air enables microbial monitoring of air through impacting microorganisms onto a standard Petri dish. High reliability of air sampling is guaranteed by an impacting speed monitoring systems.

High accuracy and reproductibility • • • • • •

Secured sampling Electronic speed and flowrate auto control Battery load indicator and secured cycle start not allowed battery power not sufficient to complete a cycle Individually tested with its grid installed, comes with a control certificate ISO/DIS 14698-1 compliant Steam sterilizable sampling grid (stainless steel)

Total traceability and documentation •

Traceability sampling location – Traceability culture media plate Sampling reports can be printed on adhesive labels without PC

•

PC printer connection

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Program, export and process sampling reports with the Sampl’air data manager software (FDA chap 21 CFR part 11 compliant)

More information on www.samplair.com

EP (European Pharmacopoeia) â&#x20AC;&#x201C; USP â&#x20AC;&#x201C; AES CHEMUNEX EP

USP

AES Chemunex

Use

TSB broth

Non selective enrichment Fertility control

TCS agar

Total flora enumeration

XIX

Sabouraud agar

Yeasts and moulds enumeration

Lactose broth

Selective enrichment for Enterobacteria and Salmonella

Potato agar Selective enrichment for the detection of Enterobacteria

EE broth Mossel

VRBL containing glucose

Detection of Enterobacteria

EMB Levine

Detection of Enterobacteria

Mac Conkey broth

Selective enrichment for the detection of E.coli

Mac Conkey agar

Detection of E.coli

TBG broth

Selective enrichment for Salmonella

XVIII

Celenite cystine

Selective enrichment for Salmonella

DCL Pharmacopoeia

Detection of Salmonella

XIV

XLD agar

Detection of Salmonella

Wilson and Blair modified agar

Detection of Salmonella

XIII

VBRP Kristensen

Detection of Salmonella

S.aureus

Detection of Salmonella

XVI

TSI agar

Salmonella confirmation

VII

Cetrimide agar

Detection of Pseudomonas

VIII

King B agar

Confirmation of Pseudomonas

King A agar

Detection of Pseudomonas

Chapman agar

Detection of S.aureus

Baird Parker agar

Detection of S.aureus

RCM

Detection of clostridies

Columbia agar

Confirmation of clostridies detection

R2A agar

Enumeration of total flora in purified waters and derivatives

Alphabetical Index 1. Diluents & sterility tests AOAC Letheen broth.........................................................................................p.13 DNP: NPD (Neutralizing Pharmacopoeia Diluent).............................................p.14 Fluid K...............................................................................................................p.16 Meat peptone â&#x20AC;&#x201C; Fluid A .....................................................................................p.17 pH7 pharmacopoeia diluent ..............................................................................p.15 Thioglycollate resazurin ...................................................................................p.19 Tryptic Soy Broth (TSB) ....................................................................................p.18

2. Analysis

Chapman ..........................................................................................................p.36 Cetrimide Pharmacopoeia Agar ........................................................................p.34 Columbia 3........................................................................................................p.43 EE Broth Mossel ...............................................................................................p.28 Hygicount and Hygicount 4N.............................................................................p.51 Mac Conkey Agar .............................................................................................p.31 Mac Conkey Broth ............................................................................................p.30 Rappaport Vassiliadis Pharma ..........................................................................p.38 R2A ...............................................................................................................p.49 RCM ...............................................................................................................p.42 Sabouraud ........................................................................................................p.25-46 Sabouraud broth ..............................................................................................p.45 TSA ...............................................................................................................p.24 TSA 4N IONISED long life ................................................................................p.50 VRBG Pharma ..................................................................................................p.29

XLD

........................................................................................................... p.39

Your contacts Technical support Name

Claire Gardyn

Job title

Microbiology Engineer

Phone number

+ 33 (0)2 99 73 36 82

E-mail c.gardyn@aeschemunex.com

Export department Name

Job title

Phone number

E-mail

Jean-Philippe Aurel

Export Manager

+ 33 (0)2 23 50 12 26

j.aurel@aeschemunex.com

AurĂŠlie Bossard

Export & Marketing Coordinator

+ 33 (0)2 23 50 12 27

a.bossard@aeschemunex.com

Emmanuelle Gonnord

Export Sales Assistant

+ 33 (0)2 23 50 12 25

e.gonnord@aeschemunex.com

LaĂŤtitia Pinel

Export Sales Assistant

+33 (0)2 23 50 12 55

l.pinel@aeschemunex.com

Fax number: + 33 (0)2 23 50 12 28 - +33 (0)2 23 50 12 00

Visit our web site: www.chromogenic-media.com