AES Culture media inserts by Innovation Diagnostics Inc.

Culture Media for the Food Industry

ESIA ®

ALOA ®

SMS ® ASAP ®

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

TA003B

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 ISO 7218 & 11133 (part 1 & 2) standards. 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 ISO 11133-2 standard. 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:

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

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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. 2

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. When a medium is at its most fragile, for example ready poured dishes of Baird Parker, we avoid transport increase such as week-ends and Bank holidays as to minimise alteration. 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:

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Name of the product, packaging and quantities

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Storage temperature,

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Reference, Batch number, expiring date.

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

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Name of the product, packaging and quantities

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Reference, Batch number, expiry date and pH.

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A conformity statement notifying that the quality control has been carried out following a Quality Control Protocol « QCP » which 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. From then on wards an e-mail will be sent to you when a modification occurs.

Moreover, the label is composed of 12 stickers on which figure the following details: Name of product, batch number, and expiring date. 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 self-adhesive 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)

Detachable sticker

Self adhesive detachable Q.C. certificate

pH measured of the batch

12 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:

Culture media for the food industry 1. Diluents & confirmation reagents Buffered Peptone Water ................................................................................... p.12 Peptone salt (Maximum recovery diluent)......................................................... p.13 Half Fraser broth .............................................................................................. p.14 Fraser broth...................................................................................................... p.15 Mucap test (adapted protocol for SMS method) ............................................... p.16 Kovacs ............................................................................................................. p.17 D-cycloserine.................................................................................................... p.18 Cryo beads. ...................................................................................................... p.19 Sterile defibrinated blood.................................................................................. p.20 R.P.F AFNOR................................................................................................... p.21

2. Analysis Total flora ......................................................................................................... p.23 PCA....................................................................................................... p.24 Yeasts and moulds........................................................................................... p.25 DRBC ................................................................................................... p.26 DG18..................................................................................................... p.28

GramEnterobacteria ................................................................................................. p.30 EE broth Mossel .................................................................................... p.32 V.R.B.G ................................................................................................. p.33 Brilliant Green........................................................................................ p.34 Coliforms .......................................................................................................... p.36 V.R.B.L.................................................................................................. p.37 V.R.B.L – MUG...................................................................................... p.38 E.coli ................................................................................................................ p.39 T.B.X .................................................................................................... p.40 See also Rebecca concept

Simultaneous detection of E.coli/enterobacteria – E.coli/coliforms ................... p.41 Rebecca™ .............................................................................................. p.42 Salmonella ....................................................................................................... p.45 RVS (Rappaport Vassiliadis Soja) ......................................................... p.46 MTTn (Müller Kauffmann tetrathionate-novobiocine)............................. p.47 ASAP™ .................................................................................................. p.48 X.L.D ISO 6579 ..................................................................................... p.49 X.L.T.4................................................................................................... p.50 Drigalski ................................................................................................ p.51 8

Wilson and Blair modified ······································································ p.52 Edel – Kampelmacher (Brilliant Green Agar ISO)·································· p.53 Kligler-Hajna·························································································· p.54 Nutrient ISO 6579·················································································· p.55 SMS™ ···································································································· p.57 SMS™ Confirmation ··············································································· p.61 SALSA™ ································································································ p.63 Shigella ············································································································ p.65 Mac Conkey agar ·················································································· p. 66 Hektoen ································································································· p.67 T.S.I······································································································· p.68 Enterobacter sakazakii ····················································································· p.69 ESIA/ESSB···························································································· p.71 mLST····································································································· p.72 Other culture media ························································································· p.73 Vibrions TCBS······················································································· p.74 Yersinia CIN ·························································································· p.75

Gram+ Staphylococci ··································································································· p.77 Baird-Parker ·························································································· p.81 Baird-Parker + RPF ··············································································· p.83 Brain Heart Infusion Broth ····································································· p.84 Lyophilised Rabbit Plasma ···································································· p.85 DNA······································································································· p.86 Giolitti Cantoni ······················································································· p.87 Clostridium ······································································································ p.88 T.S.C and Tryptose sulfite ····································································· p.91 Thioglycollate Resazurin ······································································· p.93 Listeria·············································································································· p.94 ALOA™ ·································································································· p.97 Oxford ··································································································· p.101 Palcam ·································································································· p.102 Blood Agar····························································································· p.103 T.S.Y.E·································································································· p.104 Bacillus cereus ································································································· p.105 Mossel (MYP)························································································ p.106 Bacillus Cereus Rapid Agar (BACARA®) ··············································· p.107 Campylobacter ································································································· p.108 Campylobacter according to Karmali ····················································· p.110 Brucella ································································································· p.111 Preston ·································································································· p.112 CASA® ··································································································· p.113 Lactic flora········································································································ p.115 M.R.S ···································································································· p.116 9

Diluents & confirmation reagents

Culture media for the food industry

Dilubag速 In order to optimize the sample preparation, the majority of our diluents are available in 3 or 5 litre Dilubags速 (dilution broth bags).

Dilumat 4 diluter (AESAP1056)

Dilusafe速 rack (AESDI0314)

Diluplug速: high flow rate connector (AESDI0313)

Ready to start your day !

BUFFERED PEPTONE WATER Dilution buffer In Vitro Use only

PRINCIPLE Buffered Peptone Water (BPW) is used for preenriching damaged Salmonella species from food samples to increase recovery. Its formula is in compliance with NF EN ISO 6579 and ISO/TS 22964 standards. This medium is used for the first step of the protocol when screening for Salmonella in foodstuffs before using the selective enrichment broth. The phosphate buffer in the medium therefore maintaining the pH at an adequate level throughout the incubation, thus helping the revivification of germs sensitive to acid pH. It is wiser to use this medium when preparing dilution of foodstuffs samples that have undergone stressful conditions (Example : Pasteurisation) that have could damaged the germs without eliminating then. FORMULA In grammes per litre of purified water. Peptone Sodium phosphate, Dibasic Potassium phosphate, Monobasic Sodium chloride

10,00 20,00 (1) 3,57 7,14 1,50 3,00 5,00 10,00

Final pH : 7,0 + 0,2 at 25°C (1)

: equivalent to 9 g of Sodium phosphate, Dibasic 12H2O.

BIBLIOGRAPHY 1. NF EN ISO 6579 Microbiologie-Directives générales concernant les méthodes de recherche des Salmonella. (2002) 2. ISO/TS 22964 : Lait et produits laitiers – Détection de l’Enterobacter sakazakii . (2006) PACKAGING Dehydrated medium (to be stored between 1 and 30°C) AEB140302 : 500 g AEB140303 : 5 kg Ready measured dehydrated medium (to be stored between 2 and 25°C) AEB240309 : qsp 9 litres AEB240318 : qsp 18 litres Ready to use media (to be stored between 2 and 25°C) AEB610304 : 6 Flasks of 90 ml AEB610306 : 6 Flasks of 100 ml AEB610307 : 6 Flasks of 200 ml AEB610308 : 6 Flasks of 225 ml AEB110310 : 100 tubes of 9 ml AEB110308M : 1 flask of 900ml AEB910303/4 : 4 Dilubags® (Pouchs) of 3 L AEB910305/2 : 2 Dilubags® (Pouchs) of 5 L

Note: in italic characters the formula for double concentration buffered peptone water.

Buffered peptone water double concentration (to be stored between 2 and 25°C) AEB610316 : Pack of 6 flasks 100 ml

METHOD Suspend 20,0 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.

Buffered peptone water with 0.5% of Tween 80 (to be stored between 2 and 25°C) AEB1110314M : Flask of 490 ml AEB610329L : Pack of 6 flasks of 900 ml AEB910323/4 : 4 Dilubags® (Pouchs) of 3 L

PROCEDURE In sterile conditions prepare the sample (10 or 25 g) in th order to get a 1 in 10 initial suspension. Homogenise well. Incubate round about 18 hours at 37°C, then inoculate the adequate selective enrichment broth according to the analysed product.

Made by AES CHEMUNEX - Combourg - France 140302£: 27/10/08 - Q

LIMITS AND PRECAUTIONS As to help the homogenisation, it is wise to heat slightly (70°C) the prepared BPW before proceeding to distribution in tubes or flasks prior to autoclaving.

PEPTONE SALT Isotonic broth for dilution For in vitro use

PRINCIPLE The maximum recovery diluent is an isotonic diluent containing a low level of peptone and sodium chloride. It is used for maintaining the viability of organisms during dilution procedures of food or water samples. Its formulation complies to ISO standards 6887 and 4833. The maximum recovery diluent has no lethal action on organisms, because the presence of a low level of peptone and a pH of 7,0 as protections. However, during the dilution stage (1 to 2 hours), there will be no growth of the organisms. FORMULA In grams per liter of purified water Peptone Sodium chloride

1,00 8,50

Final pH: 7,0 +/- 0,2 at 25°C

PACKAGING Dehydrated medium (Storage : between 1 & 30°C) AEB141492 : 500 g Prepared medium (Storage: between 2 & 25°C) AEB611492 : 6 flasks of 45 ml AEB611494 : 6 flasks of 90 ml AEB611494M: 6 flasks with septum of 90 ml AEB611496 : 6 flasks of 100 ml AEB611498 : 6 flasks of 225 ml AEB111500 : 100 tubes of 5 ml AEB111499 : 100 tubes of 9 ml AEB611499L: 6 flasks of 900 mL AEB611501M: 6 flasks with septum of 500 mL AEB911493/4: 4 Dilubags® of 3L AEB911495/2: 2 Dilubags® of 5L Prepared medium with 0,3 % of cystein (Storage : between 2 & 25°C) AEB111489 : 100 tubes of 9 ml

PREPARATION Suspend 9,5 grams of the powder in one liter of purified water. Mix thoroughly and warm gently until dissolution is complete. Dispense and sterilize by autoclaving at 121°C for 15 min.

Prepared medium with 2 % of polysorbate 80 (Storage : between 2 & 25°C) AEB611476 : 6 flasks of 100 ml

PROCEDURE Preparation of mother solution from dairy products Bring the medium to about 25°C Introduce 10 or 25 grams of the sample in respectively 90 or 225 ml of maximum recovery diluent. Homogenize.

141492£: 20/11/08 - H

Made by : AES CHEMUNEX - Combourg - France

Preparation of decimal dilutions Introduce 1 ml of the mother solution in a 9 ml tube of maximum recovery diluent. Homogenize and start again with the next dilution. BIBLIOGRAPHY 1. Straker R.P. and Stokes J.L. Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol. ; 1957. 5:21-25 2. Patterson J.W. and Cassels J.A. J.appl.Bacteriol. ; 1963. 26:493-497 3. International Organization for Standarization. Microbiology - General guidance for the preparation of dilutions for microbiological examination. ; 1983. BS5763 Part 6- Preparation of the dilutions. 4. ISO 6887. Directives générales pour la préparation des dilutions en vue de l'examen microbiologique. 5. ISO 4833. Directives générales pour le dénombrement de micro-organismes.

HALF FRASER BROTH Selective enrichment medium for Listeria monocytogenes In vitro use only

DESCRIPTION This complete medium is a modification of the formulation suggested by Donelly and Baigent. In order to reinforce selectivity, the nalidixic acid concentration is reduced to 20mg/l while the acriflavine concentration is increased to 25mg/l. The nalidixic acid inhibits Gram negative bacteria growth, and the acriflavine inhibits Gram positive bacteria growth. The lithium chloride inhibits the enterococcus that also hydrolyse esculin. The sodium chloride increases selectivity, and phosphate salts create a buffer effect in the tubes that allegedly contain some esculin hydrolysing bacteria, such as Listeria. Esculin is a glucoside that produces esculetin and glucose when it is hydrolysed. The esculetin reacts with ammonium ferric citrate to create a dark brown or black complex. The Half Fraser medium has been developed from this medium, reducing selectivity by including less nalidixic acid and acriflavine with respective concentrations of 10 and 12.5mg/l. FORMULA Dehydrated base for Half Fraser In grams per litre of purified water. Peptone proteose 5.00 Tryptone 5.00 Meat extract 5.00 Yeast extract 5.00 Sodium Chloride 20.00 (1) Disodium Hydrogen Phosphate, anhydrous 9.6 Potassium dihydrogen Phosphate 1.35 Esculin 1.00 Lithium chloride 3.00 Nalidixic acid 0.010 Acriflavine HCI 0.0125 Final pH: 7,2 +/- 0,2 at 25°C

PROCEDURE 25 g of test material are first enriched by adding to 225 ml of Half Fraser broth, thoroughly mixed then incubated at 30°C for 24 ± 2 hours. • Alternative method AES 10/3 – 09/00 ALOA® One Day Inoculate an ALOA® agar plate, by spreading 0,1 ml of incubated broth. • Reference standard ISO 11290/A1- 02/05: Carry out a second enrichment, by transferring 0.1ml of the initial enrichment broth into tubes containing 10ml of Fraser broth. Incubate for 48+/-2 hours at 37°C and isolate at each step by spreading on ALOA® and a second agar such as PALCAM or OXFORD. LIMITS AND PRECAUTIONS When hydrolysing esculin, Listeria and other bacteria darken the medium. Using a single medium for the exhaustive detection of specific colonies in a sample is rarely suitable. Some selective media may inhibit strains for species that are looked for, or they may allow the growth of others that are not required, especially when the latter are numerous in the sample. Using a comparative inoculation of samples is then advised, in order to obtain the maximum of details and consequently making it easier to identify the potentially pathogen colonies. BIBLIOGRAPHY 2. 3. 4.

Fraser and Sperber. 1988. Rapid detection of Listeria spp. In food and environmental samples by esculin hydrlysis. J. Food Prot. 51: 762-765. Donelly and Baigent. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52: 689-695. AFNOR NF EN ISO 11290-1 / A1. Février 2005. Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes. Partie 1 : Méthode de recherche.

(1)

equivalent to 12g of Disodium Phosphate dibasic (Na2HPO4, 2H2O)

PRESENTATION Dehydrated medium (Store between 1 and 30°C) (With antibiotics)

Supplement for Fraser and Half Fraser In grams per 10ml of purified water

AEB140412: 500g bottle AEB140413: 5 kg barrel AEB240409: QSP 9 litres AEB140417 : 10 Kg

Ammonium ferric citrate

0.5

PREPARATION Pour 55 grams of powder into 1 litre of purified water. If necessary, you may bring to the boil to obtain perfect dissolution. Spread into 225ml bottles. Autoclave for 15 minutes at 121°C. DO NOT OVERHEAT. Before inoculation, add aseptically to each flask 2,25ml of supplement for Fraser or sterile solution containing 112,5 mg of ammonium ferric citrate. Either liquid or dehydrated ammonium ferric citrate may be added before autoclaving, leading to a final concentration of 0,5 gram per litre of broth. A slight precipitate may appear. It is not prejudicial to the analysis.

Ready to use medium – complete (Store between 2 and 25°C) AEB110419: 100 Tubes of 9ml AEB610418: 6 x 225ml flasks AEB610419L: 6 x 900 ml flasks AEB910913/4: 4 Dilubags® (pouches) 3 L AEB910915/2: 2 Dilubags® (pouches) 5 L Supplement for Fraser (Store between 2 and 25°C) AEB110422S: 20 x 10 ml tubes (Store between 1 and 30°C) B1110.16 : dehydrated 1 kg barrel

Made by AES CHEMUNEX – Combourg – France 140412£: 11/06/09 - K

14 14

FRASER BROTH Selective enrichment medium for Listeria monocytogenes For In Vitro use only

DESCRIPTION This complete medium is a modification of the formulation suggested by Donelly and Baigent. In order to reinforce selectivity, the nalidixic acid concentration is reduced to 20mg/l while the acriflavine concentration is increased to 25mg/l. The nalidixic acid inhibits Gram negative bacteria growth, and the acriflavine inhibits Gram positive bacteria growth. The lithium chloride inhibits the Enterococcus that also hydrolyze esculin. The sodium chloride increase selectivity, and phosphate salts create a buffer effect in the tubes that may contain some esculin hydrolyzing bacteria, such as Listeria. Esculin is a glucoside that produces esculetin and glucose when it is hydrolyzed. The esculetin reacts with ammonium ferric citrate to create a dark brown or black complex. FORMULA Dehydrated base for Fraser In grammes per litre of purified water. Proteose peptone Tryptone Meat extract Yeast extract Sodium chloride Sodium phosphate, dibasic Potassium phosphate, monobasic Esculin Lithium chloride Nalidixic acid Acriflavine HCl

5,0 5,0 5,0 5,0 20,0 (1) 9,6 1,35 1,0 3,0 0,02 0,025

Final pH: 7,2 +/- 0,2 at 25°C (1)

equivalent to 12g of Disodium Phosphate dibasic (Na2HPO4, 2H2O) Supplement for Fraser and Fraser Demi In grammes per 10ml of purified water Ferric ammonium citrate

0,5

PREPARATION Pour 55 g of powder into 1 litre of purified water. If necessary, you may bring to the boil to obtain perfect dissolving. Spread into 10 ml tubes. Autoclave for 15 minutes at 121°C. DO NOT OVERHEAT. Before inoculation, add aseptically to each tube 0,1ml of supplement for Fraser or sterile solution containing 5mg of ferric ammonium citrate. Either liquid or dehydrated ferric ammonium citrate may be added before autoclaving, leading to a final concentration of 0,5 g per litre of broth. A slight precipitate may appear. It is not prejudicial to the analysis.

PROCEDURE ISO standard ISO 11290-1 : 25 g of test material are first enriched by adding to 225 ml of Half Fraser broth, thoroughly mixed then incubated at 30°C for 24 ± 2 hours. After incubation transfer 0,1 ml of the enriched sample to 10 ml of Fraser broth. Incubate the Fraser broth at 37°C for 48 ± 2 hours. After each step (Half Fraser and Fraser broth), subculture onto an ALOA® and a second agar such as PALCAM or OXFORD. LIMITATIONS OF THE PROCEDURE When hydrolyzing esculin, Listeria and other bacteria darken the medium. Using a single medium for the exhaustive detection of specific colonies in a sample is rarely suitable. Some selective media may inhibit some species of strains that are screened, or they allow the growth of interfering flora, especially when the latter are numerous in the sample. Using a comparative inoculation of samples is then advised, in order to obtain the maximum of details and consequently making it easier to identify the potentially pathogen colonies. BIBLIOGRAPHY 1. Fraser and Sperber. 1988. Rapid detection of Listeria spp. In food and environmental samples by esculin hydrolysis. J. Food Prot. 51: 762-765. 2. Donelly and Baigent. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52: 689-695. 3. AFNOR NF EN ISO 11290-1 / A1. Février 2005. Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes. Partie 1 : Méthode de recherche. PACKAGING Dehydrated medium with antibiotics (Store between 1 and 30°C) AEB140422: 500g bottle Ready to use medium - Complete (Store between 2 and 25°C) AEB110422 : 20 tubes of 10 ml AEB110429 : Pack of 100 tubes of 10 ml Supplement for Fraser AEB110422S: 20 tubes of 10 ml (Store between 2 and 25°C) B1110.16 : dehydrated 1 kg (Store between 1 and 30°C) Made by AES CHEMUNEX - Combourg – France 140422£ : 11/06/09 - I

MUCAP TEST (Adapted protocol for SMS© method) For In Vitro use Store between 2 and 8°C

PRINCIPLE A number of essays have been carried out on methylumbelliferone fluorescent derivative substrate. MUCAP TEST contains an organic solvent in which a height carbon chain of carbohydrate (a specific substrate of the C8-esterase enzyme) linked to methylumbellyferone is dissolved. This substrate in esterified by C8-esterase, resulting to the release umbellyferone known to by fluorescent under a Wood light (366 nm). PROCEDURE © Within the frame word of SMS method, place one drop of MUCAP TEST at the edge of the migration zone of presumptively positive in Salmonella plates. 1. Before adding the reactive, place the plate under a Wood light to look for any natural existing fluorescence. If this step show no fluorescence proceed to step 2. 2. Plate at the edge of the migration zone (or in the centre of the plate where the 3 migration zones joint up) one drop of MUP TEST. 3. Wait 5 to 8 minutes and then observe in the dark under a Wood light. RESULTS The test is considered as positive when fluorescence is seen where the drop of reactive was originally placed.

BIBLIOGRAPHY 1. Pontello M., S. Russolo, F. Carozzi and V. Bottiroli. 1987. Evaluation of a new, rapid method (Mucap Test) for the presumptive identification of Salmonella on primary isolation media. Fifth International Symposium on Rapid Methods and Automation in Microbiology and immunoIogy. Florence, 4-6 November. 2. Humbert F., G. Salvat, P. Colin, C. Lahellec and G. Bennejean. 1989. Rapid identification of Salmonella from poultry meat products by using 'Mucap test'. Int. J. Food Microbiol. 8:79-83. 3. Aguirre P.M., J.B. Cacho, L. Folgueira, M. Lopez, J. Garcia and A.C. Velasco. 1990. Rapid Fluorescence method for screening Salmonella spp from enteric differential agars. J. CLin. Microbiol. 28:148-149. PACKAGING AEB191500 : 8 ml flask for 160 tests. VL6L : Wood light battery powered VBL16006011: Dark chamber to use with VL6L light VBL16015011: Dark chamber with equipped with 4 wood lights plugged in the mains supply. Distributed by: AES Laboratoire - Combourg - France 191500SMS£ : 24/02/05 - D

MUCAP TEST does not impair on the viability of bacteria. To isolate the strain in order to identify it proceed by taking directly a sample of growth from the migration edge using an inoculating loop or a closed pipette. If MUCAP test is negative (no fluorescence after 8 minutes) perform a subculture on a selective media for Salmonella using the previously described method. After incubation, if typical colonies are seen on the plate, proceed to biochemical and serological tests. LIMITS & PRECAUTIONS The intensity of fluorescence is proportionally linked to the quality of the exciting source. Wood lights that are plugged in the mains supply guaranty a better quality than those battery powdered. A second aspect for performing the test under excellent conditions is the obscurity. The use of an enclosed chamber provided with protective window assures the best result.

KOVACS Kovacs reagent for the detection of Indole production In Vitro use only Store between 2 and 8°C

PRINCIPLE Kovacs reagent is used to reveal the presence of indole which is one of the end products of tryptophane oxidation by bacteria. The active ingredient in Kovacs reagent, pDimethylaminobenzaldehyde reacts with indole to form a pinkish red compound. FORMULA p-Dimethylaminobenzaldehyde Amyl alcohol Hydrochloric acid

5,00 g 75,00 ml 25,00 ml

Store in an amber flask at 2-8°C PROCEDURE Add two drops of Kovacs reagent at the surface of the culture. The reaction is immediate. On liquid medium: the Kovacs reagent ( not miscible) forms a ring at the surface of the broth that colours in red in presence of indole. On solid medium: The Kovacs reagent turns to red in presence of indole. RESULTS Edwersiella, E. coli, Klebsiella oxytoca, Levinea, Morganella morganii, Proteus rettgeri & vulgaris, Providencia are INDOLE +. Citrobacter, Enterobacter aerogenes & cloacae, Hafnia, Klebsiella pneumoniae, Proteus mirabilis, Salmonella, Serratia liquefaciens & marcescens, Shigella sonnei, Y. pseudotuberculosis are INDOLE -. LIMITS & PRECAUTIONS It is important to be sure that the medium used to support the test contains sufficient amino acid trytophane and no trace of indole. BIBLIOGRAPHY 1. Kovacs N. 1928. Eine vereinfachte Methode zum Nachweis der Indolbildung durch Bakterien. Z. Immunitätsforsch. 55:311-315. PACKAGING Ready to use solution AEB190504 : 25 ml flask AEB190502 : 50 ml flask Made by AES CHEMUNEX - Combourg - France 190504£ : 09/05/08 - E

D-CYCLOSERINE Culture media supplement In Vitro use only Store between 2 and 8°C

PRINCIPLE 4 % sterile D-cycloserine in solution is used as additive for T.S.C Agar. The addition of this chemical increases the selectivity of the medium towards Clostridium perfringens and helps reading the plates by lessening the black halo around the colonies. FORMULA Lyophilisated D-cycloserine

200,00 mg

METHOD Regenerate D-clycloserine with 5 ml of sterile purified water. Homogenize well, then add to 500 ml of TSC Agar base liquefied and kept at 45°C or 0.2 ml per tubes of 20 ml. LIMITS & PRECAUTIONS Once regenerated the supplement can be kept up to 12 hours if stored at 2-8°C or 2 month if frozen at –20°C. When frozen it is important to fraction the supplement in order to defreeze only the quantity needed. The regenerated supplement will suffer from cycles of defrosting/refreezing. BIBLIOGRAPHY 1. Harmon S.M., Kautter D.A. and Peeler J.T. – 1971 – Improved medium for enumeration of Clostridium perfringens. App. Microbiol. – 22:688. PACKAGING Lyophilized supplement AEB184002 : Q.S.P. 500 ml of medium base Made by AES CHEMUNEX - Combourg - France 184002 :23/05/01 - C

CRYO BEADS Preservation of reference strains In Vitro use only Store between 2 and 25°C

DESCRIPTION The Cryo-beads system is a little tube containing beads on which micro-organisms can stick. The beads are immersed in a hyper tonic cryo-preservative solution. Once inoculated , the tubes are stored between –20°C and –70°C. Each box contains 64 tubes of 25 beads, densely packed for storage in a freezer. This is a cheaper, simple and reliable method for strains preservation. It may be used for any type of microorganism.

BIBLIOGRAPHY 1. White DJ Sands R.L. 1985. Storage of Bacteria at – 76°C. Medical Laboratory Sciences. 42:289-290. 2. Feltham R.K.A., Pell P.A., Sneath P.H.A. 178. A simple method for storage of bacteria at 76°C. Journal of Apply Bacteriology. 44:313-316.

REAGENTS Each box contains 64 tubes in which 25 beads are immersed in a cryo-preservative solution.

Made by AES CHEMUNEX – Combourg –France

PRESENTATION AEB400100 : box of 64 tubes containing 25 beads.

400100£ : 07/04/08 - D PROCEDURE Strains preservation 1 – Use a permanent marker to identify the tube to be inoculated. 2 – Inoculate the tube with a fresh and pure culture that corresponds to a density of 3 or 4 on Mc Farland scale. 3 – Close the tube and turn it upside down to spread germs evenly. 4 – With a sterile pipette, remove the maximum of solution from the tube. Then place the cap back on. 5 – Store the cryo-beads tubes in a freezer. The ideal temperature is –70°C. Temperature should be at least – 20°C. Preserved strains culture. 1 - Take the tube out of the freezer. Take only one tube at a time. In this way tubes that are not used will not get warm. 2 – Open the tube and remove one bead, using sterile tweezers. 3 – Place the bead into a tube containing a broth with an appropriate medium for the species. You may also roll the bead on the surface of an agar plate. 4 – Incubate according to the species requirements. 5 – Place the tube back into the freezer as soon as possible so that the other beads do not warm up. Destroy the bead that you used in an appropriate way. LIMITS AND PRECAUTIONS Protect the tubes from any type of heat or bright light, even during inoculation. Make sure that the cryo-preservative solution is clear. Do not use the solution if it is cloudy.

STERILE DEFIBRINATED BLOOD In Vitro use only Store between 2 and 8°C

PRINCIPLE Sterile defibrinated blood is used to supplement culture medium to help the growth of fastidious germs such as pneumococci or Haemophilus. The addition of blood in a nutrient base offers an adequate medium for characterizing the hemolytic power of microorganism strains. The Blood is free from any antiseptic or anticoagulant. PROCEDURE • Fresh blood Agar. Add 2 to 10 % of sterile fresh blood to appropriate liquefied nutrient Agar base cooled to 45-50°C or a broth. Homogenise well and dispatich in tubes or plates. • Cooked Blood Agar. Add 5 % of sterile fresh blood to appropriate liquefied nutrient agar base cooled to 45-50°C. Homogenize well then heat to 80°C in a water bath for 15 minutes. Cool to 45°C, homogenize well then dispatch in to sterile Petri plates. LIMITS & PRECAUTIONS Sheep blood inhibits the growth of Haemophilus (ex : Haemophilus haemolyticus). Some strains of Enterococci β hemolytic with on horse blood plate but can be α hemolytic on sheep blood agar plates. It is recommended to use sheep blood Agar plates as first growth trials. Sterile defibrinated fresh blood can not be used for CFR tests, because spontaneous hemolysis can occur. Freezing/Defrosting triggers the hemolysis of the blood. PACKAGING Horse Blood AEB300025 : Flasks of 25 ml AEB300050 : Flasks of 50 ml AEB300100 : Flasks of 100 ml Sheep Blood AEB200025 : Flasks of 25 ml AEB200050 : Flasks of 50 ml AEB200100 : Flasks of 100 ml Distributed by AES Laboratoire - Combourg - France 300025£ : 04/05/04 - B

R.P.F AFNOR Rabbit Plasma Fibrinogen supplement In Vitro use only

PRINCIPLE RPF supplement (Rabbit Plasma Fibrinogen) is intended to be added to Baird Parker base in order to detect the presence of coagulase. Rabbit plasma is the best substrate for this enzyme; fibrinogen is added to emphasize the reaction; trypsine inhibitor is used to prevent fibrinolyses and potassium tellurite is a selective agent. COMPONENTS Fibrinogen Rabbit plasma EDTA Trypsine inhibitor Potassium tellurite

375 mg 2,5 ml 2,5 mg 2,5 mg

METHOD Add 10 mL of purified sterile water to one vial and dissolve completely its contents. Transfer the prepared solution to 90 mL of autoclaved Baird Parker base cooled to 48°C. Homogenize carefully then dispatch into sterile Petri dishes. PROCEDURE • Pour plate technique (according to ISO and French standards) : Samples are diluted as desired. Place 1 ml of the sample our its decimal dilutions in the base of a sterile Petri plate. Pour enough prepared medium as to obtain a plate 3 mm thick. Homogenize well and let set before incubating at the plates at 37°C for 24 hours or up to 48 hours if no typical colonies are seen. • Surface spreading of inoculi (according to French standards) Plates are inoculated by spreading of 0,1 ml of the sample or its dilutions. Incubate plates at 37°C for 24 hours or up to 48 hours if no typical colonies are seen. • Confirmation technique (according to French standards): According to the French Standard NF V 08-57-1 typical colonies on Baird Parker can be confirmed with Baird Parker RPF by pricking a colony into and Baird Parker RPF medium. Do not confirm more than 12 colonies per plate including controls.

BIBLIOGRAPHY 1. Baird-Parker A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. J. Appl. Bacteriol. 25:12-19. 2. Norme AFNOR NF V08-057-1. Méthode de routine pour le dénombrement des Staphylocoques à coagulase positive par technique des colonies à 37°C. Partie 1 : technique avec confirmation des colonies. 3. Norme AFNOR NF V08-057-2. Méthode de routine pour le dénombrement des Staphylocoques à coagulase positive par comptage des colonies à 37°C. Partie 2 : technique sans confirmation des colonies. 4. Norme NF EN ISO 6888-2. Méthode horizontale pour le dénombrement des Staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 2 : Technique de la gélose au plasma de lapin et fibrinogène. PRESENTATION Prepoured medium. Store between 2 & 8°C AEB520330 : Pack of 20 dishes 90 mm AEB520329 : Pack of 120 dishes 90 mm Ready to use Agar base: Store between 18 & 23°C AEB620314 : Pack of 6 x 90 ml flasks AEB620313 : Pack of 6 x 180 ml flasks Additive: Store between 2 & 8°C AEB184106: RPF AFNOR 6 qsp 100ml AEB184107: RPF AFNOR 6 qsp 200 ml AEB620337 : KIT Baird Parker base with RPF additive for 1L Made by: AES CEMUNEX - Combourg - France 184100£ : 03/10/05 -B

RESULTS Enumerate any colonies (black or not) that are surrounded by a precipitation halo. LIMITS & PRECAUTIONS Flasks of prepared complete medium should be used immediately. Ready poured plates prepared be the laboratory should be inoculated within 48 hours and stored at 2-8°C.

Analysis

Culture media for the food industry

Total flora enumeration at 30째C NF ISO 4833 V08-011 standard - July 2001

Decimal dilution of the sample in Peptone salt broth (AEB111499 - pack of 100 tubes of 9 ml)

Inoculation by incorporation on PCA agar (AEB620707 - 6 flasks of 200 ml) with 1 ml of each dilution Incubation 72+/-3 hours at 30+/- 1째C

Enumeration of the colonies and expression of the results according to the dilutions.

PCA Plate Count Agar In vitro use only To be stored between 2 and 25°C

DESCRIPTION The standard Plate Count Agar is used for aerobic microorganisms enumeration in milk, meat, products made out of meat and other foodstuff. It is also used for the analysis of pharmaceutical and cosmetic products, and their raw materials. FORMULA In grammes per litre of distilled water. Pastone Yeast extract Glucose Agar

5,00 2,50 1,00 15,00

Final pH: 7,0 +/- 0,2 to 25°C PREPARATION Pour 23.5 grammes of powder into one litre of distilled water. Bring to the boil, with frequent stirring to ensure complete dissolution. Dispense into tubes or flasks. Autoclave for 15 minutes at 121°C.

BIBLIOGRAPHY 1. FIL-IDF 49 . 1970. Méthode normalisée pour le dénombrement des germes totaux dans les poudres de lait et de lactosérum (méthode de référence). 2. FIL-IDF 61. 1971. Crèmes glacées et glaces au lait. Dénombrement des germes totaux. 3. J. O. du 27 Août 1963. Contrôle des laits concentrés sucrés et des laits secs. 4. American Public Health Association . 1960. Standard methods for the examination of dairy th prducts. 11 ed. APHA Inc. , New York. 5. Méthode officielle pour le dénombrement des germes aérobies mésophiles. Ministère de l’Agriculture. Commission XXX. Cosmétologie. 6. AFNOR V 08-011. Directives générales pour le dénombrement des micro-organismes. Techniques par comptage des colonies obtenues à 30°C.

PRESENTATION Dehydrated medium (Store between 1 & 30°C) AEB150702: 500g flask

PROCEDURE Liquefy the agar then keep the medium at a temperature of approximately 45-50°C. Place 1ml of the product to be tested or its decimal dilutions into sterile Petri dishes. Dispense 12ml of melted agar. Shake slightly to mix correctly and leave it to solidify. For mesophilic micro-organisms, incubate at 30°C for 72 hours. For thermophilic micro-organisms, incubate at 55°C. Finally, for psychrophilic micro-organisms, incubate at 7°C.

Ready to use medium AEB120709 : 100 tubes 15ml AEB620706: 6 flasks 100ml AEB620707: 6 flasks 200ml

RESULTS The enumeration should be performed on plates that present between 30 and 300 colonies.

Ready to use PCA + Skimmed milk AEB150712: 500g Flask AEB620717: 6 flask of 200ml

NOTE For dairy microbiology, it is interesting to add 1g/l of powder skimmed milk into the basic agar. In this case, caseolytic bacteries grow with a clearer halo around their colonies, indicating milk caseine proteolysis.

Pre-poured medium AEB520709: Pack of 120 dishes 90mm AEB520710: Pack of 20 dishes 90mm AEB 120711: Coffret de 10 boîtes contact 55 mm Pre-poured contact dishes AEB120710C: Pack of 10 dishes 60mm

Made by AES CHEMUNEX – Combourg – France 152852£:31/07/07 - G

Culture media for the food industry

Horizontal method for the enumeration of yeasts and moulds – Part 1: Colony count technique in products with water activity greater than 0.95 - ISO 21527-1, 2008

Weigh X g of sample in 9 times X ml of peptone water at 0.1% Or, if it is a question of a specific preparation, prepare according to the ISO 6887 standard, or the ISO 8261 or the standard specific to the tested product.

Spread 0.1 mL of the prepared sample (or the sample if it is liquid) onto the surface of a DRBC plate. (Dichloran Rose Bengale Chloramphenicol agar : AEB620547 – Pack of 6 flasks of 200mL) or 0.1 mL on 3 plates (when low contamination is estimated). Carry out in the same way with the following dilutions.

Incubate at 25°C±1°C under aerobic atmosphere for 5 days (with lids facing upwards). If necessary, leave the plates to rest for 1 to 2 days under daylight. For the samples suspected to contain mostly moulds, it is advised to incubate the plates in an open plastic bag in order to prevent dispersion dissemination.

Reading: Select the plates containing less than 150 colonies and/ or propagules. If spreading of moulds is a problem, count colonies after 2 days and again after 5 days of incubation. Carry out microscope examination in order to distinguish between colonies of yeasts and moulds or bacteria. The colonies of yeasts and propagules of moulds are counted separately, if necessary. To identify, isolate onto a isolation medium or an appropriate identification medium.

D.R.B.C Dichloran-Rose Bengal-Chloramphenicol agar Usage In Vitro To be stored between 2 and 25°C

PRINCIPLE Dichloran Rose Bengal Chloramphenicol agar is recommended by ISO 21527-1 standard for the enumeration of yeasts and moulds in products intended for human consumption or feeding of animals having a water activity (aw) greater than 0,95. The formula of this medium is a modification of the Rose Bengal agar. The joint presence of Dichloran and Rose Bengal limits the spreading of moulds, optimizing the reading of the plates. Chloramphenicol inhibits the bacterial growth. FORMULA In grams per litre of purified water Enzymatic digest of animal and plants tissues Dextrose Monopotassium phosphate (KH2PO4) Magnesium Sulfate (MgSO4,H2O) Dichloran Rose Bengal Chloramphenicol Agar

5,00 10,00 1,00 0,50 0,002 0,025 0,10 15,00

- Moulds spores spread easily in the air. Handle carefully the plates to avoid an over-estimation of the population. To avoid this phenomenon, it is recommended to incubate the plates in open plastic bags. - Others components can be added to the formula of the medium, regarding the level of bacterial contamination and / or the type of fungi present in the product to be tested. For this, refer to ISO 21527-1 standard. BIBLIOGRAPHY NF ISO 21527-1 : novembre 2008 : Microbiologie des aliments – Méthode horizontale pour le dénombrement des levures et moisissures. Partie 1 : technique par comptage des colonies dans les produits à activité d’eau supérieure à 0,95. PACKAGING Ready-to-use medium AEB620547 : Pack of 6 flasks of 200 ml Made by : AES CHEMUNEX - Combourg - France

Final pH : 5,6 + 0,2 at 25°C PROCEDURE

620547 : 08/07/09 - A

Liquefy the medium in a boiling water bath, then cool the medium to 44-47°C before dispatching into Petri plates. Onto a DRBC agar plate, spread 0,1 ml of the product to be tested for the fluid samples or 0,1 ml of the initial suspension for the solid samples. On a second plate -1 transfer 0,1 ml of the first dilution (10 ) for fluid samples -2 or 0,1ml of the 10 dilution for solid samples. Incubate the plates in an upright position at 25°C + 1°C under aerobic atmosphere for 5 days. RESULTS Count the yeasts and moulds between 2 and 5 days of incubation, on the plates containing less than 150 colonies. LIMITS AND PRECAUTIONS - AVOID EXPOSURE OF THE MEDIUM TO THE LIGHT. Use immediately or store in the dark. - A protocol using a pour plate method can be carried out if the equivalence between the two techniques has been validated for the chosen matrix. The inoculation by pour plate does not allow the differentiation between yeasts and moulds.

Culture media for the food industry

Horizontal method for the enumeration of yeasts and moulds Part 2: Colony count technique in products with water activity less than or equal to 0.95 - ISO 21527-2, 2008

Spread 0.1 mL of the prepared sample (or the sample if it is liquid) onto the surface of a DG 18 plate. (Dichloran with 18% glycerol agar : AEB611276 – Pack of 6 flasks of 100mL) or 0.1 mL on 3 plates (when low contamination is estimated). Carry out in the same way with the following dilutions.

DG18 For in vitro use only Store between 2 and 25°C

PRINCIPLE Agar DG18 allows the enumeration of yeasts and moulds in the foodstuffs intended for human consumption strongly sweetened or salted, or dry (flours, biscuits, ...) and in the products intended for the animal feeds (low mustered cereals and foods). The growth of the bacteria is inhibited by the combined action of glycerol - who allows to reduce the activity of the water in the Agar from 0,99 to 0,95 - and chloramphenicol. Dichloran limit the spreading out of filamentous fungi colonies and restricts the size most colonies, making easier the enumeration of yeasts and moulds.

PACKAGING Ready to use medium AEB611276 : Pack of 6 flasks of 100 ml Made by AES CHEMUNEX - Combourg - France 611276: 29/07/09 -C

FORMULA In grams per litre of purified water Peptone Glucose Potassium dihydrogenophosphate Sulfate de magnesium, 7H2O Dichloran (dichloro-2,6-nitro-4-aniline) Glycerol Chloramphenicol Agar Final pH at 25°C: 5,6 +/- 0,2

5,0 10,0 1,0 0,5 0,002 220,0 0,1 15,0

PROCEDURE Liquefy the medium in a boiling water bath, then cool the medium to 44-47°C before dispatching into Petri plates. Onto a DG agar plate, spread 0,1 ml of the product to be tested for the fluid samples or 0,1 ml of the initial suspension for the solid samples. On a second plate -1 transfer 0,1 ml of the first dilution (10 ) for fluid samples -2 or 0,1ml of the 10 dilution for solid samples. Incubate the plates in an upright position at 25°C + 1°C under aerobic atmosphere for 5 days. RESULTS Count the yeasts and moulds between 2 and 7 days of incubation, on the plates containing less than 150 colonies. BIBLIOGRAPHY 1.

Hocking, A.D. and Pitt, J.I. (1980). Dicloranglycerol medium for enumaration of xerophilic fungi from low moisture foods. Appl. Environm.Microbiol. 39, 488-492 NF ISO 21527-2 : novembre 2008 : Microbiologie des aliments – Méthode horizontale pour le dénombrement des levures et moisissures. Partie 2 : technique par comptage des colonies dans les produits à activité d’eau inférieure ou égale à 0,95

Gram -

Culture media for the food industry

Horizontal method for the detection and enumeration of Enterobacteriaceae Part 1: colonies enumeration method NF EN ISO 21528-1 standard - 2004 Sample (1g or 1ml) + 9ml of Buffered Peptone Water or Sample (Xg or Xml) + 9ml of Buffered Peptone Water

Incubation 18 hours +/- 2h at 37째C

1ml of culture + 10ml of EE broth

Incubation 24 hours +/- 2h at 37째C

Subculture onto VRBG

Incubation 24 hours +/- 2h at 37째C

Select the typical colonies and isolate on a nutritive agar plate

Incubation 24 hours +/- 2h at 37째C

Confirm the Enterobacteriaceae with: - Oxidase reaction (-) - Glucose fermentation (+) 30

Culture media for the food industry

Horizontal method for the detection and enumeration of Enterobacteriaceae Part 2: colonies enumeration method NF EN ISO 21528-2 standard - 2004

Preparation and dilution of the sample according to the ISO 6887 and ISO 8261 standards recommendations

Inoculation of 1 ml of sample or its decimal dilutions in VRBG agar by using pour plates technique (double layer) Incubation 24+/-2 hours at 37+/-1째C

Select 5 typical colonies (pink, red or purple with or without a halo of precipitation) and inoculate on a nutritive agar Incubation 24+/-2 hours at 37+/-1째C

Select one colony on each plate and detect their oxidase. Subculture the colonies with negative oxidase on an agar containing glucose (AEB120129A). Incubation 24+/-2 hours at 37+/-1째C

Enumerate all the typical colonies confirmed as negative oxidase or positive glucose

EE BROTH MOSSEL Enrichment broth for Enterobacteria according to Mossel In Vitro use only Store between 2 and 25°C

DESCRIPTION This liquid medium whose formulation fits with Mossel recommendations, is used for the selective enrichment of Enterobacteriaceae for the bacteriological control of 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.

PACKAGING Ready to use medium AEB111380: Pack of 100 Tubes of 10 ml Made by AES CHEMUNEX - Combourg – France 111380£ : 12/10/06 - A

FORMULA In grams per liter of purified water Enzyme digest of animal tissus Ox Bile Glucose Disodium phosphate Monopotassium phosphate Brilliant green

10,00 20,00 5,00 6,45 2,00 0,0135

Final pH : 7,2 + 0,2 at 25°C PROCEDURE In the frame word of the MPN technique inoculate the appropriate number of tubes of EE broth with the enriched buffered peptone water. (See ISO 21528-1 standard). Homogenise well. Incubate at 37 or 30°C for RESULTS When the medium becomes cloudy or change colour (yellow / green), it reveals the presumptive presence of Enterobacteriaceae. 24±2 hours. A final diagnosis should be obtained with an isolation on a selective media such as VRGB. 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. NF ISO 21528-1 (2004). Microbiologie des aliments – Méthodes horizontals pour la recherché et le dénombrement des Enterobacteriaceae – partie 1 : Recherche et dénombrement à l’aide de la technique NPP avec pré-enrichissement.

V.R.B.G. Violet crystal, neutral Red, Bile Glucose (dextrose) agar In Vitro use only

PRINCIPLE Violet crystal, neutral red bile glucose agar is used for screening and enumeration of Enterobacteria in water, dairy, and any foodstuff samples. Crystal violet and bile salts inhibit the growth of Gram positive bacteria and other Gram negative bacteria. A typical feature of Enterobacteria is the acidification of the medium due to dextrose fermentation. As a consequence the pH indicator turns to red and a precipitation of bile salts creates halos around colonies. FORMULA In grammes per litre of purified water Yeast extract Pepsic meat peptone Sodium chloride Bile salts Dextrose Neutral red Violet crystal Agar

3,00 7,00 5,00 1,50 10,00 0,03 0,002 15,00

Final pH : 7,4 + 0,2 at 25°C METHOD Suspend 41,5 grammes of powder in 1 litre of purified water. Homogenize well, then heat to boiling point until completely dissolved. DO NOT AUTOCLAVE. PROCEDURE Liquefy the medium in a boiling water bath then cool to 44-47°C. Place 1 ml of the tested product and its decimal dilutions in empty sterile Petri plates. Cover with 12 ml of the medium, homogenize well. Let the medium set then add a second layer (about 2 mm of thickness). After the second layer has set, incubate the prepared plates, reverse way up, 30 or 37°C (according to standards) for 24 hours. RESULTS Enumerate the pink to purple colonies surrounded or not by a halo of precipitation due to bile salts. The number is then expressed according to the sample of the tested product.

LIMITS AND PRECAUTIONS A classic cycle of autoclave 15 minutes at 121°C could alter the properties of the VRBG medium, nevertheless, in order to keep the ready prepared media a few days autoclave 20 minutes at 110°C. BIBLIOGRAPHY 1. Mossel D.A.A., Mengerink W.H.J. and Scholts H.H.A. 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of all Enterobacteriaceae. J. Bacteriol. 84:381. 2. Mossel D.A.A., Wisser 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 Salmonellae. J. Appl. Bact. 26:444-452. 3.NF ISO 21528-2: Décembre 2004. Méthodes horizontales pour la recherche et le dénombrement des Enterobacteriaceae. Partie 2 : Méthode par comptage des colonies 4. AFNOR NF V08-054 : Février 1999. Microbiologie des aliments. Dénombrement des entérobactéries à 30°C Méthode de routine. PACKAGING Dehydrated medium (To be stored between 1 and 30°C) AEB153202 : 500 g Ready to use medium (To be stored between 2 and 25°C) AEB623206 : Pack of 6 flasks of 100 ml AEB623207 : Pack of 6 flasks of 200 ml AEB123209 : Pack of 100 tubes of 15 ml Poured plates (To be stored between 2 and 25°C) AEB523210 : Pack of 20 plates 90 mm AEB123210C : Pack of 10 contact plates 65 mm AEB523209C : Pack of 120 contact plates 65 mm Made by : AES CHEMUNEX - Combourg - France 153202£: 26/05/08 - L

BRILLIANT GREEN Lactose Brilliant Green Bile Broth In vitro use only

DESCRIPTION The Lactose Brilliant Green Bile Broth (L.B.G.B.B.) is used for the detection, numeration and confirmation of the presence of coliforms, faecal coliforms, Escherichia coli in milk, dairy products and water. Ox bile and brilliant green, present in the medium, inhibit secondary microorganisms growth. Lactose fermentation with gas release, revealing Escherichia coli presence, is confirmed with Durham tubes. FORMULA In grams per litre of distilled water. Pastone Ox bile Lactose Brilliant green

10,00 20,00 10,00 0,0133

Final pH: 7,2 +/- 0,2 at 25°C PREPARATION Simple concentration. Pour 40 grams of powder into 1 litre of purified water. Stir gently until complete dissolution. Dispense 10ml into 16x160mm tubes with Durham tubes. Autoclave for 15 minutes at 121°C. Double concentration. Pour 80 grams of powder into 1 litre of purified water. Stir gently until complete dissolution. Dispense 13ml into 20x200mm tubes with Durham tubes. Autoclave for 15 minutes at 121°C. Make sure that the tubes do not contain air once they have cooled down. PROCEDURE MPN method Simple concentration Inoculate the tubes with 1 ml of inoculum and its decimal dilutions. Double concentration Inoculate the tubes with 10ml of inoculum. Pour several ml of sterile Pastagar B that was liquefied at 55°C. For coliforms detection, incubate at 30°C for 24 to 48 hours. For faecal coliforms detection, incubate at 44°C for 24 to 48 hours. Transfer a loop full from double concentration tubes to a simple concentration tube and incubate for 24 and 48 hours.

– Colony-count technique : Subculture 5 atypical colonies on VRBL in a BLBVB single concentration tube. Incubate at 30°C or 37°C (according to the validated procedure) for 24±2hours. Within the framework of theISO 4831 standard: standard: Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coliforms –Most probable number technique: From each tube inoculated in the first part, subculture an inoculating loop full in a BLBVB single concentration tube then incubate at 30°C or 37°C (according to the validated procedure) for 48±2hours. RESULTS Gas appearance in Durham tubes within 24 or 48 hours (according to the chosen procedure) reveals lactose fermentation and indicates the presence of coliforms. This should be confirmed with isolation and identification on an appropriate medium. LIMITS AND PRECAUTIONS Can show occasionally a slight precipitate without any incidence on the performances of the medium. BIBLIOGRAPHY 1. J.O. du 27 août 1963. Contrôle des laits concentrés sucrés et des laits secs. 2. FIL - IDF 40 : Méthode de routine normalisée pour le dénombrement des bactéries coliformes dans le lait pasteurisé. 3. J.O. du 21 septembre 1968. Méthode de prélèvement et d'analyse bactériologique des glaces et crèmes glacées. 4. FIL - IDF 62 : Crèmes glacées et glaces au lait : dénombrement des coliformes. 5. FIL - IDF 65 : Poudres de lait et de lactosérums : dénombrement des coliformes. 6. FIL - IDF 64 : Laits fermentés : dénombrement des coliformes. 7. MacKenzie E.F.W., Windle Taylor E. and Gilbert W.E. 1948. Recent experiences in the rapid identification of Bacterium coli type I. J. Gen. Microbiol. 2:197-204. 8. Norme NF EN ISO 9308-2 - 1990 – Qualité de l’eau – Recherche et dénombrement des coliformes, coliformes thermotolérants et des E.coli présumés - Méthode NPP. 9. NF ISO 4832- Juillet 2006 : Microbiologie des aliments - Méthode horizontale pour le dénombrement des coliformes. Méthode par comptage des colonies 10. NF ISO 4831- Octobre 2006 : Microbiologie des aliments - Méthode horizontale pour le dénombrement des coliformes. Technique du nombre le plus probable.

Confirmation method: Within the framework of the ISO 4832 standard: Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coliforms

BRILLIANT GREEN Lactose Brilliant Green Bile Broth In vitro use only

PRESENTATION Dehydrated medium To be stored between 1 and 30°C AEB140502 : 500 g flask Ready to use medium, with Durham tube (To be stored between 2 and 25°C) Simple concentration AEB110509 : Pack of 100 tubes of 10 ml Double concentration AEB110520 : ¨Pack of 100 tubes de 10 ml Made by AES CHEMUNEX - Combourg – France 140502: 05/01/09 –

Culture media for the food industry

Horizontal method for the enumeration of coliforms colony count technique NF ISO 4832 July 2006

Preparation and dilution of the sample according to the ISO 6887 & ISO 8261 standards

Analyze 1 ml of each dilution using poured plate method with VRBL agar (AEB623256 – 6 x 100 ml). Once the first layer is dried, pour the second one. Incubation (24 +/- 2) hours at 30 or 37 °C

Enumerate all the typical colonies (purplish, Ø > 0,5 mm, sometimes surrounded with a reddish halo Enumerate and confirm the atypical colonies (5 maximum) in a Brilliant Green broth (BLBVB - AEB110509 pack of 100 tubes of 10 ml) Incubation (24±2) hours at (30±1)°C or (37±1)°C

Consider the colonies giving gas in Durham belt as coliforms Give the result according to the enumeration of typical colonies, the not typical colonies confirmed and the dilution factors.

V.R.B.L Violet Red Bile Lactose Agar For In Vitro use only To be stored between 2 and 25°C

PRINCIPLE Violet Red Bile Lactose agar (V.R.B.L.) is used for the screening and enumeration of coliforms bacteria (Escherichia coli, Citrobacter, Klebsiella, Enterobacter) in water, dairy products, and other foodstuff. This medium is also used for the purification and isolation of presumptuous Salmonella colonies in meat samples analysis. Its formula is conform to the NF ISO 4832 standard. Crystal violet and bile salts inhibit the growth of Gram positive bacteria and other Gram negative bacteria. Lactose fermentation causes acid production, characteristic to coliforms. As a consequence the pH indicator turns to red and a precipitation of bile salts creates halos around colonies. FORMULA In grammes per litre of purified water Yeast extract Pancreatic Casein peptone Sodium chloride Bile salts Lactose Neutral Red Violet crystal Agar

3,00 7,00 5,00 1,50 10,00 0,03 0,002 15,00

pH final : 7,4 + 0,2 à 25°C METHOD Suspend 41,5 grammes of the dehydrated medium in 1 litre of purified water. Homogenize and heat to boiling point until complete dissolution. DO NOT AUTOCLAVE. PROCEDURE Liquefy the medium and cool to 45°C. Place 1 ml of the tested product and its decimal dilutions in empty sterile Petri plates. Cover with 12 ml of the medium, homogenize well. Let the medium set then add a second layer (about 2 mm of thickness). After the second layer has set, incubate the prepared plates, reverse way up, for 24 hours at 30°C for Coliforms and 44°C for faecal Coliforms.

LIMITS A classic cycle of autoclave 15 minutes at 121°C could alter the properties of the VRBL medium, nevertheless, in order to keep the ready prepared media a few days autoclave 20 minutes at 110°C. BIBLIOGRAPHY 1. Davis J.G. 1951. Milk Testing. Dairy Industries Ltd., London. 2. F.I.L.-I.D.F. 39. 1966. Méthode de routine normalisée pour le dénombrement des bactéries coliformes dans le lait cru. 3. F.I.L.-I.D.F. 62. 1971. Crèmes glacées et glaces au lait. Dénombrement des coliformes. 4. NF ISO 4832. Juillet 2006. Méthode horizontale pour le dénombrement des coliformes. Méthode par comptage des colonies. 5. AFNOR NF V08-060. Mars 1996. Dénombrement des coliformes thermotolérants par comptage des colonies à 44°C – Méthode de routine. 6. AFNOR NF V08-050. Février 1999. Microbiologie des aliments. Dénombrement des coliformes par comptage des colonies obtenues à 30°C – Méthode de routine. PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB153252 : 500 g AEB153253 : 5 kg Ready to use media To be stored between 2 and 25°C,in the dark AEB623256 : Pack of 6 flasks 100 ml AEB623257 : Pack of 6 flasks 200 ml AEB123259 : Pack of 100 flasks 15 ml Ready poured To be stored between 2 and 25°C, in the dark AEB123260C : Pack of 10 contact plates ∅ 65 mm AEB523259C : Pack of 120 contact plates ∅ 65 mm AEB523260 : Pack of 20 plates ∅ 90 mm Made by : AES CHEMUNEX - Combourg - France 153252: 28/05/08 - H

RESULTS Enumerate the pink to purple colonies of at least 0,5 mm ∅ and surrounded by halo of precipitation due to bile salts. The number is then expressed according to the sample of the tested product.

V.R.B.L – MUG V.R.B.L – MUG Agar For In Vitro use only

DESCRIPTION Violet Red Bile and Lactose agar is used for the detection and enumeration of coliforms (Escherichia col., Citrobacter, Klebsiella, Enterobacter) in water, dairy products and other foodstuffs. It may also be used for the purification and presumptive isolation of Salmonella colonies in meat products. Crystal violet and bile salts inhibit the growth of Gram positive bacteria and other Gram negative bacteria. A typical feature of coliforms is the acidification of the medium due to lactose fermentation. As a consequence the pH indicator turns to red and a precipitation of bile salts creates halos around colonies. The addition of 4-Methyl-Umbelliferyl-β-D-Glucuronide (MUG) to the medium (100mg/l), allows to distinguish Escherichia coli. The test is based on the the β-glucuronidase enzyme degrading the MUG substrate into 4methylumbelliferone that is fluorescent under an UV lamp (365 nm). This is a typical feature of Escherichia coli and other rare enterobacteria (Salmonella, Shigella, Yersinia), that is revealed after a 24 hours incubation. FORMULA In grammes per litre of purified water Yeast extract Meat peptone Sodium Chloride Bile Salts N°3 Lactose Neutral Red Crystal Violet MUG Agar

3,00 7,00 5,00 1,50 10,00 0,03 0,002 0,10 15,00

RESULTS Enumerate dark violet – red colonies with a diameter of at least 0.5mm surrounded by a halo of bile salts. Multiply the number of colonies by the dilution of the sample to determine the number of organisms in the original sample. When observed under a Wood lamp (365nm), these colonies are surrounded by a fluorescent blue area. A direct enumeration is then possible. Escherichia coli identification may be completed by the research of indole production LIMITS This medium does not have to be autoclaved. To keep it ready to use for a few days, you may autoclave it for 15 minutes at 121°C. BIBLIOGRAPHY 1. Feng P.CS and Hartman P.A. 1982. Fluorogenic Assays for immediate Confirmation of Escherichia coli Appl. Environ. Microbiol. 43:1320-1329. 2. Robison B.J. 1984. Evaluation of a Fluorogernic Assay for detection of Escherichia coli in Foods. Appl. Environ. Microbiol. 48:285-288 PRESENTATION Dehydrated medium (to be stored between 1 and 30°C) AEB1532621 : 500gr Ready to use medium (to be stored between 2 and 25°C) AEB123270C: Pack of 10 contact plates Made by : AES CHEMUNEX - Combourg - France

pH final : 7,4 + 0,2 à 25°C PREPARATION Pour 41,6 g of powder into 1 litre of distilled water. While bringing to the boil, stir slowly until powder is totally dissolved. DO NOT AUTOCLAVE.

153262£: 09/04/08-D

PROCEDURE Liquefy the medium in a boiling water bath then cool the medium to 45-47°C. Place 1ml of the product to be tested (or its decimal dilutions) into sterile Petri plates. Pour around 12ml of sterile melted medium. Mix and allow the medium to set on a cold horizontal surface. Pour a second 2mm layer of medium. Allow to solidify and then, turn the plates over for incubation. Incubate for 24 hours. For coliforms enumeration incubate at 30°C, and for fecal coliforms enumeration incubate at 44°C

Culture media for the food industry

Horizontal method for the enumeration of the beta-glucuronidasepositive Escherichia coli. Colony-count technique at 44°C using 5-bromo-4-chloro-3-indolyl beta-glucuronate NF ISO 16649-2 / NF V08-031-2 – July 2001

Prepare the mother suspension and the dilutions according to the sample

Inoculate 1 ml of the mother suspension into a Petri dish and repeat the operation for the dilutions chosen (with 2 samples). Pour 15 ml of TBX agar per plate Incubate 18-24 hours at 44+/-1°C

Enumerate the blue colonies

T.B.X. (Tryptone Bile X-Gluc) Medium for the enumeration of Escherichia coli in foodstuffs In Vitro use only

PRINCIPLE T.B.X. is a selective medium for the enumeration of Escherichia coli in Foodstuffs. Its formula complies with the ISO 16449 standard. The selectivity of the medium is due to the presence of bile salts that inhibit the growth or Gram positive. The chromogenic substrate X-GLUC (5-bromo-4-chloro-3 indolyl β-D-glucuronate) allows to screen for the presence of β-glucuronidase, this enzyme is produced by 97% of Escherichia coli strains and some other enterobacteria such as Salmonella and Shigella. FORMULA In grammme per 1 litre of purified water Tryptone Bile salts BCIG (X-Gluc) Agar

20,00 1,50 0,075 14,00

Final pH : 7,2 + 0,2 at 25°C METHOD Suspend 35,6 g of powder to 1 litre of purified water. Homogenize well, and bring to the boil under continuous homogenisation in order to dissolve the Agar. Cool to 4447°C then dispatch in tubes or flasks. Autoclave 15 minutes at 121°C.

BIBLIOGRAPHY 1. Norme NF ISO 16649-1 – août 2001 – Microbiologie des aliments – Méthode horizontale pour le dénombrement des Escherichia coli β-glucuronidase positive – Partie 2 : Technique de comptage des colonies à 44°C au moyen de membranes et de 5bromo-4-chloro-3 indolyl β-D-glucuronate. 2. Norme NF ISO 16649-2 – juillet 2001 – Microbiologie des aliments – Méthode horizontale pour le dénombrement des Escherichia coli β-glucuronidase positive – Partie 2 : Technique de comptage des colonies à 44°C au moyen de 5-bromo-4-chloro-3 indolyl β-D-glucuronate 3. Norme XP ISO/TS 16649-3 – décembre 2005 – Microbiologie des aliments – Méthode horizontale pour le dénombrement des Escherichia coli β-glucuronidase positive – Partie 2 : Technique du nombre le plus probable utilisant le 5-bromo-4-chloro-3 indolyl β-D-glucuronate PACKAGING Dehydrated culture media (to be stored between 1 and 30°C) AEB152812 : 500 g

PROCEDURE Enumeration : dispatch 1 ml of the sample or its dilutions in the bottom of a sterile Petri plate (two essays per sample). Pour 15 ml of regenerate medium, homogenize then leave to cool on a flat surface to set. Incubate the prepared plates reverse way up at 44°C for 18 to 24 hours maximum.

Ready to use medium (to be stored between 2 and 8°C) AEB622816 : Pack of 6 flasks 100 ml AEB122019 : 100 tubes of 15ml

RESULTS Escherichia coli will grow as blue colonies.

152812 : 28/04/08-C

Made by AES CHEMUNEX - Combourg - France

LIMITS & PRECAUTIONS High concentration of interfering flora (> 150 CFU/plate) can bother the enumeration. Some other strains of entrobacteria can grow as blue colonies (Salmonella, Shigella, …) Some strains of Escherichia coli, such as Escherichia coli O 157, are β-glucuronidase negative and cannot be detected on the medium.

Culture media for the food industry

Simultaneous detection of E.coli/enterobacteria â&#x20AC;&#x201C; E.coli/coliforms

REBECCA™ Rapid Enterobacteriaceae Escherichia Coli Coliform Agar Use In vitro To be stored between 2 & 8°C

ALTERNTIVE METHODS FOR AGRIBUSINESS ANALYSIS Validated by AFAQ AFNOR Certification www. afnor.fr Method REBECCA™ base or REBECCA™ + EB (Enumeration of E.coli) AFNOR VALIDATION certificate N° AES 10/06-01/08 for food and animal feeding stuffs End of validation : 17/01/2012 Method REBECCA™ + EB (Enumeration of enterobacteria) AFNOR VALIDATION certificate N° AES 10/07-01/08 For food and animal feeding stuffs End of validation : 17/01/2012

PRINCIPLE REBECCA™ is a chromogenic medium for the direct enumeration without confirmation in products for human (1) and animal consumptions and in the water of: E. coli - D-glucuronidase positive, E.coli - D-glucuronidase positive and of enterobacteria, E.coli - D-glucuronidase positive and of (2) coliformes , The enumeration of E.coli is done by the detection of βD-glucuronidase colouring the colonies in blue with or without blue halo. The screening of the other enterobacteria (not E. coli) is done by the addition to REBECCA™ base of a specific supplement that colours the colonies in pink to red (2) The medium REBECCA™ CF exist in ready to use flasks for the enumeration of both E.coli and coliforms. Coliforms expressing their β-D-galactosidase will be coloured in pink to purple due to a specific chromogenic substrate. The mixture of selective agents inhibits the growth of the interfering flora. (1)&(2) ( not in the framework of AFAQ AFNOR Certification validation) FORMULA (REBECCA base) In grammes per litre of purified water Peptones & Extracts Selective agents Chromogenic substrate Agar final pH: 7,3 + 0,2 to 25°C

18,00 5,25 0,25 14,00

FORMULA REBECCA™ CF In grammes per litre of purified water Peptones & Extracts Selective agents Chromogenic substrate Agar final pH: 7,3 + 0,2 to 25°C

18,00 5,25 0,45 14,00

PREPARATION • Dehydrated medium: See indications on the bottle. • Ready to use flasks: Regenerate REBECCA base flasks at 100°C until perfectly liquid. Cool to 44 - 47°C. Application 1 : Enumeration of E. coli Β- Dglucuronidase positive: No supplement is necessary. Application 2: Simultaneous enumeration of E. coli Β- D-glucuronidase positive and Enterobacteria (REBECCA™ supplement EB): Hydrate the supplement with 6 ml of a solution water/Ethanol (50/50) and vortex until completely dissolved. Add 1 ml of supplement per 200 ml base flasks. Homogenize gently. Application 3 : Simultaneous enumeration E. coli ΒD-glucuronidase positive and coliforms: (REBECCA™ CF): No supplement is necessary.

PROCEDURE Whatever the claim of REBECCA™ medium, several methods can be put into practice. In all the cases, to use only one plate per dilution: a) Poured plate: 1 ml of the sample or its decimal dilutions in single layer, approximately in 15ml of medium ((∅90 mm plate). b) surface inoculation: - by spreading out: of 0,1 ml of the sample or decimal dilutions. - by automated spreading using spiral. c) Membrane filtration (water – not included in validation): After filtering the sample, transfer the membrane (marking upwards) on the Agar. For this claim 55 mm plates can be also used. Incubate the plates 24 hours ± 2 hours at 37°C ±1°C.

- Supplement REBECCA™ Enterobacteriaceae (EB) Mix of selective agents and chromogenic substrates.

REBECCA™ Rapid Enterobacteriaceae Escherichia Coli Coliform Agar Use In vitro To be stored between 2 & 8°C

In all the cases, take into consideration only plates containing less than 150 typical colonies. Refer to reading and interpretation chart (Appendix 1) for analysis. Note : For poured plate method, if one suspects a strong contamination of the matrix by micro-organisms likely to invade the Agar ‘s surfaces, it can be necessary, to facilitate the reading,g to pour a double layer (approximately 5 ml) of the same medium maintained in a water bath at 44-47°C. KEEPING & STORAGE - Ready to use medium (Flasks, supplement & plates) are stored between 2 & 8°C until their expiry date. - Poured plates prepared from the dehydrated base or ready to used flasks can be kept at 2-8°C for 15 days. - Flasks prepared from the dehydrated REBECCA™ base & REBECCA™ CF can be kept one month when stored at 2-8°C. - Once regenerated, the supplement EB can be kept 24 hours at 2-8°C, or 7 days if frozen (aliquot). The supplement must not undergo more than one cycle of freezing / thawing. LIMITS & PRECAUTIONS - After regeneration, REBECCA™ EB supplement can show the presence of a slight precipitate with no consequence on the performances of the product. - REBECCA™ base or REBECCA™ CF in flasks, once liquefied can be kept in a water bath for 7 hours at 4447°C before being used. They can also undergo up to two cycles of melting procedures. - Ideally the base should be supplemented before pouring nevertheless it is possible to keep the complete medium (base + supplement) 2 hours at 44-47°C. A supplemented REBECCA™ flask cannot undergo a melting procedure. - There exist a few strains of E. coli β-D-glucuronidase negative, example: E.coli O157. - Some strains, other than E. coli have a -Dglucuronidase enzyme and therefore could grow as blue colonies. Example: Salmonella. … BIBLIOGRAPHY 1- NF ISO 16649-2 : Microbiologie des aliments – Méthode horizontale pour le dénombrement des Escherichia coli β-D-glucuronidase positive – partie 2 : Technique de comptage des colonies à 44°C au moyen de 5-bromo-4chloro-3-indolyl β-D-glucuronate. (2001).

2- NF ISO 21528-2 : Microbiologie des aliments – Méthodes horizontales pour la recherche et le dénombrement des Entérobacteriaceae – Partie 2 : méthode par comptage des colonies (2004). 3- NF ISO 4832 : Microbiologie des aliments – Méthode horizontale pour le dénombrement des coliformes – Méthode par comptage des colonies.(2006) PACKAGING Dehydrated medium (base) : (Contact us) Ready to use medium : REBECCA™ Base: AEB620027 : Pack of 6 flasks - 200 ml REBECCA™ EB supplement AEB184135 : vial for 1,2 L of REBECCA™ base REBECCA™ EB AEB520020 : Pack of 20 plates

90mm

REBECCA™ CF AEB620037: 6 Flasks of 200 ml Made by AES CHEMUNEX - Combourg – France 620027 : 17/01/08 – A

Appendix 1: Procedure and interpretation guide

MEDIUM SUPPLEMENT TO ADD TO REBECCA (BASE) INOCULATION INCUBATION

Enumeration of E. coli β-D-glucuronidase positive

Simultaneous enumeration of E.coli βD-glucuronidase positive & enterobacteria

Simultaneous enumeration of E.coli βD-glucuronidase positive and coliforms (Out of validation)

REBECCA (Base)

REBECCA Base*

REBECCA CF

None

REBECCA EB supplement*

None

See technical data sheet

24 hours ± 2 hours at 37°C±1°C

24 hours ± 2 hours at 37°C±1°C E. coli β-D-glucuronidase + : Blue colonies with or without halo

24 hours ± 2 hours at 37°C±1°C E. coli β-D-glucuronidase +: Dark blue colonies with or without

Other enterobacteria than E. coli β-Dglucuronidase +: Pink to red colonies E. coli β-D-glucuronidase + = Σ Blue colonies

Coliforms other than E. coli β-Dglucuronidase +: Pink to purple E. coli β-D-glucuronidase + = Σ Dark blue colonies

Enterobacteria = Σ Blue colonies + Σ Pink to red colonies

Coliforms = Σ Dark blue colonies + Σ Pink to purple colonies

INTERPRETATION

E. coli β-D-glucuronidase + : Blue colonies with or without halo

ENUMERATION (Method of calculation : confer with current standards)

E. coli β-D-glucuronidase + = Σ Blue colonies

Culture media for the food industry

Use of ASAP agar for Salmonella detection in food samples According to ISO 6579 – NF EN 12824 – AFNOR V 08-013 Pre-enrichment 25 g sample + 225 ml of Buffered peptone water Incubate at 37±1°C for 16 to 20 hours

Selective enrichment

0,1 ml sample + 10 ml of RVS broth

1 ml sample + 10 ml of MKTTn broth

Incubate at 41,5±1°C for 24±3 hours

Incubate at 37±1°C for 24±3 hours

Isolation

Isolate on ASAP agar and on XLD agar

Incubate at 37±1°C for 24±3 hours

Salmonella spp., Salmonella H2Snon motile, lactose+ or saccharose+ (S.arizonae, S.indiana), S.typhi, S.paratyphi A, B, C…

Incubate at 37±1°C for 24±3 hours

Klebsiella Enterobacter

Serratia

E.coli, Citrobacter, Proteus, Pseudomonas, Gram+ flora, yeasts, moulds

J + 3 : Purification (if the colonies are not well isolated) Choose 5 typical colonies on each plate. Isolate them on nutritive agar Incubate 24±3 hours at 37±1°C

RVS (Rappaport Vassiliadis Soja) Enrichment broth for Salmonella In Vitro use only To be stored between 18 and 23°C

PRINCIPLE Rappaport-Vassiliadis broth is used as a selective enrichment broth when screening for Salmonella (except S. Typhi and Paratyphi) in foods, animal feeds and biological samples (faeces). Its formula is conform to NF EN ISO 6579 and NF U 47-100. 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 litre of purified water Soy peptone 4.5 Sodium chloride 7,2 KH2PO4 1.26 K2HPO4 0,18 Magnesium chloride anhydrous 13,4 Malachite Green Oxalate 0,036 Final pH : 5,2 + 0,2 at 25°C METHOD Suspend 26,6 grammes of powder in to one liter of purified water. Homogenize slowly until complete dissolution. Dispatch 10 ml per tube. Autoclave 15 minutes at 115°C. PROCEDURE When proceeding to microbiology control of foodstuff, Rappaport-Vassiliadis Soja broth is use after a enrichment in buffered peptone water. Add 0.1 mL of the pre-enriched sample to a tube of Rappaport-Vassiliadis soja broth. Homogenize well and incubate at 41,5°C for 21 to 27 hours. For clinical samples, inoculate the tubes with 0,1 ml of faeces ( or its decimal dilutions). Homogenize well and incubate at 35-37°C for 18 to 24 hours.

LIMITS AND PRECAUTIONS Store the dehydrated medium below 30°C. The powder is very hygroscopic. Keep container tightly closed. BIBLIOGRAPHY 1. Rappaport F., Konforti N. and Navon B. 1956. A new enrichment medium for certain Salmonellae. J. Clin. Pathol. 9:261-266. 2. Vassiliadis P., Pateraki E., Papiconomou N., Papadakis J.A. and Trichopoulos D. 1976. Nouveau procédé d'enrichissement de Salmonella. Ann. Microbiol. Inst. Pasteur. 127B:195-200. 3. Vassiliadis P., Trichopoulos D., Kalapothaki V. and Serie C.H. 1981. Isolation of Salmonella with the use of 100 ml of the R10 modification of Rappaport's enrichment medium. J. Hyg. Camb. 87:35-41. 4. Harvey R.W.S. and Price T.H. 1981. Comparison of Selenite F, Müller-Kauffmann Tetrathionate and Rappaport's medium for Salmonella isolation from chicken gibbets after pre-enrichment in buffered peptone water. J. Hyg. Camb. 87:219-224. 5. Van Schothorst M. and Renaud A.M. 1983. Dynamics of salmonellae isolation with modified Rappaports's medium (R10). J. Appl. Bacteriol. 54:209-215. 6. NF EN ISO 6579. Décembre 2002 - Microbiologie – Méthode horizontale pour la recherche des Salmonella spp. 7. NF U 47-100 – Février 2005 – Méthode d’analyse en santé animale – Isolement et identification de tout sérovar ou de sérovar(s) spécifié(s) de salmonelles dans l’environnement des productions animales PACKAGING Dehydrated medium AEB140862 : 500 g Ready to use medium AEB110869 : Pack of 100 tubes of 10 ml Made by AES Laboratoire - Combourg - France 140862 : 28/06/06 - B

RESULTS Foodstuff : After incubation subculture on to an adequate selective agar (XLD, ASAP, Hektoën or XLT4 Modified). Faeces studies : After incubation, subculture on to an adapted selective agar, (ASAP, D.C.L.S., Hektoën, XLT4 Modified, S.S., etc ...).

MKTTn (Müller Kauffmann tetrathionate-novobiocine) Salmonella selective enrichment broth In Vitro use only

PRINCIPLE MKTTn broth can be used as selective enrichment broth en screening for Salmonella in foodstuff. The medium is composed of sodium thiosulfate that is oxidised into tetrathionate by a solution of iodine/iodide added to the base at the last minutes. This contributes to inhibit the growth of coliforms and most all bacteria belonging to the intestinal flora. Proteus and Salmonella can reduce tetrathionate and therefore find their intake of sulfur for their growth. Calcium neutralises the formed sulfuric acid in order to prevent any drop of pH that might compromise the growth of bacteria. Brilliant green inhibit the growth of gram positive. Oxgall and novobiocine contributes to the growth of Salmonella by slowing down the growth of interfering flora. FORMULA In grammes per litre of purified water Meat extract Enzyme digest of casein Sodium chloride Calcium carbonate Sodium Thiosulfate (5H2O) Oxgall Brilliant green (pH level of the base at 25°C : 8.0 +/-0.2) Iodine/iodide Solution Iodine Potassium Iodide Purified sterile water

4,3 8,6 2,6 38,7 47,8 4,78 9,6 mg 20,00 g 25,00 g 100,00 ml

Dissolve the potassium Iodide in 5 ml of water; add iodine and heat slightly to help to dissolve. Complete to 100 ml with purified water. PREPARATION Suspend 89,2 grammes powder into 1 litre of purified water. Boil for one minute, until completely dissolved. Cool to 45-50°C then add 20 ml of iodine/iodide solution and 4 supplements of novobiocine (each previously regenerated with 5 ml of sterile purified water) homogenise well before dispatching 10 ml per tube. PROCEDURE Add to a prepared tube 1 ml of sample primly enrich in a non-selective broth (Buffered peptone water) . Homogenise well and incubate according to specific conditions of the standard. NF EN ISO 6579 Standard : 37°C +/- 1°C for 24+/-3 hours.

RESULTS After 21-27 hours, subculture onto selective agars such as (ASAP, Hektoën, X.L.D. or modified XLT4). LIMITS & PRECAUTIONS MKTTn contains large quantities of calcium that tend to sediment in the bottom of tubes and flacks. Before using the medium it is wise to suspend this precipitate. MKTT base can be stored 4 weeks maximum at 2-8°C. Complete media prepared by the laboratory can be stored 6 days at 2-8°C. This medium is used in parallel with a second selective medium, as R.V.S.. BIBLIOGRAPHY 1. Müller L. 1923. Un nouveau milieu d'enrichissement pour la recherche du bacille typhique et des paratyphiques. Comp. rend. Soc. biol. 89:434-437. 2. Kauffmann F. 1935. Weitere Erfahrungen mit dem kombinierten Anreicherung-sverfahren für Salmonellenbacillien. Z. Hyg. Infek. - Krkh. 117:26-32. 3. Norme NF EN ISO 6579 – Décembre 2002 – Microbiologie des aliments – Méthode horizontale pour la recherche des Salmonella spp. 4. Norme NF U47-100 – Juillet 2007 – Santé animale – Recherche par l'isolement et identification de tout sérovar ou de sérovar(s) spécifié(s) de salmonelles dans l'environnement des productions animales PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB140902 : 500 g Ready to use medium (The tubes provided already added with the iodine/iodide solution and novobiocine) To be stored between 2 and 8°C AEB121609 : Pack of 100 tubes of 10 ml Novobiocine Supplement (10 mg) To be stored between 2 and 8°C AEB184150 :QSP 250 ml of medium Made by AES CHEMUNEX - Combourg – France 140902£ :27/09/07 – E

NF U 47-100 Standard : 41,5°C +/- 1°C for 24+/-3 hours. The incubation can be prolonged up to 48 hours.

ASAP™ (AES Salmonella Agar Plate) Chromogenic medium for the isolation of Salmonella For In Vitro diagnosis To be stored between 2 and 8°C

DESCRIPTION ASAP™ is a selective medium for the isolation of Salmonella from foodstuffs, clinical and environment samples. The activity of the C8-esterase, which is found in all Salmonella species, is detected using a chromogenic substrate. The enzymatic activity of Salmonella is visualized by the pink to purple colouring of their colonies. Colonies’ characteristics on ASAP™ : Salmonella spp pink to purple col. Salmonella arizonae pink to purple col. Salmonella typhi pink to purple col. Salmonella H2S pink to purple col. + + Salmonella lactose or saccharose pink to purple col. E.coli white col. Citrobacter spp. white col. Proteus spp. brown col. Klebsiella spp. blue-green col. Pseudomonas spp. inhibition Gram + bacteria inhibition Yeasts an moulds inhibition FORMULA In grammes per litre of distilled water: Peptones 10 Opaque agents 10 Chromogenic and inhibitor mix 13 Agar 15 Final pH: 7.2 +/- 0.2 at 25°C PROCEDURE Foodstuffs and environment samples Inoculate the plates from an appropriate selective enrichment culture broth. The ASAP™ medium can be used within the context of ISO 6579 standard. Incubate at 37°C+/-2°C for 24h+/- 3 hours. Clinical samples The ASAP™ medium is used for stools examination. Inoculate the plates directly from the samples or from an enrichment broth. Incubate at 37°C+/-2°C for 24h+/- 3 hours.

LIMITS When used in clinical diagnostic, some stool samples can give to the agar a pink colouring especially where the sample is first inoculated. This is due to the C8-esterase in the stool. The interpretation of the results must be based on the observation of well isolated pink colonies. According to all the studies carried out on this medium, the optimal incubation when screening for Salmonella is 24 ± 3 hours at 37°C. It is possible to prolong the incubation of 24 ± 3 hours more but this could decrease the medium’s specificity. (Growth of false positive colonies). ASAP™ plates can be kept during 48 hours in the refrigerator after incubation before being read without altering the colour of colonies. Some Salmonella are C8-esterase slow consequently they will grow as not typical colonies on ASAP (White or very weakly pink). BIBLIOGRAPHY 1. J. Minet, A. Gougeon, S. Jobert and M. Cornier (2002) - User-friendly chromogenic Salmonella media in clinical routine practice – CHU de Rennes France – ASM Congress. 2. R. Reissbrodt, B. Burhardt and H. Tschäpen (2002) – Evaluation of four chromogenic Salmonella plating media – Robert KochInstitut, Wernigerode Germany. PRESENTATION Prepoured medium To be stored between 2 and 8°C AEB520090: pack of 20 dishes 90mm AEB520089: pack of 120 dishes 90mm Made by AES CHEMUNEX – Combourg – France 520090£ : 16/12/08 - H

RESULTS Salmonella give pink to purple colonies. Confirmatory tests should be carried out for positive identification.

X.L.D. ISO 6579 Xylose Lysine Desoxycholate Agar In Vitro use only To be stored between 2 and 25°C

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 sucrose 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 gram+ micro-organisms 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

3,00 5,00 3,75 7,50 7,50 1,00 5,00 6,80 0,80 0,08 13,50

Final pH : 7,4 + 0,2 at 25°C PROCEDURE Inoculate directly the dishes with the sample and/or the selective enrich sample (ex: Müller-Kauffmann broth, Selenite broth, Rappaport Vassiliadis Soja, MKTTn, MSRV). Incubate for 18 to 24 hours at 37°C.

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 & PRECAUTIONS An excess incubation end 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 sucrose very slowly. 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. ISO 6579 (2002) : Microbiologie des aliments : Méthode horizontale pour la recherche des Salmonella spp. PACKAGING Ready to use medium AEB523400 : Pack of 20 dishes 90 mm ∅ AEB523399 : Coffret de 120 boîtes de 90 mm ∅ Ready to use medium AEB623396 : Pack of 6 flasks of 100 ml Made by : AES Chemunex - Combourg - France 523400 : 24/09/07 – D

RESULTS Enterobacteria ferment very quickly the xylose (except Edwardsellia, Proteus morganii, Proteus rettgeri, Providencia, and Salmonella paratyphi A that are xylose) This allows to differentiate 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 sucrose. Moreover these acid conditions also inhibit the H2S production.

X.L.T.4 Selective medium for screening Salmonella In Vitro use only PRINCIPLE X.LT.4 modified is a highly selective medium used for the isolation of Salmonella (except Salmonella Typhi and other related species). Most of selective culture media for Salmonella isolation allow the growth of interfering species such as Proteus, Providencia and Pseudomonas that can mask the detection of Salmonella. The modifications brought to this medium prevent the growth of interfering flora in favour of the growth of pathogens such as Salmonella Enteritidis. 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. The selectivity of the medium is increased by the addition of SEMUS ¼ supplement (Sodium-7-Ethyl-2-Méthyl-4Undecyl Sulfate at 27%). FORMULA In grammes per litre of purified water Special peptone Yeast Extract L-Lysine Xylose Saccharose Lactose Sodium Chloride Sodium thiosulfate Ferric Ammonium Citrate Phenol Red Agar

1,60 3,00 5,00 3,75 7,50 7,50 5,00 6,80 0,80 0,08 18,00

Final pH : 7,4 + 0,2 at 25°C PREPARATION Suspend 59,0 grammes of powder in 1 litre of purified water. Add 4,6 ml of SEMUS ¼ selective supplement. Heat slowly under constant agitation until the agar is completely dissolved. DO NOT OVERHEAT. DO NOT AUTOCLAVE. It is important not to boil the medium. As soon as the medium is dissolved cool to 50°C then pour into sterile Petri plates. PROCEDURE The incorporation of L-Lysine in the medium allows the differentiation of Salmonella from non pathogenic germs fermenting glucose. As soon as xylose is exhausted, Salmonella then decarboxylate lysine causing reversion to alkaline conditions (red colonies). Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions (black centred colonies).

Alkaline conditions reversion by other lysine-positive organisms is prevented by excess acid production from fermentation of lactose and saccharose. Moreover these acid conditions also inhibit the H2S production. Inoculate directly the plates with the sample and/or the selective enriched sample (MKTTn, Selenite Cystine…). Incubate for 18 to 24 hours at 37°C under aerobic conditions. If incubation is prolonged over 24 hours, the acid formed by the fermentation of sugars scatter over the plate causing on a general pH raise and a colour change of the pH indicator. RESULTS H2S+ Salmonella grow as pinkish to red black centred colonies with yellow periphery. H2S- Salmonella grow as pink to red colonies. Citrobacter are yellow opaque colonies, so are Enterobacter and Escherichia coli colonies if ever they succeed to grow on to the plates. Providencia, Proteus, Pseudomonas, Yersinia and Acinetobacter are markedly to completely inhibited. 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 and causing the dissolution of the H2S precipitate. Under those circumstances all red colonies should be considered as suspect colonies. Confirmation tests are then carried out. When samples are badly contaminated with Pseudomonas, these microorganisms manage to grow on XLT4 plates, they then show up as small red colonies. BIBLIOGRAPHY 1. Miller R.G. and C.R. Tate. 1990. XLT4: A highly selective plating medium for the isolation of Salmonella. The Maryland Poultryman. 4:2-7. 2. Miller R.G., C.R. Tate, E.T. Mallison and J.A. Scherrer. 1991. Xylose-Lysine-Tergitol 4 : An improved selective agar medium for the isolation of Salmonella. Poultry Science. 70:2429-2432. PACKAGING Dehydrated agar base Store between 1 and 30°C AEB153412: 500 g Supplement SEMUS ¼: Store between 2 and 8°C AEB180950: QSP 10 Litres Ready to use medium Store between 2 and 8°C AEB523420: Pack of 20 plates 90 mm ∅ AEB523419 : Pack of 120 plates 90 mm ∅ Made by : AES CHEMUNEX - Combourg - France 153412£:05/08/08 - F

DRIGALSKI Lactose agar for selective isolation of Enterobacteria For In Vitro use To be stored between 18 and 23°C

PRINCIPLE Drigalski is a medium used to differentiate lactose fermenting Enterobacteria from those who do not. The fermentation of the carbohydrate involves an acid production causing a colour change of the p H indicator, Bromothymol Blue, from blue to yellow. Violet crystal the growth of Gram positive strains. Drigalski is used to isolate Gram negative bacteria from urine and other samples. Being an none selective medium for Enterobacteria this makes it an ideal medium to screen all Pathogen Escherichia coli in baby’s stools. FORMULA In grammes per litre of purified water Pastone Meat extract Yeast extract Sodium Thiosulfate Bile salt Lactose Violet crystal Bromothymol blue Agar

15,00 3,00 3,00 1,00 1,00 15,00 0,005 0,08 12,00

PACKAGING Dehydrated medium AEB150952 : 500 g Ready to use medium AEB620957 : 6 Flasks of 200 ml Prepared plates AEB520960 : Pack of 20 plates ∅ 90 mm Prepared bi-plates : Drigalsky/Columb ANC SB AEB125970 : Pack of 10 bi-plates Made by : AES Laboratoire - Combourg - France 150952£: 31/05/05 - E

Final pH: 7,4 + 0,2 at 25°C METHOD Suspend 51,0 grammes of powder in one litre of purified water. Bring slowly to the boil under continuous homogenisation, until the medium is completely dissolved. Dispatch in tube or flasks. Autoclave 20 minutes at 115°C. PROCEDURE Liquefy the medium in boiling water then cool it to 4550°C in a water bath. Pour the medium into sterile Petri plates, let the plates set then dry them in an incubator with the lid slightly open. Spread the inoculum onto the surface of the plate. Incubate 37°C for 18 to 24 hours. RESULTS Colonies fermenting lactose grow as yellow colonies, the other are blue. Escherichia coli, Klebsiella & Enterobacter grow as yellow colonies. Other species of Enterobacteria and Pseudomonas grow as blue colonies. LIMITS & PRECAUTIONS As to prevent the spreading of Proteus strains, place two drops of alcohol in the lid of the plate before putting then in the incubator reverse way up.

WILSON AND BLAIR MODIFIED BISMUTH SULFITE AGAR In Vitro use only To be stored between 2 and 25°C

PRINCIPLE Bismuth Sulfite agar corresponds to a modification of Wilson and Blair’s original formula for selective isolation and primary identification of Salmonella, in particular Salmonella typhi, in water, foodstuff and human pathology samples. The medium is very rich in nutrients: two peptones, dextrose and meat extract. The selectivity towards Gram positive flora and Coliform bacteria is due to brilliant green. Its action is enhanced by the addition of Bismuth sulfite thus recommending to use this media when samples are suspected to be highly contaminated. Ferrous sulfate is for H2S production. When H2S is present, the iron in the formula is precipitated, giving positive cultures the characteristic brown to black colour with metallic sheen. FORMULA In grammes per litre of purified water Casein pancreatic peptone Meat peptone Meat extract Dextrose Disodium phosphate Bismuth sulfite Ferrous sulfate Brilliant green Agar

5,00 5,00 5,00 5,00 4,00 8,00 0,30 0,025 20,00

Final pH : 7,6 + 0,2 at 25°C METHOD Suspend 52 grammes of dehydrated medium in one litre of purified water. Heat gently to boiling point under continuous stirring until completely dissolved. Boil for a few minutes. DO NOT AUTOCLAVE. PROCEDURE Liquefy the medium then cool down to 45-50°C. homogenise well the medium to disperse the formed precipitate. Dispatch 20 ml by sterile Petri plates. Let the dishes set on a cold horizontal surface. Dry the plates in the incubator with the lid slightly open. Usually Bismuth Sulfite agar is used by isolating directly the sample or an enrichment broth onto its surface. Incubate the plates at 37°C for 24 to 48 hours. RESULTS S. typhi : Black centred colonies with black sheen, after 18-24 hours of incubation. S. derby, S. enteritidis, S. schottmuelleri : Black colonies.

S. choleraesuis, S. gallinarum, S. paratyphi : Green colonies. Shigella : Partial or even total inhibition (Brown or Greene colonies). LIMITS AND PRECAUTIONS It is recommended to inoculate in parallel less selective media as D.C.L. according to Hynes, D.C.L.S. agar, Hektoën agar, Mac Conkey agar with violet cristal, Lactose phenol red and brilliant green agar according to Kristensen or X.L.D. agar. It is recommended to use the medium when poured in Petri dishes only the day of its preparation, especially if the sample to be controlled is suspected to be highly contaminated. After to be stored 3 to 4 days at 4°C, the inhibition properties of the medium are strongly reduced, so it will be suitable for the weakly contaminated products. BIBLIOGRAPHY 1. Wilson W.J. and Blair E.M. 1926. A combination of bismuth and sodium sulphites affording an enrichment and selective medium for the typhoid-paratyphoid groups of bacteria. J. Pathol. Bacteriol. 29:310-311. 2. Wilson W.J. and Blair E.M. 1927. Use of glucose bismuth sulphite iron medium for the isolation of Bacillus typhosus and Bacillus proteus. J. Hyg.Camb. 26:374391. 3. Wilson W.J. and Blair E.W. 1937. Further experience of the bismuth sulphite media in the isolation of Bacillus typhosus and Bacillus paratyphosus B from faeces, sewage and water. J. Hyg. Camb. 31:138-161. 4. D'Aoust J.Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microb. 5:122-124. 5. AFNOR VO4-015. Février 1984. Laits de conserve Microbiologie - Dénombrement des levures et moisissures. 6. Pharmacopée américaine – Milieu XV PACKAGING Ready to use medium AEB623296 : Coffret de 6 flacons de 100 ml Made by : AES CHEMUNEX - Combourg - France 153292£ : 18/11/08 - G

EDEL-KAMPELMACHER BRILLIANT GREEN AGAR ISO In Vitro use only Store between 2 and 25°C

PRINCIPLE Originally described by Kristensen (1925), then Kauffmann (1935), Brilliant Green Phenol Red Lactose agar known as B.G.A. (Brilliant Green Agar), is used to isolate Salmonella from any type of samples. The formula was modified by a Dutch research team from Rijks Instituut voor de Volksgezondheid Utrecht, and was given the name "Edel-Kampelmacher". This new version of B.G.A. is especially used for a selective isolation of Salmonella from foodstuff (human and animal consumption). Its efficiency had made it an obligatory step in AFNOR NF V 08-013 (September 1990) general standard for Salmonella detection. This medium has a better inhibition towards Escherichia coli and Proteus, slows the growth of Pseudomonas, but more importantly has an improved recovery for Salmonella. Salmonella do not ferment lactose nor sucrose, the two sources of carbohydrate in the medium, this allows to differentiate Salmonella from other germs that ferment either one of the sugars. Finally, brilliant green inhibits the growth of secondary Gram positive flora. FORMULA In grammes per litre of purified water Peptone Beef extract Lactose Sucrose Yeast extract Disodium Hydrogen Phosphate Sodium Dihydrogen Phosphate Phenol red Brilliant green Agar

10,00 5,00 10,00 10,00 3,00 1,00 0,60 0,09 0,0047 13,00

Final pH : 6,9 + 0,2 at 25°C METHOD Suspend 52,7 grammes of dehydrated medium in one litre of purified water. Heat gently to boiling point under continuous stirring until completely dissolved. Boil for a few minutes. DO NOT AUTOCLAVE.

RESULTS Characterize the colonies : Salmonella (lactose et sucrose -) : red colonies of 1-1,5 mm ∅, surrounded by a red zone. S. typhi may grow as pink to red convex colonies of 1 mm ∅. Citrobacter, Enterobacter, E. coli, Klebsiella (lactose and/or sucrose +) : germs partially inhibited, if growth they will show up as yellowy green colonies. Proteus : germs virtually totally inhibited, might grow as red colonies. Pseudomonas : Low growth, small red colonies. LIMITS AND PRÉCAUTIONS All suspect colonies have to undergo biochemical confirmation tests (T.S.I. agar, X.L.D., UREA CHRISTENSEN, ONPG disk, CLARK ET LUBS) from fresh cultures. BIBLIOGRAPHY. 1. Kristensen, Lester and Jurgens. 1925. Br. J. Exp. Pathol. 6:291. 2. Edel W. and Kampelmacher E.H. 1968. Comparative studies on Salmonella isolations in eight European laboratories. Bull. Wld. Hlth. Org. 39:487-491. 3. Edel W. and Kampelmacher E.H. 1969. Salmonella isolations in 9 European laboratories using a standardized technique. Bull. Wld. Hlth. Org. 41:297-306. PACKAGING Dehydrated medium (To be stored between 1 et 30°C) AEB151492 : 500 g Ready to use medium AEB621497 : Pack of 6 flasks of 200 ml Ready poured medium AEB521500 : Pack of 20 plates 90 mm ∅ Made by : AES CHEMUNEX - Combourg - France 151492£:12/12/07 -F

PROCEDURE Liquefy the medium then cool down to 45-50°C. Dispatch in sterile Petri plates. Let the plates set on a cold horizontal surface. Dry the plates in the incubator with the lid slightly open. Using an inoculating loop inoculate the plate by isolation streaks with a sample from a selective enrichment broth (Rappaport-Vassiliadis-Soy broth and MKTTn broth). Incubate at 37°C for 48 hours.

KLIGLER-HAJNA Kligler-Hajna Agar In Vitro use only To be stored between 18 & 23°C

PRINCIPLE Kligler-Hajna Agar is used to differentiate the different species of Enterobacteria by identifying their ability of fermenting glucose, with or without producing gas, to screen for lactose fermenting and/or hydrogen sulfide (H2S) production. FORMULA In grammes per litre of purified water Tryptone Yeast extract Meat extract Dextrose Lactose Sodium chloride Sodium thiosulfate Ferrous citrate Phenol red Agar

20,00 3,00 3,00 1,00 10,00 5,00 0,50 0,50 0,025 15,00

Final pH : 7,4 +/- 0,2 at 25°C METHOD Suspend 58 grammes of powder in 1 litre of purified water. Bring slowly to the boil under constant homogenisation until the Agar is completely dissolved.. Dispatch in tubes. Autoclace at 121°C for 15 minutes.

Cool in slanted position, as to have a slant and a butt of about 2 to 3 cm . PROCEDURE With an inoculating needle, prick the center of wellisolated colonies obtained from a solid media. Stab the center of the medium into the deep of the tube to 3 – 5 mm from the bottom. Withdraw the needle and streak the surface of the slant. Loosen closure on the tube before incubating 24 to 48 hours at 37°C. Read tubes for acid production of slant/butt, gas and hydrogen sulfide reactions. RESULTS An alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only. An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose. An alkaline slant-alkaline butt (red/red) indicates that neither dextrose nor lactose was fermented (nonfermentation). Cracks, plits, or bubbles in the medium indicate gas production. A black precipitate in the butt indicates hydrogen sulfide production.

Citrobacter freundii Edwarsiella Enterobacter Escherichia coli Hafnia alvei Klebsiella Morganella morganii Proteus mirabilis Proteus rettgeri Proteus vulgaris Providencia Salmonella choleraesuis Salmonella enteritidis Salmonella gallinarum Salmonella paratyphi A Salmonella paratyphi B Salmonella pullorum Salmonella typhi Salmonella typhimurium Serratia marcescens Shighella

GLU. + + + + + + + + + + + + + + + + + + + + +

LAC. + + + + + -

H2S + + + + + + + + + -

GAS + + + + + + + + + + + + + + + + -

LIMITS & PRECAUTIONS Some strains of E. coli only ferment very lately lactose. In event of suspicion of Salmonella O.N.P.G. and L.D.C. test can be carried out from culture grown on KliglerHajna medium. 8 days old prepared tubes (in laboratories) should be regenerated before being used. BIBLIOGRAPHY 1. Kligler I.J. 1917. A simple medium for the differentiation of typhoid-paratyphoid group. Amer. J. Pub. Hlth. 7:1042-1044. 2. Kligler I.J. 1918. Modification of culture media used in the isolation and differentiation of typhoid, dysentery and allied bacili. J. Exper. Med. 28:319-322. 3. AFNOR V08-013. Juin 1982. Microbiologie alimentaire Directives générales pour la recherche des Salmonella. 4. AFNOR V04-015. Février 1984. Microbiologie - Laits de conserve.

PACKAGING Dehydrated medium AEB151252 : 500 g* Ready to use medium AEB121255 : 5 tubes (slant) AEB121259 : Pack of 100 tubes (slant)* Made by : AES Laboratoire - Combourg - France 151252£:07/12/03-C * : Product not CE stamped.

NUTRIENT ISO 6579 Nutrient Agar ISO 6579 In Vitro use only Store between 2 & 25°C

PRINCIPLE This nutrient Agar complies with the subculture step of characteristics colinies described in the ISO 6579 Horizontal method for the detection of Salmonella spp. This formula can also be used in the frame work of the ISO 10273 – Horizontal method for the detection of Yersinia entrocolitica. FORMULA In gramme of purified water Beef extract Peptone Agar

3,00 5,00 15,00

Final pH : 7,0 + 0,2 à 25°C METHOD Isolation of a characteristic colony onto the surface of the plate. Incubate at an appropriate temperature depending on the microorganism. Allow sufficient period of time in order to have a good culture. PACKAGING Ready to use medium AEB521970 : Pack of 20 plates∅ 90 mm BIBLIOGRAPHY ISO 6579 : Microbiologie des aliments : Méthode horizontale pour le détection des Salmonella spp. (2002) ISO 10273 – Méthode horizontale pour la recherche de Yersinia entrocolitica présumées pathogènes. (2003). ISO 8523 – Directive générales pour la recherche des Enterobacteriaceae avec pré-enrichissement. (1992). Made by AES Chemunex - Combourg - France 521910 : 12/10/06 – A

Culture media for the food industry

SMS  method (Simple method for Salmonella) AFNOR VALIDATION Certificate N° AES 10/04-05/04 for food and animal feeding stuffs and environmental samples. Limit of validation (07/05/2012)

Protocol for Salmonella detection DAY 0 : Pre-enrichment

method

25 g sample + 225 ml Buffered Peptone Water

Incubation 16 to 20 h at 37°C+/-1°C

DAY 1 : Inoculation

3 x 0,1 ml in 3 points near the side of the Petri dish

Incubation 14 to 24 h at 41°C +/- 1°C

DAY 2 : Results

Negative: no migration or migration area < 2 cm

520069 : 27/03/08 Q

Plates presenting typical colonies within 24 h are considered positive.

Presumptive positive: red migration area ≥ 2 cm

Choice of confirmation: - by any classical test from international standards - SMS Confirmation (2 options) - By any other AFNOR VALIDATION certified alternative method on a principle different from SMS

S.M.S™ (Simple Method for Salmonella) Selective medium for the detection of Salmonella In Vitro use only

ALTERNTIVE METHODS FOR AGRIBUSINESS ANALYSIS Certified by AFNOR Certification www. Afnor-validation.com SMS  method (Simple method for Salmonella) AFNOR VALIDATION Certificate N° AES 10/04-05/04 for food and animal feeding stuffs and environmental samples. Limit of validation (07/05/2012)

PRINCIPLE SMS medium is used for the detection of Salmonella spp. in food, veterinary and environmental samples. SMS principle lies on the detection of Salmonella ability to decarboxylate L-Lysine and more importantly their motility. On SMS, Salmonella produce a red and opaque halo of migration around the original point of inoculation. The medium selective agents and a 41°C incubation give to SMS a strong selectivity. The gelling base of the medium was especially optimised to authorize easy transport and handling of ready poured medium while ensuring an optimal migration of the motile Salmonella (deposited patent). PREPARATION Dehydrated medium: preparation of the media using the dehydrated base and supplements: please refer to annex I. Ready to use flasks : DO NOT BOIL THE FLASKS. Place the flasks in a water bath for at least 30 minutes at 47-50°C. After regeneration, poor 16ml (+/- 1ml) into 90 mm plates. Place the dishes on flat surface and let the plates set. The prepared plates can be used immediately or can be stored for one week at 2-8°C before to being used. PROCEDURE SMS method (food, animal feeding stuffs and environmental samples) – Refer to annex II : • The SMS is inoculated after a pre-enrichment phase of 16 to 20 hours at (37±1)°C in buffered peptone water or any other diluent required by the screened sample (as described in the standards). • Inoculate aseptically 0.1 ml of the broth culture at 5 mm from the edge of a SMS plate. This part of the procedure is essential since capillarity forces could occur thus leading to unwanted spread of the specimen. Repeat this step twice as to form a triangle on the SMS plate with the 3 inoculated drops.

• Incubate at (41±1)°C, do not turn over the plate. The incubation of the plate should not exceed 24 hours. The plate can be read before the end of the incubation (as from 14 hours of incubation). In presence of a typical profile of Salmonella (see results), it is not necessary to continue the incubation up to 24 hours before carrying out the confirmation tests. Otherwise, plates must be incubated until the end of the 24 hours (+/- 1hour) before a second reading. RESULTS On SMS, Salmonella produce arcs of migration superior to 2 cm (from the inoculation point). These arcs usually induce colour change of the medium to red and are preceded by a turbid zone easily observable against the light. Plates which present this profile have to be regarded as presumptively positive and will have to be submitted to confirmation tests. Plates which do not present that profile (absence of migration) have to be considered as negative (absence of Salmonella in the analysed sample). Within the framework of the AFNOR VALIDATION label (SMS method), all positive results have to be confirmed by one of the following methods : 1 - By tests described in the standardized methods. An initial isolation and purification on a suitable selective agar is necessary. Proceed by taking directly a sample of growth from the migration edge using an inoculating loop or a closed pipette. 2 – By the two protocols of the SMS CONFIRMATION method (See SMS CONFIRMATION technical data sheet) 3 - By any other AFNOR VALIDATION validated method from which the principle is different from the SMS method. The full procedure of this second validated method will have to be followed. If this method has common steps with SMS method, start the procedure at the last common step to both methods. (for example, buffered peptone water). Conservation of incubated buffered peptone water cannot exceed 48 hours. In the event of discordant results (positive result with SMS method not confirmed by the selected confirmation method), the laboratory must carry out sufficient means to be ensured of the validity of the result. Note: It is also possible to confirm presumptive positive plates by using MUCAP test (OUT OF THE FRAMEWORK OF AFNOR VALIDATION LABEL). Tests carried out throughout the trial periods, preliminary and collaborative studies, showed 100 % specificity and sensitivity for these tests carried out during ISO 16140 procedure. (A report is available upon request).

S.M.S™ (Simple Method for Salmonella) Selective medium for the detection of Salmonella In Vitro use only

PROCEDURE LIMITATIONS • Non motile Salmonella cannot be detected with SMS method. These Salmonella are non pathogenic and very seldom met in foodstuffs. • It is not advised to use cold SMS plates. It is wise to take out the plates 15 minutes before proceeding to the analysis, as to let the plates acclimatise to the room temperature. • In order to prevent any unwanted spreading of the sample, it is wise to wait 15 minutes after the inoculation before transferring the plates into the incubator. • The plates of SMS must be handled with care after their incubation. The high incubation temperature tends to slightly liquefy the gelling base. It is strongly recommended to maintain the plates a few minutes at room temperature before their examination. • Any plate showing a migration zone superior to 2 cm extending out from the inoculated drop, must be considered as presumptively positive and undergo a confirmation test. The absence of colour change is due to the fact that some Salmonella have L-lysine decarboxylase enzyme in fewer numbers making it virtually impossible to have a variation of the pH indicator. • As to help the reading of the plates, particularly those that have totally turned red, we advise to compare them to a negative or not inoculated one. • Presumptive positive plates are characterised by strong opacity due to the migration of Salmonella in the medium. • After incubation, SMS plates can be stored at refrigerated temperatures (48 hours maximum) before interpretation and confirmation.

PACKAGING Pre-poured medium AEB520069 : Pack of 120 plates (90 mm diameter) AEB520070: Pack of 20 plates (90 mm diameter) Ready to use complete medium AEB620067 : Pack of 6 flasks 200ml (for about 72 tests) Dehydrated media and supplements: AEB141062: SMS base – 500 g (570 tests) AEB684155 : SMS lyophilised supplement Pack of 6 vials (for 5 litres of medium) AEB180252 : Ready to use SMS supplements (Vials for 3 litres of medium) Possibility of special packaging for the supplements, please contact us. SMS  CONFIRMATION: Milieu SALSA AEB125980 : Pack of 10 bi-plates ∅ 90 mm AEB526760 : Coffret de 20 bi-plates ∅ 90 mm BHI broth : AEB110103 : Pack of 100 tubes - 3 ml Test Latex : MGNF42 : Pack of 50 Tests Mucap test* AEB191500 : Pack of 160 tests *: MUCAP TEST See technical data sheet for its use with SMS plates. Made by AES CHEMUNEX - Combourg - France 520069£ : 27/03/08 - Q

• The use of the media prepared from the dehydrated base and supplements, requires some precautions (annex I) • Within the framework of the analysis of acidifying samples such as described in the paragraph 9.1.2.2 of the ISO 6579 standard, it is necessary to assure that the pH level does not lower more than 4,5 during pre enrichment step. Such low pH level can interact on the motility of Salmonella this making the migration difficult with SMS™. The use of a buffered peptone water with double strength buffer is recommended to stabilize the pH.

Culture media for the food industry SMS  Simple Method for Salmonella 5 hours protocol

SMS inoculating Confirmation Using an loop or a closed Pasteur pipette, take a sample of 5 hours culture directly protocol in the SMS  at the edge of the migration zone.

Enrichment :

SMS Simple method for Salmonella: Validation: AES10-4 05-04

Subculture the sample from SMS in a tube of 3 ml BHI broth (AEB110103 – Pack of 100 tubes). Incubate the prepared tube 5 h ± 1 h at 37 ± 1°C. Carry out latex agglutination test directly from the enriched tube.

Latex test : Microgen Latex agglutination test for Salmonella (MGNF42 – Pack of 50 tests)

Homogenise the latex reagent before each use. Put a drop of the latex reagent in the centre of the disk on the reaction card. Add to the latex one drop of the enriched BHI broth.

Interpretation • •

Homogenise the two drops using the spatula. Rotate the card for about 2 minutes.

Disk n°1 : Positive control (Agglutination) Disk n°2 : Positive test (Agglutination) Disk n°3 : Negative test (Absence of agglutination) For each series of confirmation, carry out positive control using the control solution and negative control by testing for auto-agglutination with the saline buffer.

If a characteristic agglutination is observed within 2 minutes, the result is positive and the tested strain belongs to Salmonella. If no characteristic agglutination is observed, presence of discordant result, the laboratory must carry out sufficient means to ensure the validity of the result.

Culture media for the food industry SMS  Confirmation SMS  Simple Method for Salmonella hours protocol 2424 hours protocol Using an inoculating loop or a closed Pasteur pipette, take a sample of culture directly in the SMS  at the edge of the migration zone. SMS Simple method for Salmonella: Validation AES10-4 05-04

Inoculation :

C8 esterase+ strain (pink to purple colonies), LDC + & H2S+ (Black centred red colonies). Characteristic profile (majority of cases) of Salmonella, carry out a latex test.

Subculture the sample onto the surface of the two sides of a SALSA plate. Incubate SALSA 24h ± 2h at 37°C ±1°C.

Slow C8 esterase strain, LDC+ & H2S+ (Black centred red colonies). Characteristic profile of Salmonella, carry out a latex test. il caractéristique d’une

C8 esterase+ strain (Pink to purple colonies), Lactose+ (Yellow and H2S- colonies). Characteristic profile of Salmonella, carry out a latex test.

Absence of colonies or absence of characteristic colonies on the two sides of the bi-plate. Discordant result*.

Latex test : Microgen Latex agglutination Salmonella (MGNF42 – Pack of 50 tests)

Homogenise the latex reagent before each use. Put a drop of the latex reagent in the centre of the disk on the reaction card and a drop of saline buffer. Choose one isolated characteristic colony on either side of the bi-plate and delay it in the drop of saline buffer.

Homogenise the two drops using the spatula. Rotate the card for about 2 minutes.

Interpretation Profile 1 2 3 4* 5*

XLD ASAP  Pink Red + H2S Not typical Red + H2S Pink Not typical Profile 1, 2 or 3 or SALSA Absence of typical colony

LATEX + + + NA

Conclusion Salmonella Salmonella Salmonella Discordant Discordant

*: In the event of discordant results (presumptive positive SMS and not typical within the frame work of SMS CONFIRMATION 24 hours protocol), the laboratory must carry out sufficient means to ensure the validity of the result. 125980 : 02/02/07 - A

saline buffer. Once the colony is dissociated add CONFIRMATION on drop ofSMS™ the latex solution

Methods of confirmation of Salmonella after detection on SMS  In Vitro use only

PRINCIPLE SMS CONFIRMATION protocols allow to confirm the presence of Salmonella exclusively after SMS method (AFAQ AFNOR Certification: AES 10-4 05-04) option N°2. Chose between 2 options : - 5 hours protocol: by a subculture in BHI broth of a sample from a positive presumptive SMS plate incubated 5 +/-1 h and followed by a latex agglutination test. - 24 hours protocol : By subculture on a SALSA plate of a sample from a positive presumptive SMS plate and followed by a latex agglutination test. SALSA is a bi-plate composed of two selective media specific for Salmonella. ASAP (white) lies on the detection of C8-esterase cleaving specifically the chromogenic substrate thus pigmenting the colonies in pink to purple. XLD (red), Salmonella will grow as black centred red colonies due to the production of hydrogen sulphide (H2S) and Lysine decarboxylation activity. The combination of these two media enables SALSA to be highly specific (including Lactose positive or slow C8esterase Salmonella). PROCEDURE From a presumptive positive SMS plate (ie : Showing a migration superior to 2 cm from the inoculation point. These arcs usually induce colour change of the medium to red and are preceded by a turbid zone easily observable against the light) carry out confirmation test as described below: - 5 hours protocol: Using an inoculating loop or a closed Pasteur pipette, take a sample of culture directly in the SMS  at the edge of the migration zone and subculture in a tube of 3 ml of BHI broth Incubate the prepared tube 5 h ± 1 h at 37°C ± 1°C Carry out agglutination latex test directly from the BHI enriched broth. Put one drop of the broth in contact with a drop of latex reagent first prepared on the reaction card. Homogenise the two drops using the spatula then rotate the card for two minutes.

and homogenise using the spatula, then rotate the card for two minutes. RESULTS 5 hours protocol: If a characteristic agglutination is observed within two minutes, the result is positive: the incriminated strain is a Salmonella. If no agglutination is observed then presence of discordant result*. 24 hours protocol : If characteristic colonies are observed on at least one of the two sides of SALSA and the agglutination test is positive within two minutes then the result is positive: the incriminated strain is a Salmonella. The interpretation of this protocol can be summed : Profile 1

ASAP  Pink

XLD

LATEX

Red + H2S

Not typical

Red + H2S

Pink

Not typical

4* 5*

Profile 1, 2 or 3 on SALSA Absence of typical colony

Conclusio n Salmonell a Salmonell a Salmonell a Discordan t* Discordan t*

*In the event of discordant results (presumptive positive SMS and not typical within the frame work of SMS CONFIRMATION 5 hours or 24 hours protocols), the laboratory must carry out sufficient means to ensure the validity of the result. STORAGE: Microgen Salmonella latex agglutination kit and SALSA plates must be stored between 2 & 8°C until the end of their shelf life. BHI tubes can be stored at room temperature.

- 24 hours protocol: Using an inoculating loop or a closed Pasteur pipette, take a sample of culture directly in the SMS at the edge of the migration zone and subculture onto a SALSA plate. Incubate SALSA 24 h ± 2h at 37°C ± 1°C On at least one isolated typical colony present on either side of the plate, perform a latex agglutination test. From this colony, prepare on one disk of the reaction card a suspension in the

SMS™ CONFIRMATION Methods of confirmation of Salmonella after detection on SMS  In Vitro use only

LIMITS & PRECAUTIONS The two SMS CONFIRMATION protocols (5 and 24 hours) can only be used after the SMS method. SMS CONFIRMATION protocols can be used on positive presumptive SMS plates stored up to 48 hours. In all cases, always perform all controls of the Microgen Salmonella latex agglutination kit (see technical sheet). With the latex agglutination kit use only freshly grown colonies. Old colonies of Salmonella could give false negative results. It is not possible to confirm several samples on one SALSA plate. In the SMS CONFIRMATION 24 hours protocol, latex agglutination test must only be carried out on one well isolated colony on SALSA. If no isolated colony is obtained during the subculture from the SMS, carry out a second subculture on a SALSA plate from one of the sides. PACKAGING SALSA AEB125980 : Pack of 10 bi-plates ∅ 90 mm AEB526760 : Pack of 20 bi-plates ∅ 90 mm BHI broth : AEB110103 : Pack of 100 tubes - 3 ml Test Latex : MGNF42 : Pack of 50 Tests Made by : AES CHEMUNEX– Combourg – France 125980£ : 02/07/07-A

Sodium chloride Sodium thiosulfate

5,00

SALSA™ 6,80

Bi-plate for the detection of Salmonella & Shigella In Vitro use only Store between 2 & 8°C

PRINCIPLE The bi-plate associates two selective media, for the simultaneous detection of Salmonella & Shigella. On the one hand the chromogenic medium ASAP (white) that allows the detection of Salmonella by revealing the presence of C8-esterase whose activity is characterised by the staining of the colonies from pink to purple. On the other hand XLD (red) that allows the presumptive screening Salmonella & Shigella. Shigella sp. gow as red coloured colonies due to the absence of the fermentation of xylose and of hydrogen sulphide production. Salmonella grow as red back centred colonies due to L-Lysine decarboxylation en the production of hydrogen sulphide (H2S). Clinical application : SALSA is used within the framework of stools analysis of patients with diarrhoea for the simultaneous detection of Salmonella & Shigella. As to raze the level of sensitivity, for the detection of Salmonella, an enrichment broth can be used. Industrial application : SALSA integrates the SMS confirmation protocol (Solution n°2 : SMS confirmation). METHOD For an optimal use dry the plates before inoculation. Protocole pour les échantillons cliniques : Subculture the sample or its enrichment onto the surface of the two sides of the SALSA plate. Incubate at 37°C±1°C for 24 ± 3 hours. SMS confirmation 24 hours protocol : By using a sterile inoculating loop or a strilised closed glass pipette take some culture at the edge of the migration zone directly from the SMS plate. Subculture onto the 2 sides of the SALSA plate. Incubate at 37°C±1°C for 24 ± 2 hours. FORMULA In gramme per litre of purified water ASAP  Peptones Opacifying agent Chromogenic substrate and inhibitor mix Agar pH = 7,2+/- 0,2 at 25°C XLD Yeast extract L - Lysin HCl Xylose Saccharose Lactose monohydrate Sodium Desoxycholate

10 10 13 15

3,00 5,00 3,75 7,50 7,50 1,00

Ammoniacal ferric citrate Phenol red Agar pH: 7,4 + 0,2 at 25°C

0,80 0,08 13,50

RESULTS Bi-plate ASAP: Salmonella form pink colonies. Bi-plate XLD: Shigella and H2S- Salmonella form red colonies. H2S+ Salmonella form black centred red colonies. Clinical application : In the case of presence of suspect colonies, carry out the usual tests of confirmation (biochemical and immunological) starting from the typical colonies observed. Industrial application : Refer to SMS confirmation technical data sheet. LIMITS AND PRECAUTIONS A prolonged incubation of XLD medium can cause the dilution of the formed acids resulting by a colour variation of the pH indicator. Some Salmonella arizonae and Shigella sonnei can ferment lactose and give non typical colonies on XLD (yellow colonies). On ASAP medium, in clinical bacteriology, some stools can cause the Agar to take on a pinkish tint at the inoculation spot. This colouring is due to the presence of C8-esterase in these samples. The interpretation of the results must in all cases be based on the observation of the colonies. Moreover, all the studies carried out on medium ASAP show that an incubation of (24±3) hours with 37°C is optimal and sufficient for the detection of Salmonella. Any prolongation of the incubation can reduce the specificity of the medium (appearance of false positive). Some Salmonella are C8-esterase slow consequently they will grow as none typical colonies on ASAP (White or very weak pink). 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

SALSA™ Bi-plate for the detection of Salmonella & Shigella In Vitro use only Store between 2 & 8°C

4. J. Minet, A. Gougeon, S. Jobert and M. Cornier (2002) - User-friendly chromogenic Salmonella media in clinical routine practice – CHU de Rennes France – ASM Congress 5. R. Reissbrodt, B. Burhardt and H. Tschäpen (2002) – Evaluation of four chromogenic Salmonella plating media – Robert Koch-Institut, Wernigerode Germany. PACKAGING Ready to use medium AEB125980: Pack 10 Bi-plates 90 mm AEB526760: Pack 20 Bi-plates 90 mm Made by : AES CHEMUNEX - Combourg – France AEB526760 : 24/01/08

Culture media for the food industry

Horizontal method for the detection of Shigella spp NF EN ISO 21567 standard project

Dilution of 25g of sample in 225 ml of broth (Shigella + 0,5 µg/ml of novobiocine). Adjust the pH to 7,0 if necessary. Incubate in anaerobic atmosphere 16-20 hours at 41,5 +/-1°C

Isolate on a Mac Conkey agar Incubate 20 -24 hours at 37+/-1°C (Incubate an additional 24 h if the result is negative )

Colourless to pale pink and translucent colonies

Isolate on XLD agar

Isolate on Hektoen agar

Incubate 20 -24 hours at 37+/-1°C

Incubate an additional 24 h if the result is negative

Translucent colonies with a red/ cherry centre

Green and wet colonies

Purify the selected colonies on a nutritive agar Incubate 20-24 hours at 37+/-1°C

Carry out biochemical confirmations (identification test)

Carry out serological confirmations

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

RESULTS Lactose – germs grow as colourless colonies as oppose to lactose + germs that grow as pink to brick-red 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

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.

151602: 06/03/09-I * : Products not

stamped.

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 (45-57°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.

HEKTOEN Hektoen Agar In Vitro use only To be stored between 18 and 23°C

PRINCIPLE Hektoen Agar is used to isolate and differentiate pathogen Enterobacteria. The chose of peptone and the bile salts (inhibitor) makes it an ideal medium to detect Salmonella & Shigella. Proteus do not spread on this medium. The main principle of this medium relies on the fermentation of three carbohydrates : salicine, lactose and saccharose. FORMULA In grammes per litre of puriified water Meat peptone Yeast extract Bile salts Lactose Saccharose Salicine Sodium chloride Sodium thiosulfate Ferric citrate Bromothymol blue Acid Fuchsine Agar

12,00 3,00 9,00 12,00 12,00 2,00 5,00 5,00 1,50 0,064 0,10 15,00

Final pH : 7,6 + 0,2 at 25°C METHOD Suspend 76,6 grammes of powder in one litre of purified water. Bring to the boil under completely dissolved, and boil for one minute. DO NOT AUTOCLAVE. PROCEDURE Liquefy the medium then cool to 45-50°C. Poor into sterile Petri plates, let them set on a flat surface. Dry in an incubator with the lids slightly open. Inoculate the plate with the sample then incubate them 24 to 48 hours at 37°C.

LIMITS & PRECAUTIONS Some strains of Vibrio can grow on this medium as salmon yellow colonies. BIBLIOGRAPHY 1. King S. and Metzger W.I. 1968. A new plating medium for the isolation of enteric pathogens, II. Comparison of Hektoën Enteric Agar with SS and EMB Agar. Appl. Microbiol, 16:579-581. 2. Taylor W.I. and Schelhaut D. 1971. Comparison of Xylose Lysine Desoxycholate Agar, Hektoën Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with stool specimens. Appl. Microbiol. 21:3237. 3. King S. and Metzger W.I. 1968. A new plating medium for the isolation of enteric pathogens. I. Hektoën Enteric Agar. Appl. Microbiol. 16:577-578. PACKAGING Dehydrated AEB151152 : 500 g Ready to use medium AEB621157 : Flacon de 200 ml Ready to use plates AEB521160 : Coffret de 20 boîtes de 90 mm AEB52159 : Coffret de 120 boîtes de 90 mm Made by : AES Laboratoire - Combourg - France

151152: &13/03/06 -G

RESULTS Microorganisms that ferment one of the 3 carbohydrates will grow as salmon pink colonies, those who do not will grow as blue colonies. Sulfide hydrogen production is reveal by the presence of ferric citrate. Colonies characteristics : * Salmon / yellow : Arizona, Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia, Y. enterocolitica. * Black centred Salmon / yellow: Proteus vulgaris. * Black centred green colonies: Proteus mirabilis, Salmonella. * Green colonies : Morganella morganii, Proteus rettgeri, Providencia, Salmonella H2S -, Shigella. * Small blue colonies : Pseudomonas.

T.S.I Triple Iron Sugar Agar In Vitro use only

PRINCIPLE Triple Iron Sugar, also known as T.S.I., is used for differentiating gram-negative enteric bacilli based on the fermentation of dextrose, lactose and sucrose and on hydrogen sulfide production. FORMULA grammes per liter of purified water Mix of peptone Dextrose Lactose Sucrose Sodium chloride Sodium thiosulfate Ferrous sulfate Phenol red Agar

20,00 1,00 10,00 10,00 5,00 0,20 0,20 0,025 13,00

Final pH: 7,3 + 0,2 at 25°C PREPARATION Suspend 59,4 grammes in 1 liter purified water. Heat to boiling to dissolve completely. Dispense into tubes with closures. Autoclave at 118°C for 15 minutes. Cool in slanted position, as to have a slant and a butt of about 2 to 3 cm . PROCEDURE With an inoculating needle, prick the center of wellisolated colonies obtained from a solid media. Stab the center of the medium into the deep of the tube to 3 – 5 mm from the bottom. Withdraw the needle and streak the surface of the slant. Loosen closure on the tube before incubating 24 to 48 hours at 37°C. Read tubes for acid production of slant/butt, gas and hydrogen sulfide reactions. RESULTS An alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only. An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose and/or sucrose. An alkaline slant-alkaline butt (red/red) indicates that neither dextrose nor lactose was fermented (nonfermentation). Cracks, plits, or bubbles in the medium indicate gas production. A black precipitate in the butt indicates hydrogen sulfide production. REMARKS This medium, is similar to Kligler Iron Agar, it is added with sucrose that can be fermented faster than lactose by some gram-negative enteric bacilli. This allows to differentiate Proteus from Salmonella.

It is common to sample a T.S.I. and a urea broth at the same time when wanting to identify Proteus or any other species that has an active urease.

When T.S.I. has been prepared for more than a week before being used it is best to regenerate the medium by letting T.S.I. agar dissolve in hot water then cooling the tube as describe in the preparation paragraph.

Citrobacter Edwardsellia Enterobacter aerogenes Enterobacter cloacae Escherichia coli Klebsiella Proteus mirabilis Proteus vulgaris Proteus morganii Proteus rettgeri Shigella dysenteriae Shigella sonnei Salmonella typhi Salmonella paratyphi A Salmonella cholereasuis Salmonella enteritidis Salmonella typhimurium AG : A: NC : ALk : +: -:

Butt AG A/AG AG AG AG AG AG AG A/AG A A A A AG AG AG AG

Slant A A A A A A A A NC/ALC NC NC/ALC NC/ALC NC/ALC NC/ALC NC/ALC NC/ALC NC/ALC

H2S + + + + + + +

acid production (yellow) and gas acid production (yellow) No colour change (red) Alkaline reaction (red) Hydrogen sulfide production No hydrogen sulfide production

BIBLIOGRAPHY 1 American Public Health Association. 1963. Diagnostic Procedures and Reagents. 4th Ed. pp. 150 and 294-295. A.P.H.A. Inc., New York. 2. American Public Health Association. 1966. Recommended Methods for the Microbiological Examination of Foods. 2nd Ed. pp. 158 and 185, A.P.H.A. Inc., New York. 3. Hajna A.A. 1945. Triple-Sugar Iron Medium for the Identification of the Intestinal Group of Bacteria. J. Bact., 49:516-517. 4. US Pharmacopoeia. XVI medium. 5. European Pharmacopoeia. M medium.

PACKAGING Dehydrated medium (Store between 1 & 30°C) (1) DIF26540 : 500 g Prepared medium in flask (Store between 2 & 25°C) (2) AEB122909 : Pack of 100 slanted tubes Made by AES CHEMUNEX - Combourg - France 152902 : 02/08/07 - H

Culture media for food industry

Milk & Milk products Detection of Enterobacter Sakazakii ISO/PFR TS 22964, September 2004 Pre-enrichment : Weigh X g of the sample in 9 times X ml Buffered Peptone Water (BPW) (Dilubag : AEB910303/4 (4 x 3L) & AEB910305 (2 x 5L)) Incubate at (37 ± 1)°C for (18 ± 2) hours

Selective enrichment : Transfer 0,1 ml from the cultured BPW into 10 ml of mLST selective broth (AEB 110549 : 100 tubes of 10 ml). Incubate at (44 ± 0,5)°C(1) for (24 ± 2) hours. (1) : It is recommended to use a water bath as incubation temperature should never go over 44,5°C.

Selective isolation : Streak a 10 µl loopful of the incubated mLST onto the surfate ESIA (AEB520010: Pack of 20 plates Ø 90 mm). Incubate at (44 ± 1)°C for (24 ± 2) hours. Typical colonies: Green-blue, Ø 1 to 3 mm None typical colonies: white to purple (to mauve)

Typical colonies

None typical colonies: Purple to mauve colonies

Confirmation: Select 1 to 5 presumptive colonies(2) and subculture onto TSA (AEB522860 : Pack of 20 plates Ø 90 mm) Incubate at (25 ± 1)°C for (46 ± 2) hours. Examine the TSA-plates for the presence of yellow-pigmented, pursue by carrying out biochemical tests on one colony. (2) : If no yellow-pigmented colony is seen on TSA, after the first subcultured colony, continue screening with the 4 remaining presumptive colonies from ESIA.

Culture media for the food industry

How to use ESIA agar for the detection of Enterobacter sakazakii

Homogenize X g of sample In 9 X ml of ESSB broth Incubate 24±2 hours at 37±1°C

Isolate on ESIA agar Incubate 21±3 hours at 44±1°C

Presence of typical colonies (blue colonies)

Absence of typical colonies (purple colonies)

Confirmation on microwell or automated identification system

Absence of Enterobacter sakazakii in X gram of sample

ESIA Enterobacter sakazakii isolation agar In Vitro use

PRINCIPLE Enterobacter sakazakii is an enterobacteria which was blamed in cases of neonatals infections conveyed by infantile dried milk and resulting in serious enterocolitis or meningitis. It also can be responsible for hospital acquired infections. It is considered as a thermotolerant coliform, meaning that it may grow at a temperature of 44°C. Initially regarded as Enterobacter cloacae with yellow pigments, the Enterobacter sakazakii species was isolated in 1980 on the basis study relating in particular to RNA-DNA hybridation. The virulence factors of this germ remain not elucidated to this day. ESIA chromogenic Agar is used for specific detection of Enterobacter sakazakii in foodstuffs such as powdered milk. On this medium E.S. grow as blue colonies. FORMULA In grammes per litre of purified water Peptone Yeast extract Sodium chloride Sodium desoxycholate Crystal violet X-alpha-glucopyranoside Agar

7 3 5 0.6 0.002 0.15 15

Final pH : 7,0 + 0,2 à 25°C METHOD Suspend 30.8 g in 1 litre of purified water. Bring to the boil until completely disloved. Autoclave 15 minutes at 121°C. Cool to 44-47°C then pour in Petri plates. PROCEDURE Detection of Enterobacter sakazakii according to standarded procedure: Refer to ISO/TS 22964 standard.

LIMITS & PRECAUTIONS Some coliforms can grow on ES isolation Agar. They can easily be differentiated from Enterobacter sakazakii as they grow as purple colonies. BIBLIOGRAPHY 1. Simmons, B.P., Gelfand, M.S., Haas, M., Metts, L. and Ferguson, J. 1989. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect Control Hosp Epidemio. 10:398401. 2. Van Acker, J., De Smet, F., Muyldermans, G., Bougatef, A., Naessens, A. and Lauwers, S. 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J Clin Microbiol. 39:293297. 3. ISO/TS 22964 : Milk and milk products – Detection of Enterobacter sakazakii. PACKAGING Dehydrated (to be stored between 1 and 30°C) AEB150002 : 500 g Ready to use ESIA (to be stored between 2 and 25°C) AEB520010 : Pack of 20 plates 90 mm Ready to use ESSB (to be stored between 2 and 25°C) AEB611448 : Pack of 6 flasks 225 ml Made by AES CHEMUNEX - Combourg - France 520010£ : 28/04/08 - C

Detection of Enterobacter sakazakii according to ESSB/ESIA method developed by AES/CHEMUNEX): ESSB/ESIA was developed by AES/CHEMUNEX for the specific detection of Enterobacter sakazakii in foodstuffs samples, such as milk powder, and foodstuffs prepared from milk powder. ESSB broth was specially formulated as to guaranty an optimal growth of Enterobacter sakazakii, and an inhibition of interfering flora making the screening simple on the ESIA Agar. (See protocol) RESULTS Typical colonies of Enterobacter sakazaki. appear blue. Using well isolated typical colonies Enterobacter sakazakii procedure to confirmation tests.

m LST Modified Lauryl sulfate with Vancomycine In Vitro use only To be stored beteen 2 and 8

PRINCIPLE Modified Lauryl sulfate with Vancomycin (m LST) was specially formulated for the detection of Enterobacter sakazakii in the framework of the technical specification : ISO/ TS 22964 : Milk and milk products – Detection of Enterobacter sakazakii. Modifications carried out on the lauryl sulfate broth, such as increase of the sodium chloride contents on the one hand and addition of Vancomycin on the other hand associated to an incubation at 44°C, make this medium an ideal enrichment broth for Enterobacter sakasakii while inhibiting the growth of interfering germs present in this category of sample.

LIMITS AND PRECAUTIONS In the refrigerator (2-8°C), the sodium lauryl sulphate could precipitate. The reaction is reversible with increasing of temperature. This will not affect the properties of the broth.

FORMULA In grams per litre of purified water

Made by AES CHEMUNEX - Combourg - France

Tryptose Lactose Sodium chloride Potassium dihydrogen phosphate Dipotassium hydrogen phosphate Sodium lauryl sulfate Vancomycin

20,00 5,00 34,00 2,75 2,75 0,10 0.01

BIBLIOGRAPHY ISO TS 22964: Milk and milk products – Detection of Enterobacter sakazakii (2006). PACKAGING Ready to use medium AEB110549 : Pack of 100 tubes of 10 ml AEB610544 : Pack of 6 flasks of 50 ml

110549£ : 18/06/09 - E

Final pH : 6,8 + 0,2 at 25°C PROCEDURE Transfer 0.1 ml of the enrichment broth (buffered peptone water) into 10 ml of m LST broth. Incubate at 44°C +/-0.5°C for 24 hours +/- 2 hours. It is recommended to incubate the tubes in a water bath since an incubation higher than 44,5°C can cause damage during this step of the detection. RESULTS After incubation, steak a 10 µL loopful of the selective enrichment broth onto the surface of the selective isolation agar for Enterobacter sakasakii ® ESIA . Incubate the plates at 44°C +/-1°C for 24 hours +/- 2 hours. Presumptive colonies of Enterobacter sakazakii are green blue and of have a diameter of 1 to 3 mm.

Culture media for the food industry

Other culture media •

Vibrions TCBS

•

Yersinia CIN

VIBRIONS TCBS Selective medium for Vibrio cholerae For in vitro use To be stored between 2 and 25°C

PRINCIPLE The formulation of the T.C.B.S. medium (Thiosulphate Citrate Bile Salts Sucrose) corresponds to the one described by Nakanishi, then modified by Kobayashi. The medium is used for the isolation of Vibrio cholerae, Vibrio parahaemolyticus and most of the other Vibrio species. It allows to detect the V. parahaemolyticus in fishes and seafood. The thiosulfate and sodium citrate, present in high concentration (10 g/l) in the medium and the basic pH inhibit the enterobacteriacea growth. The biliary salts slow down the enterococci development and the Gram positive germs while helping the Vibrio growth. The fermentation of saccharose by the Vibrio species produces an acid production causing the pH indicators the change of colour, from blue to yellow.. FORMULA In grammes for 1 litre of purified water Yeast Extract Peptone Sodium Thiosulfate Sodium Citrate Biliary salt Saccharose Sodium Chloridea Ferric Citrate Bromothymol Blue Thymol Blue Agar

5,00 10,00 10,00 10,00 8,00 20,00 10,00 1,00 0,04 0,04 16,00

Final pH : 8,4 + 0,2 à 25°C PREPARATION Suspend 90,0 grammes of powder in 1 litre of purified water. Bring slowly to the boil, shaking until complete dissolution DO NOT AUTOCLAVE PROCEDURE Liquefy the medium in a boiling water bath then cool to around 45-50°C. Pour in sterile Petri dishes and let the medium solidify on a cold and horizontal surface. Dry the plates with an incubator, half-open lids Inoculate the medium directly with the stools, or any other sample taking suspected to contain Vibrio or from an enrichment liquid medium. Incubate at 37°C for 18 - 24 hours.

RESULTS During the first 24 hours, most of the other bacteria are totally inhibited. However, after 24 hours, some strains of Proteus and Streptococcus faecalis can appear. These colonies can be easily differentiated from those of Vibrio. Vibrio alginolyticus gives wide yellow colonies with a diameter of 3 - 5 mm. Vibrio cholerae, fluvialis et vulnificus give yellow colonies with a diameter of 2 - 3 mm. Vibrio metschnikovii gives yellow colonies with a diameter of 2 - 4 mm. Vibrio mimicus et vibrio vulnificus gives green colonies with a diameter of 2 - 3 mm. Vibrio parahaemolyticus gives blue or green colonies with a diameter of 3 - 5 mm. The small colonies which can appear (medium not yellow coloured) correspond to E. coli, Klebsiella, Salmonella typhi, Shigella, Proteus, Providencia, Pseudomonas aeruginosa. LIMITS AND PRECAUTIONS The cultures on T.C.B.S. medium must be rapidly examined after their withdrawal from the incubator. Indeed, the yellow colonies of Vibrio (for example V. cholerae) tend to change to green if the storage at room temperature is prolonged. Confirm the diagnosis of V. cholerae by the usual techniques using the specific agglutinating serums. BIBLIOGRAPHY 1. Nakanishi Y. 1963. An isolation agar medium for cholera and enteropathogenic halophilic vibrios. Modern Media 9:246. 2. McCormack W.M., De Witt W.E., Bailey P.E., Morris G.K., Socharjono P. and Gangarosa E.J. 1974. Evaluation of Thiosulphate-Citrate-Bile Salts-SucroseAgar a selective medium for the isolation of Vibrio cholerae and other pathogenic vibrios. J. Inf. Dis. 129:497-500. 3. AFNOR V08-024. 05/91. Directives générales pour la recherche des Vibrio parahaemolyticus PACKAGING Dehydrated medium AEB153182 : Flask of 500 g Made by AES CHEMUNEX - Combourg - France 153182: 17/04/08 - E

YERSINIA C.I.N Yersinia Selective Agar In vitro use only

PRINCIPLE Yersinia C.I.N. Agar (Cefsulodin Irgasan Novobiocin), complies with Schiemann description. It is used for the selective isolation of Yersinia enterocolitica in Foodstuff and clinical samples. The mix of antibiotics, and the action of inhibitors such as violet crystal, inhibits the growth of interfering flora. The fermentation of mannitol by the microorganisms causes a colour change to red of the pH indicator due to acid metabolites. This acid production also cause the desoxycholate to precipitate. FORMULA In grammes per litre of purified water Peptone Yeast extract Mannitol Sodium Pyruvate Sodium chloride Magnesium sulfate Sodium desoxycholate Irgasan Neutral red Violet crystal Agar

20,00 2,00 20,00 2,00 1,00 0,01 0,50 0,004 0,03 0,001 12,00

C.N. (Selective supplement) For 5 ml of purified water Cefsulodin 7,50 mg Novobiocin 1,25 mg Final pH : 7,4 + 0,2 at 25°C METHOD Suspend 57,5 grammes of powder in one litre of purified water. Bring slowly to the boil under constant homogenisation until completely dissolved. Boil for one minute. DO NOT AUTOCLAVE. PROCEDURE Liquefy the medium then cool to 45-50°C. Add one vial of regenerated supplement to 500 ml of prepared Agar base. Homogenise well then pour into sterile Petri plates. Inoculate de plates directly with the sample or with an enrichment culture (P.B.S: Peptone - Bile salts-Sorbitol incubated at 22-25°C for 48 to 72 hours) Incubate the plates at 30°C for 24 to 48 hours.

After 48 hours : Colonies are much bigger and can show desoxycholate precipitations zones LIMITS & PRECAUTIONS Other strains of Gram negative bacilli (Citrobacter freundii, Enterobacter agglomerans & Serratia liquefaciens) that ferment mannitol might grow on Yersinia CIN Agar, may interfere in the ready of the plates. All colonies that grow on this medium should under go biochemical confirmation tests (Kligler-Hajna, urease, saccharose, saliciline). BIBLIOGRAPHY 1. Schiemann D.A. 1979. Synthesis of a selective agar medium for Yersinia enterocolitica. Can. J. Microbiol. 25:1298-1304. 2. Devenish J.A. and Schiemann D.A. 1981. An abbreviated scheme for identification of Yersinia enterocolitica isolated from food enrichments on CIN (cefsulodin-irgasan-novobiocin) agar. Can. J. Microbiol. 27:937-941. 3. Head C.B., Whitty D.A. and Ratnam S. 1982. Comparative study of selective media for recovery of Y. enterocolitica. J. Clin. Microbiol. 16:615-621. 4. Schiemann D.A. 1982. Development of a two step enrichment procedure for recovery of Yersinia enterocolitica from food. Appl. Environ. Microbiol. 43:14-27. 5. Mossel D.A.A. 1987. Cefsulodin Irgasan Novobiocin (C.I.N.) agar. Inter. J. Food Microbiol. 5:208-209. 6. NF EN ISO 10273 – Microbiologie des aliments – Méthode horizontale pour la recherche de Yersinia enterocolitica présumées pathogènes. Déc. 2003. PACKAGING Dehydrated agar base (To be stored between 1 and 30°C) AEB153452 : 500 g CN. Selective supplement (To be stored between 2 and 8°C) AEB184011 : q.s.p. 500 ml, lyophilised* Ready to use medium (To be stored between 2 and 8°C) AEB123460 : Coffret 10 boîtes de 90 mm Fabriqué par AES CHEMUNEX - Combourg - France

RESULTS After 24 hours : Yersinia enterocolitica colonies show up as small red centred colonies with irregular and transparent edges.

153452:20/08/07 -F * : Product none CE stamped.

Gram+

Culture media for the food industry

Enumeration of Staphylococci aureus NF EN ISO 6888-1 standard ; NF EN ISO 6888-2 standard - 2003

Decimal dilution of the water sample in a Peptone salt broth (AEB111499 - pack of 100 tubes of 9 ml)

6888-1 standard

Inoculation by spreading on a Baird Parker medium (AEB520320 - 20 plates 90 mm) 0,1 ml of each dilution Incubation 24 - 48 hours at 35+/- 1°C or 37 +/- 1°C

6888-2 standard

Inoculation by incorporation or on surface of Baird Parker RPF agar (AEB520220—pack of 20 plates) with 1 ml or 0,1 ml of each dilution. Incubation 18 - 24 hours at 37+/- 1°C

Mark the typical colonies (dark or grey surrounded by a lightening halo) on the back of the plate after 16 to 24 hours of incubation Re-incubation of all the plates 22 - 24 hours at 35+/- 1°C or 37+/- 1°C

Enumeration of the typical and non typical colonies (dark without lightening halo) on the plates containing between 15 and 150 colonies and expression of the results according to the dilutions, and then selection of 5 colonies of each plate for confirmation

Enumeration of the typical colonies (colonies surrounded by an opaque or blurred zone) and expression of the results according to the dilutions.

Culture media for the food industry

Research of the Catalase on 3 selected colonies, then inoculation of a BHI broth from the positive catalase selected colonies Incubation 20 - 24 hours at 35+/- 1°C or at 37°C +/-1°C

Carry out a Coagulase test by adding 0,1 ml of the BHI broth to 0,3 ml of rabbit plasma (AEB1840088 - flask of 2,5 ml) Incubation 4 - 6 hours or up to 24 hours at 37+/- 1°C

Confirmation Check if the coagulation occured (the coagulum takes up more than 3/4 of the volume taken up by the liquid initially) If there is no coagulation, re-incubate at 37+/-1°C up to 24 hours for confirmation

Culture media for the food industry

Horizontal method for the enumeration of the coagulase-positive Staphylococci (Staphylococcus aureus and other species) Part 3: detection and MPN technique for low numbers NF EN ISO 6888-3 - June 2003

Research method

Dilute 1 g or 1 ml of sample in 9 g or 9 ml of single concentration Giolitti Cantoni (or 10g or 10 ml in 10 g or 10 ml of double concentration Giolitti Cantoni broth. Incubation 24+/-2 hours at 37°C +/-1°C with a layer of paraffin or agar OR in anaerobic atmosphere (jars, …)

Enumeration method (NPP)

Prepare a mother solution according to the ISO 6887 or ISO 8261 standards recommendations

Inoculate 3 tubes of double concentration Giolitti Cantoni broth with 10 ml of the mother solution and dilute these tubes in series. Inoculate 3 tubes of single concentration Giolitti Cantoni broth with 10 ml of the mother solution and dilute these tubes in series. Incubation 24+/-2 hours at 37+/-1°C with a layer of paraffin or agar OR in anaerobic atmosphere (jars, …)

Culture media for the food industry

Appearance of a black precipitate or broth blackening

No black precipitate or broth blackening Re-incubation 24+/-2 hours at 37+/-1°C

Subculture on Baid Parker agar Incubation 24+/-2 hours at 37°+/-1°C

Subculture on Baird Parker agar with RPF Incubation 24+/-2 hours at 37°+/-1°C

Mark the typical colonies (black to grey, brilliant and convex, surrounded with a clear halo and possibly with an opalescent ring on contact with colonies, 1 to 1,5 mm of diameter).

Presence of small black to white colonies surrounded by a precipitate halo.

Incubation 24+/-2 hours at 37+/-1°C

Mark the new typical colonies and the non typical colonies (black and brilliant colonies without bright area or opalescent ring or grey colonies without bright area.

Carry out a confirmation test with rabbit plasma on each marked colony. If one of the confirmation is positive:

Presence of coagulase-positive Staphylococcus in the sample analyzed

Calculate the result of the enumeration according to the number of positive tubes per dilution and to the MPN technique

BAIRD –PARKER Base for Baird-Parker Agar In Vitro use only

DESCRIPTION Baird Parker Agar is a highly specific and selective medium for isolation and enumeration of coagulasepositive staphylococci from food. It may also be used for identification of staphylococci on the basis of their ability to clear egg yolk. Baird-Parker Agar contains sodium pyruvate in order to stimulate the growth of Staphylococcus aureus without destroying the selectivity. The tellurite additive is toxic to egg yolk clearing strains other than Staphylococcus aureus and imparts a black color to the colonies. The egg yolk additive, in addition to being an enrichment, aids in the identification process by demonstrating lecithinase activity (egg yolk reaction). Glycine and lithium chloride have inhibitory action for organisms other than Staphylococcus aureus. The addition of sulfamethazine at 50 mg/l inhibit the growth of Proteus. Other microorganisms, like Bacillus, yeasts, Micrococcus, Stahylococcus epidermidis or saprophiticus may grow sparsely but give no typical colonies. Nevertheless, typical, confirmation tests, like search of free coagulase, thermonuclease or Dnase should be performed to confirm findings. FORMULA In grammes per litre of purified water Tryptone Meat extract Yeast extract Sodium pyruvate Glycine Lithium chloride Agar

10,00 5,00 1,00 10,00 12,00 5,00 17,00

Final pH : 7,2 + 0,2 at 25°C Final pH: 6,8 ± 0.2 at 25°C for Baird Parker used in Pharmacopoeia. PREPARATION Suspend 60 g of the powder in 1 litre of purified water. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. Dispense and sterilize by autoclaving at 121°C for 15 minutes. PROCEDURE Liquefied the medium in a boiling water bath then cool to 45-50°C and add 50 ml of sterile tellurite yolk egg solution and if needed, 25 ml of 0,2 % sterile sulfamethazin solution. Mix thoroughly but gently and pour into sterile Petri plates. Samples can be inoculated directly onto the surface of a plate or on after an enrichment step (chapman broth or Giolitti cantoni). Incubate plates at 37°C for 24 hours and 48 hours if no typical colony grows in 24 hours.

RESULTS Typical colonies of Staphylococcus aureus are black, shiny, convex and with a diameter of 1 to 1,5 mm. They are surrounded by: • a clear halo due to the action of a lipoproteinase • opaque zones, due to the action of a lecithinase, which appears in the clear halo, sometimes only after 48 hours of incubation. LIMITS AND PRECAUTIONS Addition of sulfamethazin is recommended in order to inhibit the growth of Proteus. Bacillus give brown colonies, yeast white colonies and Micrococcus brown to black colonies all with no typical clear zone. Coagulase-negative Staphylococcus are inhibited but may grow sparsely. They give more irregular colonies with an opaque halo and, very rarely, a clear zone. Plates prepared by the laboratory must be used within two days. BIBLIOGRAPHY 1. Zebovitz E., Evans J.B. and Niven C.F. Jr. 1955. Tellurite-glycine agar, a selective plating medium for the quantitative detection of coagulase positive staphylococci. J. Bacteriol. 70:686-690. 2. Baird-Parker A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. J. Appl. Bacteriol. 25:12-19. 3. Baird-Parker A.C. 1963. A classification of micrococci and staphylococci based on physiological and biochemical tests. J. Microbiol. 30, 409-427. 4. Smith B.A. and Baird-Parker A.C. 1964. The use of sulphamethazine for inhibiting Proteus spp. on Baird-Parker's isolation medium for Staphylococcus aureus. J. Appl. Bacteriol. 27:78-82. 5. Baird-Parker A.C. and Davenport E. 1965. The effect of recovery medium on the isolation of Staphylococcus aureus after heat treatment and after storage of frozen or dried cells. J. Appl. Bacteriol. 28:390-402. 6. AFNOR V08-014-1 ISO 6888-1 Octobre 1999. Microbiologie des aliments : méthode horizontale pour le dénombrement de Staphylocoques à coagulase positive. (Staphylococcus aureus et autres espèces) partie 1 : Technique utilisant le milieu gélosé de Baird-Parker. 7. AFNOR V08-014-2 ISO 6888-2 Octobre 1999. Microbiologie des aliments : méthode horizontale pour le dénombrement de Staphylocoques à coagulase positive. (Staphylococcus aureus et autres espèces) partie 2 : Technique utilisant le milieu gélosé de au plasma de lapin et fibrinogène. 8. AFNOR V08-014-3 ISO 6888-3 Juin 2003. Microbiologie des aliments : méthode horizontale pour le dénombrement de Staphylocoques à coagulase positive. (Staphylococcus aureus et autres espèces) partie 3 : Recherche et méthode NPP pour les faibles nombres 9. European Pharmacopoeia. O medium.

BAIRD –PARKER Base for Baird-Parker Agar In Vitro use only

PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB150302 : 500 g Ready to use base. To be stored between 2 and 25°C AEB620316 : 6 flasks of 100 ml AEB620317 : 6 flasks of 200 ml AEB620314 : 6 flasks of 90 ml AEB620313 : 6 flasks of 180 ml Ready to use Baird Parker (Pharma.) base. To be stored between 2 and 25°C AEB620316P: pack of 6 flasks of 100 ml Ready poured medium. To be stored between 2 and 8°C AEB520320 : Pack of 20 plates 90 mm ∅ AEB520319 : Pack of 120 plates 90 mm ∅ Ready poured Baird Parker with Sulfamethazine To be stored between 2 and 8°C AEB520320S: Pack of 20 plates 90 mm ∅ AEB520319S: Pack of 120 plates 90 mm ∅ Ready poured Baird Parker RPF To be stored between 2 and 8°C AEB520330 : Pack of 20 plates 90 mm ∅ AEB520329 : Pack of 120 plates 90 mm ∅ Ready to use sterile tellurite egg yolk. To be stored between 2 and 8°C AEB180052 : 50 ml flasks AEB680052 : 6 flasks of 50 mL AEB680057 : 6 flasks of 200 mL Ready to use sterile solution of sulfamethazine at 0,2 %. To be stored between 2 to 8°C. AEB180403 : 20 tubes 2,5 ml tube RPF supplement To be stored between 2 and 8°C AEB184106: 6 qsp 100 ml AEB184107: 6 qsp 200 ml Made by : AES CHEMUNEX - Combourg - France 150302£ : 21/11/08 - N

BAIRD-PARKER + RPF Baird-Parker Agar with R.P.F For In Vitro diagnosis

DESCRIPTION Baird Parker Agar with Rabbit Plasma Fibrinogen (RPF) is a highly selective and specific solid medium for isolation and enumeration of positive coagulase Staphylococci (particularly Staphylococcus aureus) in foodstuffs. The direct detection of a coagulase on the medium is possible due to the presence of R.P.F. FORMULA In grammes per litre of purified water Tryptone Meat extract Yeast extract Sodium pyruvate Glycine Lithium chloride Fibrinogen Rabbit plasma + EDTA Trypsine inhibitor Potassium tellurite Agar

10,00 5,00 1,00 10,00 12,00 5,00 3,75 25,00 ml 0,025 0,025 17,00

pH final : 7,2 + 0,2 à 25°C PREPARATION Liquefy a flask of BP agar base in a boiling water bath, then cool to 45-47°C. Reconstitute one vial of RPF lyophilized supplement with 10ml of sterile purified water for QS 100ml flasks and 20ml for QS 200ml flasks. Mix gently until complete dissolution. Poor the reconstituted supplement in the bottle of BP agar base sterilized and cooled to 48°C. Mix well. PROCEDURE • Poured plate technique (according to ISO 6888-2 standard) : Samples are diluted as desired. Place 1 ml of the sample or its decimal dilutions in the base of a sterile Petri plate. Pour enough prepared medium as to obtain a plate 3 mm thick. Homogenize well and let set before incubating the plates at 37°C for 24 hours or up to 48 hours if no typical colony is seen. • Selective isolation (research and NPP methods according to ISO 6888-3 standard) From a tube of GIOLITTI CANTONI broth simple or double concentration, spread one loop on the surface of a poured plate. Incubate at 37°C for 24h, then 48h if no typical colony is seen. Plates showing coagulase activity (opaque halo) have to be considered as positive.

LIMITS & PRECAUTIONS Flasks of prepared complete medium should be used immediately. Ready poured plates prepared by the laboratory should be inoculated within 48 hours and stored at 2-8°C. BIBLIOGRAPHY 5. Baird-Parker A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. J. Appl. Bacteriol. 25:12-19. 6. Norme NF EN ISO 6888-2. Méthode horizontale pour le dénombrement des Staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 2 : Technique de la gélose au plasma de lapin et fibrinogène. 7. Norme NF EN ISO 6888-3. Méthode horizontale pour le dénombrement des Staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces). Partie 3 : Recherche et méthode NPP pour les faibles nombres PACKAGING Poured plates : Store between 2 & 8°C AEB520330 : Pack of 20 plates 90 mm AEB520329 : Pack of 120 plates 90 mm Ready to use agar base: Store between 2 & 25°C AEB620314 : Pack of 6 x 90 ml flasks AEB620313 : Pack of 6 x 180 ml flasks RPF lyophilized supplement: Store between 2 & 8°C AEB184106: RPF AFNOR 6 qsp 100ml AEB184107: RPF AFNOR 6 qsp 200 ml AEB620337 : KIT Baird Parker base with RPF supplement for 1L Made by: AES CHEMUNEX - Combourg - France 520330 : 19/03/08- G£

RESULTS Enumerate colonies (with black coloration or not) with a precipitation halo.

BRAIN HEART INFUSION BROTH B.H.I BROTH For In Vitro diagnosis To be stored between 2 and 25°C

DESCRIPTION The B.H.I. broth is used for the cultivating fastidious aerobic and anaerobic micro-organisms, such as Neisseria meningitides, pneumococci, Streptococci. Its formula is compliant to the French food standard AFNOR NF EN ISO 6888-1. B.H.I. broth is recommended for blood culture, and the growth of pathogen fungi. B.H.I. broth in used for the culture of Staphylococcus strains when performing test for the detection of coagulase.

PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB140102 : 500 g Ready to use medium AEB110109: Pack of 100 tubes 5 ml AEB110110 : Pack of 100 tubes 10 ml AEB610104 : Pack of 6 flasks 90 ml* AEB110103 : Pack of 100 tubes of 3ml* Made by : AES CHEMUNEX - Combourg - France

FORMULA In grammes per litre of distilled water

140102£ : 29/08/07 -I 200 grs of infusion from calf brains 250 grs of infusion from beef heart Proteose peptone Dextrose Sodium chloride Disodium phosphate

12,50 5,00 10,0 2,0 5,0 2,50

* : Product not

stamped.

Final pH : 7,4 + 0,2 to 25°C PREPARATION Suspend 37 grams of medium in 1 litre of purified water. Homogenise until completely dissolved. Autoclave at 121°C for 15 minutes. Cool to room temperature. Dispense in flasks or tubes. PROCEDURE Inoculate the broth with micro organism strain or sample according to the laboratory specification. REFERENCES 1. Rosenow E.C. 1919. Studies on selective localisation. Focal infection with special reference to oral sepsis. J. Dental Research 1:205-249. 2. Chapman G.H. 1946. Isolation and testing of faecal Streptococci. Am. J. Digestive Diseases 13:105-107. 3. AFNOR NF EN ISO 6888-1. 1999. Microbiologie alimentaire – Méthode horizontale pour le dénombrement des Staphylocoques à coagulase positive. Partie 1 : Technique utilisant le milieu gélosé Baird Parker. 4. AFNOR V59-105 Octobre 1982. Gélatine alimentaire Recherche de Staphylococcus aureus.

LYOPHILISED RABBIT PLASMA Detection of free coagulase In Vitro use only To be stored between 2 and 8°C

PRINCIPLE Screening for free coagulase, extra cellular enzyme that In Vitro is able to coagulate rabbit plasma, help to differentiate Staphylococcus aureus from Staphylococcus epidermis, Staphylococcus saprophyticus and Micrococcus, since only pathogen strains of Staphylococcus aureus are capable within 24 hours to coagulate rabbit plasma. METHOD Under sterile environment regenerate the rabbit plasma with 7.5 ml of sterile purified water. The lyophilised product in stored at 2-8°C until the end of its shelf life. Once regenerated the product can be kept 15 days at 2-8°C or frozen (-20°C) allowing then a shelf life of 1 month.

PACKAGING Lyophilised rabbit plasma AEB184088 : Flask of 7,5 ml for 15 tests Ready to use BHI broth (to be stored between 1823°C) AEB110110 : Coffret de 100 tubes de 10 ml Made by : AES Laboratoire - Combourg - France 184087£: 07/04/05 – D

PROCEDURE Inoculate a BHI broth with the tested strain. Incubate at 37°C for 20 to 24 hours. Homogenise 0.5 ml of the incubated broth to 0.5 ml of the regenerated rabbit plasma in a sterile 5 ml tube. Incubate at 37°C for up to 24 hours. Intermediate reading can be done after 4 and 6 hours to detect fast coagulase strains. RESULTS Coagulation of the plasma (usually total) is seen during the first 4 hours of incubation. A positive result is given when a coagulum is observed even when late after 24 hours incubation. LIMITS & PRECAUTIONS Other strains of Staphylococci might give positive coagulase test such as Staphylococcus intermedius & Staphylococcus equi. When screening for coagulase positive strains, one can come accross coagulase negative strains that could potentialy be pathogen. Carrying out other confirmation tests (Dnase, thermonuclease, phosphatase) could confirm the dangerousness of the strain. Auto coagulation due to citrate is prevented by the presence of EDTA in the product. BIBLIOGRAPHY 1. AFNOR NF V08-057-1. Méthode de routine pour le dénombrement des staphylocoques à coagulase positive par comptage des colonies à 37°C.

D.N.A DeoxyriboNucleic Acid agar For In Vitro use

PRINCIPLE The DNA agar is a solid culture medium which formula, based on the works of Jeffries, Holtman and Guse and those of Di Salvo, is the one of the Tryptic Soy agar with 2 g/l DeoxyriboNucleicn Acid added. It allows to detect the activity of the bacteria DNase and particularly to identifiy the pathogenic Staphylococci. The DeoxyriboNucleic Acid, present in the medium, is depolymerized by the DNase in a mix of mono and polynucleotides with short chain. Positive reactions will be revealed by using chlorhydric acid (1N) or toluidine blue 0,1% solution. Around the DNase + colonies, the digested D.N.A will not precipitate with the chlorydric acid or will not be stand in blue by the colouring but pink. make the blue colour of the polychrome colorant (which is the toluidine) turn pink. FORMULA In grammes for 1 litre of purified water Peptones Deoxyribonucleic Acid Sodium Chloride Agar

20,00 2,00 5,00 15,00

Final pH: 7,3 + 0,2 at 25°C PREPARATION Suspend 42,0 grammes of powder in 1 litre of purified water. Bring slowly to the boil, shaking until complete dissolution. Dispense in tubes or flasks. Autoclave at 121°C for 15 minutes PROCEDURE Liquefy the medium at about 50°C. Pour in sterile Petri dishes. Inoculate the suspected colonies (taken on Chapman agar or on Baird-Parker agar, in the case of Staphylococci) in single streaks of 2 cm of length or by Ø 1 cm spots of diameter at the agar surface. Several strains (4 to 5 maximum) can be inoculated on the same plate simultaneously. Incubate at 37°C for 24 hours. RESULTS Spread at the surface of the agar a solution of chlorhydric acid 1 N or of a toluidine blue solution (0,1%). The reagent surplus will be sucked. After 5 to 10 minutes of contact, note the colonies appearance. In presence of chlorhydric acid (1 N) * clear zone around the streak, the rest of the agar is opaque: DNase + * absence of zone around the streaks : DNase In presence of toluidine blue at 0,1% * pink zone around the streak, the rest of the agar is blue: DNase + *absence of zone around the streak: DNase –

The micro-organisms that are Cocci, Gram +, catalase + and giving a positive reaction could be identified as presumptuous. Staphylococcus aureus Other complementary tests such as detection of the free coagulase or of the thermonuclease should be carried out to confirm the diagnosis. N.B. The differentiation of the Staphylococci will be easier by adding mannitol (10 g/l) and phenol red (0,025 g/l) as pH indicator, before the medium sterilization. The species which deteriorate the mannitol develop yellow colonies, surrounded by a yellow halo. The detection of DNase is an important identification test for the Staphylococci, but also for the differentiation of Serratia marcescens and liquefaciens (DNase +) from the DNase – enterobacteria (particularly Enterobacter and Klebsiella). LIMITS AND PRECAUTIONS Other micro-organisms, such as Aeromonas, some strains of Proteus-Providencia, some Xanthomonas maltophilia and Vibrio have a DNase. BIBLIOGRAPHY 1. Jeffries C.D., Holtman D.F. and Guse D.G. 1957. Rapid method for determining the activity of micro-organisms on nucleic acid. J. Bacteriol. 73:590-591. 2. Di Salvo J.W. 1958. Deoxyribonuclease and coagulase activity of micrococci. Med. Techns. Bull. Suppl. to U.S. Armed Forces Med. J. 9:191-196. 3. Schreier J.B. 1969. Modification of Deoxyribonuclease Test Medium for rapid identification of Serratia marcescens. Amer. J. Clin. Pathol. 51:711-716. 4. Smith P.B., Hancock G.A. and Rhoden D.L. 1969. Improved Medium for Detecting Deoxyribonuclease-Producting Bacteria. Appl. Microbiol. 18:991-993. PACKAGING Dehydrated medium (To be stored between 1 to 30°C) AEB150052 : 500 g Ready to use medium (To be stored between 2 to 25°C) AEB120059 : 100 tubes of 20 ml Prepared medium (To be stored between 2 to 25°C) AEB121061 : Pack of 10 plates 55 mm Made by AES CHEMUNEX - Combourg - France 150052: 11/03/08 - G

GIOLITTI-CANTONI Giolitti-Cantoni broth (base) In vitro use only

PRINCIPLE Giolliti-Cantoni broth is an enrichment medium used to enrich small quantities of Staphylococcus aureus in foods. The concentrations of lithium chloride and potassium tellurite prevent the growth of interfering Gram negative and positive flora. Pyruvate and glycine are added as growth factor to enhance the growth of staphylococci that is revealed by a blackening of the medium (reduction of tellurite). FORMULA In grammes per litre of purified water SC DC Tryptone 10,00 20,00 Meat extract 5,00 10,00 Yeast extract 5,00 10,00 Lithium chloride 5,00 10,00 Mannitol 20,00 40,00 Sodium Chloride 5,00 10,00 Glycine 1,20 2,40 Pyruvate de sodium 3,00 6,00 Final pH : 6,9 + 0,2 at 25°C METHOD Suspend 54,2 grammes of powder in one litre of purified water. Add 1 gram of polysorbate (Tween) 80. Bring slowly to the boil under continuous homogenisation. Dispatch 10 ml per tubes of 16 x 160 mm. Autoclave 20 minutes at 115°C.

RESULTS After 24 h of incubation, subculture onto Baird Parker or Baird Parker RPF plates all tubes that show a dark precipitate or blackening of the medium. After 48 h of incubation, subculture all tubes onto onto Baird Parker or Baird Parker RPF. LIMITS AND PRECAUTIONS The prepared tube (base) can be stored for up to 15 days at 2-8°C. Once add with the tellurite solution, the tubes have to be used in the day. BIBLIOGRAPHY 1. Giolitti G. and Cantoni C. 1966. A medium for the isolation of Staphylococci from Foodstuffs. J. Appl. Bacteriol. 29:395-398. 2. FIL-IDF 60. 1971. Recherche des staphylocoques à coagulase positive dans les poudres de lait. 3. Norme ISO 6888-3 – Microbiologie des aliments – Méthode horizontale pour le dénombrement des staphylocoques à coagulase positive (Staphylococcus aureus et autres espèces) – Partie 3 : recherche et méthode NPP pour les faibles nombres. PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB140392 : 500 g Prepared medium base (with polysorbate and without potassium tellurite) – single concentration (SC) (Store between 2 and 25°C) AEB110399 : Pack of 100 tubes of 10 ml

Note: As to prepare one litre of Giolitti Cantoni double concentration one needs to suspend double quantities of powder (108,4 grammes) and Polysorbate 80 ( 2 ml). Dispatch in tubes of 20 x 200 mm.

Prepared medium base (with polysorbate and without potassium tellurite) –double concentration (DC) (Store between 2 and 25°C) AEB110400 : Pack of 100 tubes of 10 ml

PROCEDURE Regenerate prepared tubes at 100°C for 15 minutes. Cool then add 0.1 ml of a 1% tellurite solution to each tube (single concentration). Add 1 g or 1 ml of the tested product or its decimal dilution. Homogenise well without adding any air. Add to each tube 1 cm thick cork of sterile liquefied AgarAgar (AEB175356 – 500g Dehydrated Agar). When using double concentration broth, proceed as with single concentration broth but add 0,2 ml of tellurite solution and 10 g or10 ml of the tested product. Incubate at 37°C for 24 to 48 hours

Sterile potassium tellurite solution (1 %) (Store between 2 and 8°C) AEB180452 : 20 Tubes of 5 ml Made by: AES CHEMUNEX - Combourg - France

140392: 26/11/08 - G

Culture media for the food industry

Horizontal method for the Clostridium perfringens enumeration NF EN ISO 7937 standard - 2005

Preparation of the mother suspension and of its dilutions

Inoculate 2 plates of TSC agar in the mass and in double layer with 1 ml of the mother suspension or of its dilution Incubate in anaerobic atmosphere 20+/-2 hours at 37째C

Enumerate the black colonies as presumptive C.perfringens and carry out confirmation tests (you can choose between 3 protocols) on 5 colonies by plate.

Confirmation with biochemical identification test

Subculture the selected colonies in a Thioglycollate broth. Incubate in anaerobic atmosphere 18 - 24 hours at 37째C

If the black colonies are not well isolated, inoculate 5 typical colonies in a Thioglycollate broth. Incubate in anaerobic atmosphere 18 - 24 hours at 37째C

Culture media for the food industry

Transfer 5 drops of inoculated broths in tubes of Lactose-sulfite broth

Isolate on TSC agar plates Incubate in anaerobic atmosphere 18-24 hours at 37°C

Incubate 18-24 hours at 46°C

All the tubes considered as positive are those presenting a black colouring of which the Durham bell is at least filled at 1/4. In case of doubt on the gas production, transfer some drops of the culture in a new tube of Lactose sulfite broth and incubate again 18 24 hours at 46°C and then carry out another reading

Inoculate by central prick in nitratemotility medium. Incubate in anaerobic atmosphere 24 hours at 37°C

Note the motility from the central prick Pour 0,2 to 0,5 ml of reagent for the detection of nitrites. Note the appearance of a red colouring (transformation of the nitrates in nitrites). Any pink colouring must not be taken into account. If no colouring appears within 15 minutes, add a pinch of zinc and wait 10 minutes. If a red colouring appears, there was no nitrates reduction.

Inoculate the gelatine lactose broth.

Incubate in anaerobic atmosphere 24 hours at 37°C

Note the gas production and the lactose fermentation (yellow colouring). Place the tubes 1h at 5°C. If the medium solidifies (gelatine liquefaction), reincubate an additional 24 hours and confirm the gelatine liquefaction.

Consider as Clostridium perfringens the black colonies in TSC medium which are non motile and which reduce the nitrates in nitrites or produce acid or gas, and liquefy the gelatine within 48 hours.

Culture media for the food industry

Horizontal method for the ASR bacteria enumeration NF ISO 15213 standard - September 2003

Preparation and dilution of the sample according to the NF ISO 6887-1 or 8261 standards (it is possible to detect only the spores of the ASR bacteria making the mother supension subjected to a thermic treatment (for example 20 minutes at 75°C )

Inoculation by incorporation in the tryptose sulphite agar (AEB622896 – flaks of 100 ml) with 1 ml of each dilution (2 plates per dilution) + addition of a second layer of tryptose sulphite agar OR Inoculation by incorporation in regenerated tubes of tryptose sulphite agar with 1 ml of each dilution (2 tubes per dilution) + addition of 2-3 ml of a layer of tryptose sulphite agar (AEB122899 – 100 tubes of 20 ml). Incubation 24 -48 hours at 37+/-1°C (in anaerobic atmosphere for the Petri dishes)

Enumerate the dark colonies on the plates containing less than 150 characteristic colonies or the tubes containing colonies well separated. Give the result according to the number of colonies enumerated and to the dilution factor.

T.S.C and Tryptose sulfite Base for Tryptone Sulfite Cycloserine agar In Vitro use

DESCRIPTION Tryptose Sulfite agar with or without D-cycloserine, as described by Harmon, Kautter and Peeler, is used for selective isolation and enumeration of anaerobe sulfitoreducing germs in particular Clostridium perfringens in animal and human foodstuffs. Tryptose sulfite agar (without egg yolk) has the advantage of producing small colonies that are easy to count (especially plates with numerous colonies). Sodium sulfite is reduced to sulfide which with ferric citrate forms a black precipitate around the colonies. D- Cycloserine, added to the base medium, gives a higher sensitivity to Clostridium perfringens than antibiotics such as polymyxin or kanamycin. It also reduces the size of the black halo around the colonies. The International Commission on Microbiological Specifications for Foods (I.C.M.S.F.) recommends T.S.C. agar , considering that has higher performances with vegetative cells and sporulating Clostridium perfringens than agars such as S.P.S. (Angelotti’s method ) and T.S.N. (Marshall’s method). FORMULA In grams per litre of purified water Tryptose Papaic digest of soybean meal Yeast extract Sodium metabisulfite Ferric ammonium Citrate Agar

15,00 5,00 5,00 1,00 1,00 15,00

Add 0,2 ml of 4% D- Cycloserine solution in each tube. It is essential to mix properly the agar and the sterile additive solution. Heat up the sample to be analysed in order to destroy vegetative cells and activate spores. Technique using a tube: Introduce 1 ml of each decimal dilution from the sample into the tubes. Make sure that no air bubbles are created. After homogenisation, cool the tubes down in a bath of icy water. Incubate at 37°C or 46°C for 24 hours. Technique with a Petri plate: Place 1 mL of each decimal dilution into a sterile Petri plate. Pour 15 to 20 ml of T.S.C. Let this first layer set then add another 5 mL to form the second layer. When the plates are set, incubate the plates, reverse way up, at 37°C or 46°C for 24 hours under anaerobic atmosphere. Note: A 46°C incubation reinforces the medium selectivity for Clostridium perfringens. Water analysis with tryptose sulfite agar: Heat the sample 15 minutes at 75 ±5 °C as to destroy vegetative forms and activate spores. Filter 50 or 100 mL of sample. Transfer the membrane to a ready prepared plate of Tryptose sulfite. Incubate under anaerobic atmosphere for 24 to 48 hours at 37°C.

Final pH : 7,6 + 0,1 at 25°C

RESULTS Sulfito-reducing colonies are surrounded by a black halo, due to sulfite reduction that creates an iron sulfide precipitate. To confirm Clostridium perfringens presence use usual confirmation tests.

PREPARATION Pour 42 grams of powder in 1 litre of purified water or equivalent. Bring slowly to the boil and stir until powder is completely dissolved. DO NOT OVERHEAT THE MEDIUM. Dispense 20 ml per tube (20 x 200 mm) Autoclave for 15 minutes at 121°C.

LIMITS AND PRECAUTIONS Avoid heating inoculated tubes. Either ready to use or prepared in laboratory, the base medium should be regenerated at 100°C for 20 minutes before use.

PROCEDURE Foodstuff analysis with T.S.C. agar: Liquefy the medium at 100°C (ex: water bath) then cool to around 45-50°C. Reconstitute a tube of D-cyclosérine supplement with 5 ml of purified water, in order to obtain a 4% solution (1 tube of reconstituted supplement gives 500 ml of medium).

T.S.C and Tryptose sulfite Base for Tryptone Sulfite Cycloserine agar In Vitro use

BIBLIOGRAPHY 1. AFNOR T90-415. Essais des eaux - Recherche et dénombrement des spores de bactéries anaérobies sulfito-réductrices et de Clostridium sulfito-réducteurs. Méthode générale par incorporation en gélose en tubes profonds. 2. Norme NF EN 26461-2. Essais des eaux - Recherche et dénombrement des spores de bactéries anaérobies sulfito-réductrices de Clostridium sulfito-réducteurs. Méthode générale par filtration sur membrane. 3. NF EN ISO 7937 : Microbiologie des aliments – Méthode horizontale pour le dénombrement de Clostridium perfringens. Méthode par le comptage des colonies. 4. NF EN ISO 15213 : Microbiologie des aliments Méthode horizontale pour le dénombrtement des bactéries sulfito-réductrices se développant en conditions anaérobies PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB152892 : 500 g bottle Ready to use medium (without D-cycloserine) for foodstuffs testing (Store between 2 and 25°C) AEB122899 : 100 tubes of 20 ml AEB622896 : Pack of 6 bottles of 100 ml AEB622897 : Pack of 6 flasks of 200 mL AEB622906 : Kit composed of 5 flasks of 100 ml + 1 vial of supplement Ready to use medium (without Cycloserine) for water analysis (Controlled according to NF T 90-461 standard) (Store between 2 and 25°C) AEB122899E: Pack of 100 tubes of 20 mL AEB622896E: Pack of 6 flasks of 100 mL Lyophilised D-Cycloserine - 200 mg/vial (Store between 2 and 8°C) AEB184002 : q.s.p. 500 ml Made by AES CHEMUNEX - Combourg - France 152892£: 05/01/09 - L

THIOGLYCOLLATE RESAZURIN Fluid Thioglycollate medium For In Vitro use

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 oxidationreduction 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 regenerated in a boiling water bath. Incubate 21h+/-3h at 37°C.

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* 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 100 ml 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.

Culture media for the food industry

Detection of Listeria monocytogenes NF EN ISO 11290-1 February 1997 V08-028-1 Amendment February 2005 Dilution of Xg (or ml) of sample in 9X ml (or g) of Half Fraser broth (AEB610418 - 6 flasks of 225ml AEB910915 - Dilubag of 5 L) Incubation 24±2 hours at 30±1°C

Subculture 0.1ml of the previous culture in 10ml of Fraser broth (AEB110429- 100 tubes of 10ml) Incubation 48± 2 hours at 37±1°C

Isolation of the previous culture on ALOA agar (AEB 520080- 20 plates Ø 90 mm) and onto a second agar and complementary to the first agar for example : Oxford agar (AES522000- 20 plates Ø90 mm)or Palcam (AEB522050- 20 plates Ø90 mm) with an inoculating loop. Incubation 24 h±3h at 37±1°. Incubation de 24 h ± 3h if necessary

Isolation of the previous culture on ALOA agar (AEB 520080- 20 plates Ø 90 mm) and onto a second agar and complementary to the first agar for example : Oxford agar (AES522000- 20 plates Ø90 mm) or Palcam (AEB522050- 20 plates Ø90 mm) with an inoculating loop.

Subculture 5 presomptive colonies (on ALOA: blue green colonies with an opaque halo) onto a TSYE agar to perform confirmation test (AEB522865 – 20 plates Ø90 mm)

Incubation 24 h±3h at 37±1°. Incubation de 24 h ± 3h if necessary

Incubation 18 – 24 hours at 35 ± 1°C or at 37 ± 1°C

Culture media for the food industry

Subculture 5 presomptive colonies (on ALOA : blue green colonies with an opaque halo) onto a TSYE agar to perform confirmation test (AEB522865 – 20 plates Ø90 mm) Incubation 18 – 24 hours at 35 ± 1°C or at 37 ± 1°C

Confirmation : 1 – Catalase test 2 – Gram colouring 3 – Motility examination (optional test) 4 – Research of haemolysis 5 – Carbohydrates use 6 – CAMP test

Results expected : 1 - Catalase : positive reaction (Catalase +) 2 - Gram colouring: small gram positive bacilli -Gentian violet staining (AEB070600- flask of 1 litre) -Lugol (AEB040600– flask of 1 litre) -Gram differentiating reagent (AEB090600- flask of 1 litre) -Fuschine de Ziehl (AEB030600– flask of 1 litre) 3 - Motility : motile bacteria -motility agar or fresh microscopy observation (in TSYE broth) 4 - Haemolysis : positive Haemolysis (Haemolysis+) -sheep blood agar : (AEB152452 blood agar base 500 g) (AEB200025 Defibrinated sheep blood 25 ml) 5 - Carbohydrates use : Rhamnose +, Xylose 6 - Camp test : S. aureus +, R. equi -

Culture media for the food industry

Enumeration of Listeria monocytogenes NF EN ISO 11290-2 August 1998 V08-028-2 Amendment February 2005

Dilution (of your choice) of the sample in Buffered Peptone Water* (AEB910303 – Dilubag de 3 L AEB910305 - Dilubag de 5 L) Reviving 1 hour at 20°C * : or in Fraser medium without anitibiotics

Spread 0,1 ml on 2 plates of ALOA agar Ø 90 mm first dried with an incubator or for more precision 1 ml on 2 plates Ø 140 mm (option : you can spread on 6 plates Ø 90 mm) Incubation 24 ± 3 hours at 37°C

Enumerate the typical colonies (if present) (Blue green with an opaque halo)

In case of low growth or if no typical is observed. Continue the incubation 24 ± 3 hours at 37°C

Presence of typical colonies ? (Blue green with an opaque halo)

If yes, carry out a confirmation test

Result : If no typical colony has been enumerated or confirmed in 48h: give Less of (1 / dxV) Listeria monocytognes per gramme of product Otherwise : The number of Listeria monocytogenes to take into account for the enumeration is equal to the colonies pointed after 24 hours of incubation on ALOA agar (or after 48 hours of incubation according to the growth intensity) and whose membership to the Listeria monocytogenes species has been confirmed. 96

ALOATM (Agar Listeria according to Ottaviani & Agosti) Selective medium for the detection & the enumeration of Listeria monocytogenes In Vitro use Store between 2 & 8°C

ALTERNATIVE METHODS FOR AGRIBUSINESS ANALYSIS Validated by AFAQ AFNOR Certification www. afnor.fr ALOA COUNT™ Method (Enumeration) AFNOR VALIDATION Certificate N° AES 10/5-09/06 For foodstuffs analysis. End of validation: 15/09/2010 ALOA ONE DAY™ (Detection) AFNOR VALIDATION Certificate N° AES 10/3-09/00 For foodstuffs analysis and environment samples. End of validation : 27/09/2012

PRINCIPLE ALOA™ is a selective medium used for the detection and enumeration of L .monocytogenes in foodstuffs samples (AFNOR VALIDATION certified methods – see enclosed protocols). This medium is also used to carry out screening and enumeration of L.monocytogenes & L.sp in foodstuffs and any kind of samples (ISO 11290 standard protocols). On this medium Listeria grow as blue-green regular round colonies (detection of β-glucosidase by using a specific chromogenic substrat). Listeria monocytogenes also shows an opaque halo this helps to easily differentiate them from other species of Listeria. The halo is due to the activity of a phospholipase involved in the infection process of pathogenic Listeria. The selectivity is obtained by the combination of lithium chloride, anti microbial and antifungical components. FORMULA (IN G/L OF MEDIUM) Animal tissue enzyme digest Caseine enzyme digest Yeast extract Sodium pyruvate Glucose Magnesium glycerophosphate Magnesium Sulfate Sodium chloride Lithium chloride Disodium hydrogen phosphate anhydrous X-glucoside Nalidixic acid Ceftazidime Polymyxin B Amphotericin B Phosphatidylinositol Agar Final pH 7,2 +/- 0,2 at 25°C

18 g 6g 10 g 2g 2g 1g 0,5 g 5g 10 g 2,5 g 0,05 g 0,02 g 0,02 g 76700 U 0,01 g 2g 13,5 g

PROCEDURE Detection and enumeration of Listeria monocytogenes according to standard methods : Refer to standards ISO 11290-1 & -2 amendment 1 (2004). Alternative methods AES Chemunex AFNOR VALIDATION certified : • Detection of Listeria monocytogenes according to ALOA ONE DAY™ Inoculate the plates directly with the sample or an appropriate incubated enrichment culture medium (to perform the isolation, see the enclosed procedure AFNOR VALIDATION certified ALOA ONE DAY™ protocol). Incubate at 37°C and read plates after 24 to 48 hours. It is not necessary to prolong incubation to 48 hours for the plates screened at 24 hours what ever the result of the screening. • Enumeration of Listeria monocytogenes according to ALOA COUNT™ Spread 0.1 ml of the revived primary dilution onto the surface of a ∅ 90 mm ALOA™ plate (option to spread 1ml onto a ∅ 140 mm ALOA™ plate or split onto 3 ∅ 90 mm plates as to increase the accuracy) or 1 ml using poor-plate method (see the enclosed ALOA COUNT™ protocol). Incubate at 37°C+/-1°C for 48±3 hours when absence of typical colonies or low growth. Note: A first reading can be carried out after 24 hours of incubation, as to detect faster any sample with high levels of contamination nevertheless final results should be given only after 48 hours of incubation. RESULTS Note the characteristics of the colonies : Listeria sp.: blue to blue-green colonies, round, regular, without any opaque halo, diameter from 1 to 2 mm. Listeria monocytogenes: colonies with Listeria sp. characteristics and surrounded by an opaque halo. Listeria monocytogenes strains grow as typical colonies in 24 hours. Within the framework of the AFNOR VALIDATION certification (ALOA ONE DAY™ & ALOA COUNT™), all positive results have to be confirmed by one of the following methods : 1 – By traditional tests described in the standard methods ISO, CEN or AFNOR on colonies isolated from ALOA plates. An initial purification of isolated colonies is necessary. 2 - ALOA Confirmation™ (see ALOA confirmation™ technical data sheet).

ALOATM (Agar Listeria according to Ottaviani & Agosti) Selective medium for the detection & the enumeration of Listeria monocytogenes In Vitro use Store between 2 & 8°C

3 – For the ALOA ONE DAY protocol only, by any other AFNOR VALIDATION certified method from which the principle is different from ALOA ONE DAY method. The full procedure of this second validated method will have to be followed. If this method has common steps with ALOA ONE DAY method. Start the procedure at the last common step to both methods. (for example, Half Fraser Broth). Keeping of incubated Half Fraser broth cannot exceed 48 hours In the event of unmatched results (positive result with AES Chemunex alternative method not confirmed by the selected confirmation method), the laboratory must carry out sufficient means to be ensured of the validity of the result In the frame work of ALOA COUNT™, when in presence of a positive result, confirmation tests do not need to be carried out if confirmation tests were carried out during detection step. PROCEDURE LIMITATIONS In presence of highly contaminated plates, the reading can be helped by comparing the opacity of the medium between the centre and the sides of the plate (confer to the protocol) or by comparing to a non inoculated ALOA plate. L.monocytogenes presence, even in great numbers, is characterised by an intense opacity of the medium. This allows to easily differentiate plates where L.monocytogenes are present (opaque medium) from those where they are absent (clear medium). In case of doubt, subculture onto a second ALOA plate. After 24 hours of incubation, some strains of Listeria ivanovii can show a very tight light halo. After 48 hours of incubation, Listeria ivanovii can show the same characteristics as Listeria monocytogenes. Under the two circumstances, carrying out confirmation tests allow to differentiate the two species without hesitation. ALOA plates can be placed in the refrigerator after incubation. The halo and the colour of the colonies will not be altered at those low temperatures. Some strains of B.cereus can grow as flat, rough colonies with irregular outline, non homogeneous white to blue colour and a large and intense halo. When using ALOA Kits (base + supplements), a flask of liquefied base not supplemented, can be cooled and liquefied a second time without altering the properties of the medium. Supplemented ALOA base can be kept liquefied at 47 ± 2°C for up to 7 hours before being used. Poured plates using ALOA Kit can be stored in the dark at a refrigerated temperature (2-8°C) for a week.

BIBLIOGRAPHY 1. Ottaviani, F., Ottaviani, M., Agosti, M. (1997) Differential agar Medium for Listeria monocytogenes. In "Quimper froid. Symposium proceedings" P6 A.D.R.I.A. Quimper (F) 16-18 June, 1997. 2. Ottaviani, F., Ottaviani, M., Agosti, M. (1997) Esperienza su un agar selettivo e differenziale per Listeria monocytogenes. Industrie Alimentari 3. Vlaemynck, G., Lafarge, V., Scotter, S. (2000) Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium. Journal of Applied Microbiology, 88 : 430-441. 4. Artault, S., Bind, J.L., Delaval, Y., Gaillard, N. Validation AFNOR de la méthode ALOA pour la détection de Listeria monocytogenes dans les produits alimentaires. Colloque Société Française de Microbiologie, 19-20 octobre 2000. 5. ISO 11290 partie 1 et 2 – Amendement 1 (2004) : Microbiologie des aliments – Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes. PACKAGING Ready to use media: AEB520080 : Pack of 20 plates 90 mm AEB520079 : Pack of 120 plates 90 mm AEB120082 : Pack of 10 plates 140 mm AEB620088 : Kit for 1 litre (bases + supplements) 5 x 200 ml base + supplements Dehydrated medium + supplements: contact us. Made by AES CHEMUNEX – Combourg – France 520080£: 30/06/08 – S

Culture media for the food industry

Listeria monocytogenes detection in food samples Method Analytical protocol (AFNOR Validation n°10/3-09/00) J 0: Enrichment X g or X ml of sample + 9 X ml ½ FRASER ( for ex. 25 g of sample + 225 ml of ½ FRASER)

During AFNOR validation of the ALOA ONE DAY method, only samples of up to 25 g (± 5 %) were tested.

Incubation 24 ± 2 hours at 30 ±1 °C J + 1: Inoculation

Spread 0.1 ml on ALOA Incubation 24 to 48 hours at 37 ± 1 °C

Advice: Keep an area not inoculated at the edge of the plates as to help the visualisation of the opaque halo on plates with confluent growth (Contrast centre/periphery)

J+2: Results

Absence of typical colonies

Typical colonies

Absence of Listeria monocytogenes

Presumptive presence* of Listeria monocytogenes

Confirmation

520080: 30/06/08 - S

* In compliance with AFNOR Validation certification, any positive result obtained with an alternative method must be confirmed

Culture media for food industry

Enumeration of Listeria monocytogenes ALOA COUNT™

Dilute X g or X ml of sample in 9 X ml of buffered peptone water Revivify 1 hour ± 5 minutes at 20 ± 2°C (Proceed to 1/10th dilution if necessary)

Surface spread method

Pour-plate method

Spread 0.1 ml* onto the surface of a dried plate of ALOA™ agar Ø 90 mm As to raise the accuracy of the enumeration spread 1 ml* onto the surface of a dried plate of ALOA™ agar Ø 140 mm.

(Option: split the 1 ml inoculum onto 3 plates Ø 90 mm)

Add 1 ml* into a sterile Ø 90 mm plate and poor 15 ml of melted ALOA™ cooled to 47°C ± 2°C. Homogenise and let the prepared plate set before incubation 48 ± 3 hours at 37±1°C

Incubate 48 ± 3 hours à 37±1°C

Typical colonies ?

Less than X L monocytogenes per gramme of product

Note: A first reading can be carried out after 24 hours of incubation, as to detect faster any sample with high levels of contamination nevertheless final results should be given only after 48 hours of incubation.

Yes

Listeria monocytogenes Presumption

Confirm one colony ALOA Confirmation™ Or standard Tests X L. monocytogenes per gramme of product

* : Revived primary dilution, if necessary proceed in the same way with the 1/10th dilutions 100

LIMITS AND PRECAUTIONS OXFORD THE GROWTH OF MOST OF THE GRAM + STRAINS IS INHIBITED, NEVERTHELESS, A FEW ENTEROCOCCUS STRAINS monocytogenes ARE PARTIALLY Isolation of Listeria INHIBITED AND GIVE COLONIES WITH SLIGHT For in vitro use only HALO AFTER A 40 HOURS INCUBATION. Some of Staphylococcus strains grow without halo.

PRINCIPLE The OXFORD medium, which is based on Curtis' formulation, is recommended for the isolation of Listeria monocytogenes in biological and food samples. It contains a nutritive base and inhibitors : lithium chloride inhibits Enterococcus’ growth, acriflavin inhibits Gr am - species and most of the Gram + species. Colistin sulfate, cycloheximide, cefotetan and fosfomycin improve the selectivity of the medium. Esculin acts as a differential indicator, esculine is hydrolysed in esculetine by Listeria. The combination between esculetine and ammonium ferric citrate produces black halos around the colonies. FORMULA In grammes per litre of purified water: Peptones 23,00 Starch 1,00 Sodium chloride 5,00 Esculin 1,00 Ammonium ferric citrate 0,50 Lithium chloride 15,00 Agar 12,00

BIBLIOGRAPHY 1. Curtis G.D.W., Mitchell M.G., King A.F. and Griffin E.J. 1988. Personal Communication. John Radcliffe Hospital, Oxford, U.K. PACKAGING Dehydrated medium (To be stored between 1 and 30°C) AEB151992N: 500g Pre-poured medium (To be stored between 2 and 8°C) AEB522000: pack of 20 plates 90mm AEB521999: pack of 120 plates 90mm AEB122002: Pack of 10 plates 140 mm CCCFA supplement (To be stored between 2 and 8°C) AEB184122N : QS for 500 ml Made by: AES CHEMUNEX – Combourg – France 151992N: 31/07/08 – H

Selective supplement (for 5ml of a mix of 50% ethanol/water) Cycloheximide 200,00 mg Colistin sulphate 10,00 mg Cefotetan 1,00 mg Fosfomycin 5,00 mg Acriflavin 2,50 mg pH: 7,0 +/- 0,2 at 25°C PREPARATION Suspend 57,5 grammes of powder in one litre of purified water. Bring slowly to the boil, under constant stirring until completely dissolved. If needed dispatch in flasks. Autoclave the prepared base 15 minutes at 121 C. Melt the base then cool to 44-47°C, add two CCCFA supplements per litre of prepared base. Each vial of supplement must be dissolved with 5 ml of a 50% ethanol sterile solution. Homogenize the complete medium before dispatching in sterile Petri plates. PROCEDURE Inoculate the plates directly from the sample or from an appropriate enrichment culture medium. Incubate at 37°C for 24 to 48 hours. RESULTS Listeria produces grey or greenish-grey colonies with brownish-black halo. Definitive identification tests must be conducted on suspect colonies.

101

PALCAM Listeria monocytogenes selective medium In Vitro use only

PRINCIPLE PALCAM medium is appropriate for the detection and enumeration of Listeria monocytogenes in foodstuffs and all sorts of samples even in high levels of contamination. To the nutrient base are added selective agents to inhibit the growth of interfering flora. • Lithium chloride: Enterococci. • Acriflavin: most Gram- and unwanted Gram +. Listeria spp. (except L. grayiandt murrayi) grow as : Colonies surrounded by an olive-green to black halo due to the hydrolysis of esculin into dihydroxycoumarin that reacts with the iron. The medium can change of colour to brown when high concentration s of Listeria growth on the plate. Mannitol strains of Staphylococci and enterococci that manage to grow on this hostile medium grow as yellow colonies.

Note the characteristics of the colonies: Listeria monocytogenes: greenish colonies, of Ø 1,5-2 mm, surrounded by a black halo. Enterococi : small white to grey colonies, diameter < 1 mm surrounded by a green halo. Staphylococci : white or yellow colonies, diameter : 1,53 mm, surrounded by a white halo. Identification of Listeria monocytogenes typical colonies will be carried out according to ISO standard specifications.

BIBLIOGRAPHY 1.

FORMULA In grammes per litre of purified water Peptones Corn starch Yeast extract Sodium chloride D- Glucose Mannitol Esculin Ferric ammonium citrate Lithium chloride Phenol red Agar

23,00 1,00 3,00 5,00 0,50 10,00 0,80 0,50 15,00 0,08 15,00

Selective supplement (for 5 ml of purified water) Acriflavin HCl 2,5 mg Ceftazidim 10,0 mg Polymyxin B Sulfate 5,0 mg

Final pH : 7,2 à 25°C

METHOD Suspend 73,9 grammes of powder in one litre of purified water. Bring to the boil under constant homogenisation until completely dissolved. Autoclave 15 min at 121°C

PROCEDURE Liquefy the medium then cool to 45-50°C. Add per 500 ml of medium base, one vial of regenerated selective supplement. Homogenise well then pour into Petri plates. Prepared plates can be kept up to 30 days when stored in the dark at +4°C.

Van Netten P., Van De Ven A., Perales I. and Mossel D.A.A. 1988. A selective and diagnostic for use in the enumeration of Listeria spp. in foods. Int. J. Food Microbiol. 6:187-198. Van Netten P., Perales I. and Mossel D.AA. 1988. An improved selective and diagnosic medium for isolatin and counting of Listeria spp. in heavily contaminated foods. Lett. Appl. Microbiol. 7:17-21. Van Netten P.,, Perales I., Van De Moosdijk A., Curts G.D. W. and Mossel D.A.A. 1989. Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food microbiol. 8:299-316. Norme NF V08-055. Décembre 1993. Microbiologie alimentaire. Recherche de L. monocytogenes. Méthode de routine.

PACKAGING Dehydrated medium Store between 18 & 23°C AEB152042 : 500 grs Selective supplement Store between 2 & 8°C AEB184042: 500ml – lyophilised vial Ready to use plates Store between 2 & 8 °C in the dark AEB522050: Pack of 20 plates ∅ 90mm AEB522049: pack of 120 plates ∅ 90mm AEB122052: pack of 10 plates ∅ 140mm Made by: AES Laboratoire – Combourg – France 152042: 15/10/03-I

RESULTS Inoculate directly the plates with the sample or with the appropriate enrichment culture. Incubate at 37°C for 24 to 48 hours. AES Laboratoire PALCAM as been optimised to enhance the growth of L. monocytogenes.

102

BLOOD AGAR Base for Blood Agar In Vitro use only To be stored between 2 and 8°C

PRINCIPLE The medium with blood is used within the framework of confirmation tests such as the search for the type haemolysis or to carry out test. FORMULA In grammes per litre of purified water Proteose peptone Liver digestion Yeast extract Sodium chloride Agar

15,00 2,50 5,00 5,00 13,00

Final pH : 7,2 + 0,2 at 25°C Addition of 5% of fresh defibrinated sheep’s blood to the base (liquefied and cool to 44 - 47°C) PROCEDURE • .Determination of haemolysis Swab a purified culture of the strain onto the surface of the agar. Incubate the prepared plate using the optimal conditions for the growth of the microorganism. • CAMP test: Carry out CAMP test according to ISO 11290 standard’s procedure. Inoculate by a single streak Staphylococcus aureus and Rhodococcus equi. The streaks are parallel and diametrically opposite. The tested strains will be inoculated be streaks parallel to each other but perpendicular the reference strains. BIBLIOGRAPHY 1. Waterworth P.M. 1955. Brit. J. Exp. Path. 36(2):186-194. 2. NF EN ISO 11290: Méthode horizontale pour la recherché et le dénombrement de Lisetria monocytogenes. PACKAGING Dehydrated base AEB120650: Pack of 10 plates ∅ 90 mm Made by : AES CHEMUNEX - Combourg - France 120650£ : 29/11/06 - A

103

T.S.Y.E Listeria monocytogenes isolation medium In Vitro use only To be stored between 2 and 25°C

DESCRIPTION T.S.Y.E. agar is made by adding yeast extract and agar to trypticase soy broth. Its formula is conform to the one described in the standards ISO 11290-1 and -2. FORMULA In grammes per litre of purified water Tryptone Soytone Sodium chloride Dipotassium Phosphate Dextrose Yeast extract Agar

17,0 3,0 5,0 2,5 2,5 6,00 15,00

final pH : 7,3 + 0,2 at 25°C PROCEDURE Subculture well-isolated-suspect colonies provided from a Listeria selective agar. Incubate at 30 or 37°C for18 to 24 hours. RESULTS Listeria colonies are see-through, without pigment, diameter of about 1mm. Under Henry lighting, colonies take on a bluish shade of colour and show a granular surface. To confirm Listeria monocytogenes specie, follow by a Gram coloration, a catalase enzyme characterisation, a subculture on a blood agar and the study of the xylose and rhamnose fermentation. BIBLIOGRAPHY 1.

Norme ISO 11290. Décembre 1993. Microbiologie alimentaire. Méthode horizontale pour la recherche et le dénombrement de L. monocytogenes.

PACKAGING Pre-poured dishes AEB522865 : Pack of 20 dishes (90 mm) AEB522864 : Pack of 120 dishes (90 mm) Made by AES CHEMUNEX - Combourg - France 122865£ : 30/08/07-E

104

Culture media for the food industry

Horizontal method for the enumeration of presumptive Bacillus cereus - colony-count technique at 30째C NF EN ISO 7932 - 2005

Decimal dilution of a sample in Peptone water (AEB111499 - pack of 100 tubes of 9 ml)

Inoculation by spreading on surface a Mossel agar (AEB521740 - plates 90 mm) with 0,1 ml of each dilution (2 plates per dilution)

Incubation 18 - 48 hours at 30+/-1째C

Enumeration of the typical colonies on plates containing between 15 and 150 colonies. Typical colonies: big, pink colonies that can develop a precipitate.

Confirmation: Carry out haemolysis testing using a sheep blood agar (AEB120650: 10 plates of 90 mm) on at least 5 isolated colonies. (If necessary, re-isolate colonies onto Mossel agar plates and incubate for 18-24h at 30째C). Incubate for 24+/-2 hours at 30째C

Presumptive Bacillus cereus = Pink colonies with a halo of precipitation that give a positive reaction to haemolysis testing.

105

MOSSEL Bacillus cereus Agar according to Mossel specifications In Vitro use only

PRINCIPLE Bacillus cereus Agar is used for the detection and enumeration of spores and vegetative cells of Bacillus cereus in foodstuffs. A presumptuous identification is carried out through the screening of typical colonies after incubation. * B. cereus does not ferment mannitol. This characteristic helps to differentiate B. cereus from contaminating microorganisms which ferment mannitol, causing phenol red to turn yellow. * B. cereus synthesize lecithinase, its action on the egg yolk lecithin produces insoluble breakdowns that accumulate around the colonies, forming a whitish precipitation. Finally, Polymyxin B can be added to inhibit accompanying microflora when the tested sample is heavily contaminated.

RESULTS Bacillus cereus colonies are large flat, rough irregular and pink (Mannitol -) surrounded by an opaque halo due to the presence of the lecithinase. These colonies are flat rough and tend to spread.

FORMULA Bacillus cereus agar base In grammes per litre of medium

BIBLIOGRAPHY 1. Donovan K.O. 1958. A selective medium for Bacillus cereus in milk. J. Appl. Bacteriol. 21:100-103. 2. Mossel D.A.A., Koopman M.J. and Jongerius E. 1967. Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15:650-653. 3. AFNOR NF EN ISO 7932 Juillet 2005. Microbiologie des aliments - Directives générales pour le dénombrement de Bacillus cereus. Méthode par comptage des colonies à 30°C.

Peptone Beef extract Sodium chloride Mannitol Phenol red Agar

10,00 1,00 10,00 10,00 0,025 15,00

Final pH : 7,2 + 0,2 at 25°C 50% egg yolk solution 40 ml (Sterile egg yolk diluted at 50% with physiological water (9g/L)). Polymyxin B sulfate 10 ml (50.000 UI/qs 500 ml to be regenerated with 5 ml of sterile purified water, use 2 vials to finish 1L de Mossel complete agar) METHOD Suspend 46,0 g of powder in 950 ml of purified water. Heat slowly up to boiling point until completely dissolved under constant agitation. Dispense in flasks and autoclave 15 minutes at 121°C. PROCEDURE Liquefy the medium then cool to 44-47°C. As to prepare 1 litre of medium add to 950 ml of base 40 ml of the 50 % egg yolk solution, and if necessary 2 doses of Polymyxin B (50 000 UI/vial) previously hydrated with 5 ml of purified water. It is important to homogenize well before dispensing in plates. Spread 0,1 ml of the prepared sample or its decimal dilutions at the surface of a prepared plate. Incubate at 30°C for 48 hours with a daily observation.

LIMITS AND PRECAUTIONS Other microorganisms such as Staphylococcus aureus, Serratia marcescens and Proteus vulgaris are known to use the egg yolk. Continue identification by looking at the bacterial morphology, testing dextrose fermentation, nitrate reduction and the production of acetylmethylcarbinol (VP). Once opened a flask of egg yolk emulsion can be used until its expiry date if it was manipulated according to good laboratory practice (ie: Use in aseptic conditions and storage at 2 to 8°C).

PACKAGING Dehydrated medium To be stored between 1 and 30°C AEB151732 : 500 g 50% egg yolk solution ready to use To be stored between 2 and 8°C AEB180102 : 50 ml flask AEB680102: 6 flasks of 50 ml AEB680107 : 6 flasks of 200 ml Polymyxin B sulfate supplement (50 000 UI/vial) To be stored between 2 and 8°C AEB184001 : q.s. 500 ml Ready to use medium base without supplements To be stored between 2 and 25°C AEB621736 : 6 Flasks of 100 ml Ready poured medium To be stored between 2 and 8°C AEB521740 : Pack of 20 plates 90 mm ∅ Made by : AES CHEMUNEX - Combourg - France 151732£ : 13/02/09-K

106

BACILLUS CEREUS RAPID AGAR (BACARA®) Selective medium for Bacillus of cereus group For In Vitro use only

PRINCIPLE Bacillus cereus is responsible for food-borne outbreaks. It produces thermoresistant spores that make it particularly adapted to foodstuffs submitted to thermal treatment. Some strains of B. cereus can grow at refrigeration temperature, which is an emerging risk for ready-to-use products. They represent by themselves 5% of the collective food poisoning in France and are also involved in a lot of opportunistic infections on inpatients. Bacillus cereus belongs to the Bacillus cereus group within can be found B. thuringiensis, B. Weihenstephanensis, B. mycoides, B. pseudo-mycoides et B. anthracis. Except the last-mentioned, the phenotypic differentiation of species between Bacillus cereus is impossible with actual culture methods. ® BACARA agar is a selective chromogenic medium that allows the enumeration of Bacillus of the cereus group without confirmation. On this medium, typical colonies of B. cereus show a pink / orangey colour due to the metabolism of the substrate and are surrounded with an opaque halo due to the phospholipase activity. ® The selectivity of BACARA agar has been especially optimized to prevent growth of interfering flora and thus to allow an easy interpretation of plates even when matrix highly contaminated with competitive flora are analysed. FORMULA In grams per litre of purified water Special mix of peptones Yeast extract Sodium chloride Phosphate buffer Agar Mix of antibiotics Chromogen substrate Phospholipids Final pH : 7,2 + 0,2 at 25°C

10,00 4,00 4,00 10,00 12,00 0,26 0,05

PROCEDURE Spread 0,1ml of the revived primary solution onto the ® surface of a Ø 90mm BACARA plate (option to spread ® 1ml onto a Ø 140mm BACARA plate or split onto 3 Ø ® 90mm BACARA plates as to increase the accuracy)

RESULTS After incubation, Bacillus from cereus group present large pink/orangey colonies surrounded with an opaque halo.

Pink orangey colour

Opaque halo : Phospholipase activity

LIMITS AND PRECAUTIONS Thanks to the high specificity and selectivity of ® BACARA , it is not necessary to carry out confirmation tests on typical colonies. All the strains belonging to the Bacillus cereus group will give characteristic colonies. Some bacteria can also show an orangey colour on ® BACARA medium but without expression of the phospholipase activity ; they will be thus easily distinguishable from the Bacillus cereus. BIBLIOGRAPHY 1- AFNOR NF EN ISO 7932 Juillet 2005. Microbiologie des aliments – Méthode horizontale pour le dénombrement de Bacillus cereus présomptifs. Technique par comptage des colonies à 30°C. 2- N.A LOGAN, P.C.B. TURNBULL : Actualité permanente en Bactériologie Clinique – Bactéries aérobies sporulées 3- F.A DROBNIEWSKI : Bacillus cereus and related species – Clin Microbiol Rev 1993 October Species ; 6 (4): 324-338.

PACKAGING :

Pre-poured complete medium To store between 2 and 8°C AEB520100 : Pack of 20 plates of 90 mm AEB120102: Pack of 10 plates of 140 mm

Incubate the BACARA plates for 24 +/-2h at 37°C +/1°C.

Manufactured by AES CHEMUNEX - Combourg - France 520100 : 21/10/08- A

107

Culture media for the food industry

Horizontal method for the detection and enumeration of Campylobacter spp. Part 1 : Detection method NF EN ISO 10272-1 (V08-26-1) – April 2006

Enrichment Sample preparation of x grammes of product in 9x ml of Bolton broth Incubate in microaerophilic atmosphere (5+/-1) hours at (37+/-1)°C, then (44+/- 4) hours at (41,5+/-1)°C. (Campypack H2CO2 – BBL71034 - 10 envelopes without catalyst – GasPak Campy for jar – BBL60680 - system without water - 20 units)

Isolation Inoculate by isolation a mCCD agar plate and another selective culture medium such as the Karmali agar (AEB120380 – 10 plates 90 mm) Incubate 44 hours +/- 4 hours at (41,5+/-1)°C in microaerophilic atmosphere (Campypouch System – BBL60656 – 25 pouches GasPak Campy in bags – BBL60685 - system without water - 20 units)

Confirmation Isolate the suspected colonies (1 per agar, 4 additional if the first one is not confirmed) on Columbia with blood agar Incubation in microaerophilic atmosphere 24 to 48 hours at (41,5±1)°C From the cultures obtained/get from Blood Columbia agar, carry out the following tests: - microscopic test - growth on Columbia agar at (25+/-1)°C for (44±4) hours in microaerophilic atmosphere - growth on Columbia agar at (41,5+/-)°C for (44±4) hours in anaerobic atmosphere - detection of oxidase The Campylobacter spp. give the following results: Small bacilli, curved with a characteristic motility « in spiral », giving nor culture at 25°C in microaerophilic atmosphere nor at 41,5°C in anaerobic atmosphere and at positive oxidase. The identification of the Campylobacter species described in this standard is optional.

108

Culture media for the food industry

J+3 - J+8: Confirmation

From the colonies isolated on Blood Columbia agar, carry out: - an oxidase detection (Oxidase test â&#x20AC;&#x201C; MGNMID61G) - the inoculation of a TSI (Triple Sugar Iron) agar - 100 tubes in slopes Incubate 1 - 5 days at 42Â°C in microaerophilic atmosphere

Interpretation of the results

The thermotolerant Campylobacter give the following results: Oxidase + Glucose Lactose Saccharose Gas You can conclude that there is a presence of Campylobacter if at least one colony presents these features.

109

CAMPYLOBACTER according to KARMALI Selective medium for Campylobacter according to Karmali In Vitro use only Store between 2 and 8°C

PRINCIPLE Karmali plates are used for the selective isolation of Campylobacter. Toxique metabolites are neutralised by the addition of activated charcoal or haematin. This allows not to use fresh blood traditionally used. As to improve the Campylobacter tolerance to oxygen the following substances have been added: ferrous salts, sodium metabisulfite and pyruvate. Cefoperazone is added as to prevent the growth of Gram negative strains, Vancomycine for Gram positive strains and Cycloheximide for yeasts. This selectivity does not implies on the growth of Campylobacter coli sensitive to antibiotics. FORMULA In grammes per liter of purified water Special mix of peptone Sodium chloride Corn starch Activated charcoal Haematin Sodium pyruvate Cycloheximide Tris Buffer Agar Cefoperazon Vancomycin

23,00 5,00 1,00 4,00 0,032 0,10 0,10 1,00 14,00 0,032 0,02

BIBLIOGRAPHY 1. Bolton F.J. and Coates D. 1983. Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance. J. Appl. Bacteriol., 54:115-125. 2. Bolton F.J., Coates D. and Hutchinson D.N. 1984. The ability of Campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. J. Appl. Bacteriol., 56:151-157. 3. Karmali M.A., Siumor A.E., Roscoe M., Fleming P.C., Smith S.S. and Lane J. 1986. Evaluation of a Blood-Free, Charcoal-Based, Selective Medium for the isolation of Campylobacter Organisms from Feces. J. of Clinical Microbiology, 23:456-459. 4. Pener J.L. 1988. The Genus Campylobacter : a Decade of Progress. Clin. Microbiol. Rev., 1:157-172. PACKAGING Ready to use plates AEB120380 : Pack of 10 plates ∅ 90 mm

Made by AES Laboratoire - Combourg - France 140412:07/04/06-G

Final pH : 7,4 + 0,2 at 25°C PROCEDURE Isolate the sample (stools, pre-enrichment broth, dilution) directly onto the surface of the plate. Incubate the plate under microaerophilic atmosphere. RESULTS This medium is used to isolate Campylobacter. Colonies characteristics: not rough, greyish colour, translucent, round with clear edges or colonies that smear following the isolation streaks. Observation of the germs through a microscope help to orientate the diagnose since Campylobacter are small curved bacilli can also look like pig tales with a darting motility.

110

BRUCELLA Brucella Broth In Vitro use only To be stored between 1 and 30°C

PRINCIPLE Brucella broth is used for the culture of fastidious germs such as Brucella. The addition of 10% of fresh Sheep blood enhances the growth of fastidious germs such as Brucella, anaerobic germs and Campylobacter. Thus, it can be used as enrichment broth. Campylobacter fetus ssp. jejuni can be transported in this medium at 25°C for more than 3 weeks. FORMULA In grammes per litre of purified water Pancreatic digest of casein (tryptone) Peptic digest of animal tissues Dextrose Yeast extract Sodium chloride Sodium bisulfite

10,00 10,00 1,00 2,00 5,00 0,10

Final pH : 7,0 + 0,2 at 25°C METHOD Suspend 28,1 grammes of powder in one litre of purified water, homogenize well until completely dissolved. Dispatch in appropriate containers then autoclave at 121°C for 15 minutes. PROCEDURE Inoculate tubes or flasks with sample then incubate them at 37°C for 7 days under aerobic atmosphere or enriched with CO2 gas. RESULTS Tubes or flasks showing sign of growth will be subcultured on appropriate solid media. BIBLIOGRAPHY 1. Wang W.L.L., Leuchtefeld N.W., Reller L.B. and Blaser M.J. 1980. J. Clin. Microbiol. 12:479-480. PACKAGING Dehydrated medium AEB140072 : 500 g Made by : AES CHEMUNEX - Combourg - France 140072£ : 20/03/08 - F

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PRESTON Preston selective supplement In Vitro use only To be stored between 18 and 23°C PRINCIPLE Preston selective supplement formula is based on the one described by Bolton et Robertson. It is used to make Preston agar, specially elaborated medium for direct screening (without primary enrichment) of Campylobacter from various origin of sampling (human, animal, avian or environment). It is also used to prepare Preston broth know to be strongly recommended for selective enrichment of samples suspected to be highly contaminated by interfering flora and/or knowingly to have few viable targeted bacteria (Campylobacter). This broth is also added with F.B.P. growth supplement (i.e.: sulphate Ferrous, sodium métaBisulphite, sodium Pyruvate) this allows to incubate under aerobe atmosphere. FORMULA For 2 ml of acetone 50% (in purified water) Actidione Polymyxin B Rifampicin Trimethoprim

50,00 mg 2500 UI 5,00 mg 5,00 mg

PREPARATION PRESTON AGAR (DIRECT SCREENING) Suspend 12,5 g of 2,5% nutritive broth and 5 to 7,5 g of agar (depending on the strength of the agar) in 475 ml of purified water. Heat to boiling to dissolve. Autoclave 15 minutes at 121°C. Cool to reach 42 + 1°C and add 25 ml of sterile haemolysed horse blood cells, and one vial of Preston selective supplement prepared with 2 ml of sterile acetone 50% (in purified water). Mix carefully. Dispense in dishes. All these procedures have to be done under aseptic environment. PRESTON BROTH (SELECTIVE ENRICHIMENT) Suspend 12,5 g of 2,5% nutritive broth in 475 ml of purified water. Heat to boiling to dissolve. Autoclave 15 minutes at 121°C. Cool to reach 42 + 1°C and add 25 ml of sterile haemolysed horse blood cells, one vial of Preston selective supplement prepared with 2 ml of sterile acetone 50% (in purified water), and one vial of growth supplement F.B.P., prepared with 5 ml sterile purified water. Mix carefully. Dispense 5 ml in sterile tubes with closures. All these procedures have to be done under aseptic environment. As to keep a microaerobe atmosphere the remaining space left in the tube must be

as minimum as possible. Preston broth can be kept 7 days at a temperature between 2 and 8°C. PROCEDURE Using an enrichment step : Emulsify the sample in Preston broth. Incubate at 42°C for 24 hours in aerobe atmosphere. Subculture the enriched specimen on an adequate selective solid medium (for example Preston agar). Direct screening : Emulsify the sample in a sterile saline solution. Inoculate the prepared specimen directly onto the surface of a Preston agar solid medium as to have isolated colonies after growth. Incubate at 42°C for 24 to 48 hours (according to the presumed contamination) under a special atmosphere containing 5-6% of oxygen, 10 % of carbon dioxide and 84-85 % of nitrogen. RESULTS Look for typical colonies and undergo Campylobacter authentication tests. BIBLIOGRAPHY 1. Smibert R.M. 1978. Ann. Rev. Microbiol. 32:673-709. 2. George H.A., Hoffman P.S., Smibert R.M. and Kreig N.R. 1978. Improved media for growth and aerotolerance of Campylobacter fetus. J. Clin. Microbiol. 8:36-41. 3. George H.A., Hoffmann P.S., Kreig N.R. and Smibert R.M. 1979. Studies on the microaerophilic nature of Campylobacter fetus subsp. jejuni. II. Role of exogenus superoxide anions and hydrogen peroxide. Can. J. Microbiol. 25:8-16. 4. Bolton F.J. and Robertson L. 1982. A selective medium for isolation Campylobacter jejuni/coli. J. Clin. Pathol. 35:462-467. 5. Bolton F.J., Coates D., Hinchliffe P.M. and Robertson L. 1983. Comparison of selective media for isolation of Campylobacter jejuni/coli. J. Clin. Pathol. 36:78-83. PACKAGING Preston supplement AEB184017 : Lyophilised, vial to make 500 ml of medium 2,5% nutritive broth dehydrated AEB140852 : 500 g F.B.P. growth Supplement AEB184021 : Lyophilised, vial to make 500ml of medium Made by AES Laboratoire - Combourg – France 184017£:22/09/93-A

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CAMPYLOBACTER SELECTIVE AGAR (CASA®)

Selective medium for the detection and enumeration of Campylobacter In Vitro use only Store between 2-8°C

PRINCIPLE Campylobacter is considered as the genus of bacteria that causes the majority of gastro-intestinal diseases in human kind. Campylobacteriosis in humans are generally caused by thermotolerant species of Campylobacter such as C. jejuni, C.coli, C.lari and even C. fetus. with an incidence in Europe of 46,1 for 100.000 inhabitants in 2006. Thermotolerant Campylobacter spp. are widespread in nature. The principal reservoirs are the alimentary tracts of wild and domesticated animals, the environment and foodstuffs from animal origin. ® CASA is a selective medium for the detection and enumeration of thermotolerant Campylobacter in foodstuffs and specimen from patients. ® On CASA the majority of Campylobacter will grow as brick red colonies in 24 to 48 hours of incubation while other bacteria, yeasts and moulds are inhibited by the presence of a mix of highly selective chemicals. FORMULA In grams per litre of purified water Special mix of peptone Starch Sodium chloride Phosphate buffer Agar Mix of antibiotics Chromogenic substrate Final pH : 7,3 + 0,2 at 25°C

20,00 1,00 5,00 10,00 12,00 0,08 0,025

PROCEDURE Food testing : The medium can be used within the framework of ISO 10272-1 standard and ISO/TS 10272-2 technical specification in parallel to mCCDA plates for the detection or the enumeration of Campylobacter. It can also be used within the framework of procedures validated by the laboratory. Campylobacter detection: Subculture the enriched sample ® in Bolton broth onto a CASA plate and the second selective medium (mCCDA) then incubate at 41,5°C+/1°C for 44 hours +/-4 hours under micro-aerobic atmosphere. Campylobacter enumeration : Spread 0,1 ml of the initial suspension and its successive 1 in 10th dilutions (2 plates ® per dilution), then incubate CASA plates at 41,5°C for 44 hours +/-4 hours under micro-aerobic atmosphere.

Medical diagnosis : ® Subculture by swabbing onto CASA plate liquid stools from a patient either directly or after proceeding to an initial suspension or after an enrichment period using an appropriate broth. Incubate the plates at 37°C or 42°C (according to the chosen analytical procedure) for 24 to 48 hours under micro-aerobic atmosphere. INTERPRETATION : After incubation, Campylobacter grow as red brick ® colonies on CASA plates with variable morphology (small with regular outlines or spread out with a red centre). Food testing : Typical colonies of Campylobacter must undergo confirmation tests according to standard procedures or by any other equivalent method. Subculture 5 colonies onto Columbia with sheep’s blood plates. On the colonies obtained after purification, carry out confirmation tests described in the standard procedure. Miniaturized identification system or immunological agglutination tests can be used. Medical diagnosis : From the typical colonies carry out microscope observation with and/or without staining this will allow to orientate easily towards the Campylobacter genus : small incurved bacilli, in the shape of a comma or a spiral with a characteristic motility. Coccoides shape of bacilli can also be observed and are in particular associated with a loss of the capacity of the microorganism to grow (viable but not cultivable phenomenon). Others tests by culture, biochemical or immunological may be carried out to complete the diagnosis. LIMITS AND PRECAUTIONS ®

• CASA is a very selective medium that allows nearly exclusively only the growth of Campylobacter. The majority of Campylobacter can be detected only after 24 hours of incubation. In the event of the absence of colonies after 24 hours of incubation, it is recommended to prolong the incubation period up to 48 hours. As any method to detect pathogens it is strongly recommended to carry out biochemical or immunological confirmation tests on at least one typical ® colony obtained on CASA .

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CAMPYLOBACTER SELECTIVE AGAR (CASA®)

Selective medium for the detection and enumeration of Campylobacter In Vitro use only Store between 2-8°C

BIBLIOGRAPHY 4-

78-

NF EN ISO 10272-1 (2006) : Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement des Campylobacter spp. Partie 1 : méthode de recherche. ISO/TS 10272-2 (2006) : Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement des Campylobacter spp. Partie 2 : technique par comptage des colonies. EFSA Journal (2007) : The community summary report on trends and sources of zoonoses, zoonotic agents, antimicrobial resistance and foodborne outbreaks in European Union in 2006 FEDERIGHI M. Campylobacter et Hygiène des aliments, 1999. KING L.. LEHOURS P., MEGRAUD F : Bilan de la surveillance des infections à Campylobacter chez l’homme en France en 2007.(INVS)

PACKAGING : Ready to use plates : AEB520270 : Pack of 20 plates Ø 90 mm Made by AES CHEMUNEX - Combourg - France 520270 : 03/07/09- A

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Culture media for the food industry

Lactic flora enumeration NF ISO 15214 standard â&#x20AC;&#x201D; September 1998

Decimal dilution of the sample in Peptone salt broth (AEB111499 - pack of 100 tubes of 9 ml)

Inoculation by incorporation on MRS pH 5,7 agar (AEB621756V - 6 flasks of 100 ml) with 1 ml of each dilution Incubation 72+/- 3 hours at 30 +/- 1Â°c

Enumeration of the colonies and expression of the results according to the dilutions

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M.R.S Man, Rogosa and Sharpe agar In Vitro use only To be stored between 2 and 25°C

PRINCIPLE Man, Rogosa et Sharpe agar (M.R.S.) is recommended for isolation and enumeration of Lactobacillus species in milk, milk products and any foodstuffs. MRS agar contains peptone and dextrose that supply nitrogen, carbon and other elements necessary for growth. Polysorbate 80, acetate, magnesium and manganese provide growth factors for cultivating varieties of lactobacilli. Incubating the sample under an enriched carbon dioxide atmosphere will encouraged the Lactobacillus growth. Organisms other than lactobacilli such as Pediococcus and Leuconostoc may grow on this medium, therefore isolates must be conformed as lactobacilli by appropriate biochemical testing. FORMULA In grammes per litre of purified water. Proteose petone Yeast extract Dextrose Potassium phosphate, dibasic Beef extract Sodium acetate Ammonium citrate Magnesium sulfate Manganese sulfate Polysorbate 80 Agar

10,00 5,00 20,00 2,00 10,00 5,00 2,00 0,20 0,05 1,00 15,00

METHOD OF PREPARATION Suspend 70,0 grammes in 1 litre of purified water. Heat to boiling to dissolve completely. Adjust the final pH according to the specimen tested or the flora screened. Autoclave 15 minutes at 121 °C. PROCEDURE Inoculate 1 ml of the specimen or its decimal dilutions in a sterile dish. Then pour 15 ml of the liquefied medium (45-50°C), homogenise well. Let the medium set then turn over the plates before incubation. • ISO 15214 protocol (MRS pH 5,7): Incubate at 30°C for (72+/-3) hours under aerobic atmosphere. Possible inoculation on surface : incubation under anaerobic atmosphere or micro-aerobic atmosphere. • ISO 7889 protocol (MRS pH 5,4): Incubate at 30°C for (72+/-3) hours under anaerobic atmosphere. • NF V04-503 protocol (MRS pH 5,7): Incubate at 25°C for (72+/3) hours under aerobic atmosphere.

LIMITATIONS OF THE PROCEDURE When Lactobacillus are associated with other interfering flora, it is best to use a more selective medium such Rogosa agar. BIBLIOGRAPHY 1. De Man J.C., Rogosa M. and Sharpe M.E. 1960. An improved medium for the cultivation of Lactobacilli. J. Appl. Bact. 23:130-135. 2. Briggs M. 1953. An improved medium for Lactobacilli. J. Dairy Res. 20:36-40. 3. Sharpe M.E., Freyer T.F. and Smith D.G. 1966. Identification of the lactic-acid Bacteria. In : Identification Methods for Microbiologists. Part A. (Gibbs B.M. and Skinner F.A. Ed. London and New York, Academic Press. Pages 65-79. 4. Cox G.P. and Briggs. 1954. Experiments on growth media for Lactobacilli. J. Appl. Bact. 17:18. 5. AFNOR V 04-503. Viandes et produits à base de viande. Dénombrement des bactéries lactiques. 6. ISO 15214 Dénombrement des bactéries lactiques mésophiles. 7. ISO 7889. Yogurt –Enumeration of characteristic microorganisms- Colony-count technique at 37°C. PACKAGING Dehydrated medium (Store between 1 and 30°C) AEB151752 : 500 g MRS agar (acid) pH 5,4 AEB621756A : Pack of 6 flasks of 100 ml AEB621757A : Pack of 6 flasks of 200 ml MRS agar pH 5,7 AEB621756V : Pack of 6 flasks of 100 ml MRS agar (neutral) pH 6,4 AEB121758N : Pack of 100 tubes of 20 ml AEB621756N : Pack of 6 flasks of 100 ml AEB621757N : Pack of 6 flasks of 200 ml AEB521760 : Pack of 120 dishes ( 90 mm) Made by AES CHEMUNEX - Combourg - France 151752£:21/02/08 -K

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Alphabetical Index

1. Diluents Buffered Peptone Water .............................................................................. p.12 Cryo beads .................................................................................................. p.19 D-cycloserine ............................................................................................... p.18 Fraser broth ................................................................................................. p.15 Half Fraser broth.......................................................................................... p.14 Kovacs ......................................................................................................... p.17 Mucap test (adapted protocol for SMS method) .......................................... p.16 Peptone salt (Maximum recovery diluent).................................................... p.13 RPF AFNOR ................................................................................................ p.21 Sterile defibrinated blood ............................................................................. p.20

2. Analysis

ALOA™ ......................................................................................................... p.97 ASAP™ ......................................................................................................... p.48 Bacillus Cereus Rapid Agar (BACARA®) ..................................................... p.107 Baird Parker................................................................................................. p.81 Baird Parker + RPF...................................................................................... p.83 BHI (Brain Heart Infusion)............................................................................ p.84 Blood Agar ................................................................................................... p.103 Brucella........................................................................................................ p.111 Brilliant Green .............................................................................................. p.34 Campylobacter according to Karmali ........................................................... p.110 CASA® ......................................................................................................... p.113 DG18 .......................................................................................................... p.28 DRBC .......................................................................................................... p.26 DNA .......................................................................................................... p.86 Drigalski ....................................................................................................... p.51 Edel Kampelmacher (Brilliant Green Agar ISO)........................................... p.53 EE broth Mossel .......................................................................................... p.32 ESIA / ESSB ................................................................................................ p.71 117

Giolitti Cantoni ............................................................................................. p.87 Hektoen ....................................................................................................... p.67 Kligler-Hajna ................................................................................................ p.54 Lyophilised Rabbit plasma ........................................................................... p.85 Mac Conkey................................................................................................. p.66 mLST .......................................................................................................... p.72 MKTTn ......................................................................................................... p.47 Mossel (MYP) .............................................................................................. p.106 MRS .......................................................................................................... p.116 Nutrient ISO 6579 ........................................................................................ p.55 Oxford .......................................................................................................... p.101 Palcam......................................................................................................... p.102 PCA .......................................................................................................... p.24 Preston ........................................................................................................ p.112 Rebecca™ .................................................................................................... p.42 SALSA™ ....................................................................................................... p.63 SMS™ .......................................................................................................... p.57 SMS™ Confirmation ..................................................................................... p.61 TBX .......................................................................................................... p.40 Thioglycollate resazurin ............................................................................... p.93 TSC and Tryptose sulfite ............................................................................ p.91 TSI .......................................................................................................... p.68 TSYE .......................................................................................................... p.104 RVS (Rappaport Vassiliadis Soja) ............................................................... p.46 Vibrions TCBS ............................................................................................. p.74 VRBG .......................................................................................................... p.33 VRBL .......................................................................................................... p.37 VRBL + MUG ............................................................................................... p.38 Wilson and Blair modified ............................................................................ p.52 XLD ISO 6579.............................................................................................. p.49 XLT4 .......................................................................................................... p.50 Yersinia CIN................................................................................................. p.75

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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

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