aes culture media inserts

AES CHEMUNEX Rue Maryse Basti 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 [email protected]

Culture Media Culture Media Culture Media Culture Media for the Food Industryfor the Food Industryfor the Food Industryfor the Food Industry

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ALOA

ASAP

ESIA

TA003B

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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:

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

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

Our media carry a label or a stamp on which is indicated a preservation temperature. This information is only given as a guide line figure as being the optimum condition for storage established by our laboratory. More importantly, an isolated derive or a moderate variation in temperature that could occur during transport or incubation does not affect (only in scares exceptions) the quality of our products.

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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:

Name of the product, packaging and quantities Storage temperature, Reference, Batch number, expiring date.

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

Name of the product, packaging and quantities Reference, Batch number, expiry date and pH. A conformity statement notifying that the quality control has been carried out following a

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 radio-sterilised media (for more details please contact us).

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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

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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 medias 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.

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 : [email protected]

QQCCPP aanndd ccoonnttrrooll cceerrttiiffiiccaattee mmaannaaggeemmeenntt

Detachable sticker

Self adhesive detachable Q.C. certificate

12 individual self adhesive & detachable stickers

pH measured of the batch

Products abbreviation

Quality Control Protocol reference and index revision letter (Download Quality Control Protocol (QCP) from our web site: www.aeschemunex.com)

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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:

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Culture media for the food industry

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

Total flora ......................................................................................................... p.23 PCA....................................................................................................... p.24

Yeasts and moulds........................................................................................... p.25 DRBC ................................................................................................... p.26 DG18..................................................................................................... p.28

Gram-

Enterobacteria ................................................................................................. 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 (Mller Kauffmann tetrathionate-novobiocine)............................. p.47 ASAP .................................................................................................. p.48 X.L.D ISO 6579 ..................................................................................... p.49 X.L.T.4................................................................................................... p.50 Drigalski ................................................................................................ p.51

1. Diluents & confirmation reagents

2. Analysis

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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

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Dilubag

In order to optimize the sample preparation, the majority of our diluents are available in

3 or 5 litre Dilubags (dilution broth bags).

Ready to start your day !

Dilumat 4 diluter (AESAP1056) Dilusafe rack (AESDI0314)

Diluplug: high flow rate connector (AESDI0313)

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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 10,00 20,00 Sodium phosphate, Dibasic 3,57(1) 7,14 Potassium phosphate, Monobasic 1,50 3,00 Sodium chloride 5,00 10,00

Final pH : 7,0 + 0,2 at 25C

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

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

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 121C.

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

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

BIBLIOGRAPHY 1. NF EN ISO 6579 Microbiologie-Directives gnrales concernant les mthodes de recherche des Salmonella. (2002) 2. ISO/TS 22964 : Lait et produits laitiers Dtection de lEnterobacter sakazakii . (2006)

PACKAGING Dehydrated medium (to be stored between 1 and 30C) AEB140302 : 500 g AEB140303 : 5 kg

Ready measured dehydrated medium (to be stored between 2 and 25C) AEB240309 : qsp 9 litres AEB240318 : qsp 18 litres

Ready to use media (to be stored between 2 and 25C) 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

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

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

Made by AES CHEMUNEX - Combourg - France

140302: 27/10/08 - Q

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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 1,00 Sodium chloride 8,50

Final pH: 7,0 +/- 0,2 at 25C

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 121C for 15 min.

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

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 gnrales pour la prparation des dilutions en vue de l'examen microbiologique.

5. ISO 4833. Directives gnrales pour le dnombrement de micro-organismes.

PACKAGING Dehydrated medium (Storage : between 1 & 30C) AEB141492 : 500 g

Prepared medium (Storage: between 2 & 25C) 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 & 25C) AEB111489 : 100 tubes of 9 ml

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

Made by : AES CHEMUNEX - Combourg - France

141492: 20/11/08 - H

PEPTONE SALT

Isotonic broth for dilution For in vitro use

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HALF FRASER BROTH

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 Disodium Hydrogen Phosphate, anhydrous 9.6 (1) 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 25C

(1) equivalent to 12g of Disodium Phosphate dibasic

(Na2HPO4, 2H2O)

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

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 121C. 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.

PROCEDURE 25 g of test material are first enriched by adding to 225 ml of Half Fraser broth, thoroughly mixed then incubated at 30C 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 37C 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. Fraser and Sperber. 1988. Rapid detection of Listeria spp. In

food and environmental samples by esculin hydrlysis. J. Food Prot. 51: 762-765.

3. Donelly and Baigent. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52: 689-695.

4. AFNOR NF EN ISO 11290-1 / A1. Fvrier 2005. Microbiologie des aliments. Mthode horizontale pour la recherche et le dnombrement de Listeria monocytogenes. Partie 1 : Mthode de recherche.

PRESENTATION Dehydrated medium (Store between 1 and 30C) (With antibiotics) AEB140412: 500g bottle AEB140413: 5 kg barrel AEB240409: QSP 9 litres AEB140417 : 10 Kg Ready to use medium complete (Store between 2 and 25C) 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 25C) AEB110422S: 20 x 10 ml tubes (Store between 1 and 30C) B1110.16 : dehydrated 1 kg barrel

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

Selective enrichment medium for Listeria monocytogenes In vitro use only

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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 5,0 Tryptone 5,0 Meat extract 5,0 Yeast extract 5,0 Sodium chloride 20,0 Sodium phosphate, dibasic 9,6(1) Potassium phosphate, monobasic 1,35 Esculin 1,0 Lithium chloride 3,0 Nalidixic acid 0,02 Acriflavine HCl 0,025

Final pH: 7,2 +/- 0,2 at 25C

(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 121C. 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 30C 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 37C 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. Fvrier 2005. Microbiologie des aliments. Mthode horizontale pour la recherche et le dnombrement de Listeria monocytogenes. Partie 1 : Mthode de recherche.

PACKAGING Dehydrated medium with antibiotics (Store between 1 and 30C) AEB140422: 500g bottle

Ready to use medium - Complete (Store between 2 and 25C) 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 25C) B1110.16 : dehydrated 1 kg (Store between 1 and 30C)

Made by AES CHEMUNEX - Combourg France

140422 : 11/06/09 - I

FRASER BROTH

Selective enrichment medium for Listeria monocytogenes For In Vitro use only

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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.

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.

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 (Adapted protocol for SMS method)

For In Vitro use Store between 2 and 8C

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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, p-Dimethylaminobenzaldehyde reacts with indole to form a pinkish red compound.

FORMULA p-Dimethylaminobenzaldehyde 5,00 g Amyl alcohol 75,00 ml Hydrochloric acid 25,00 ml

Store in an amber flask at 2-8C

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. Immunittsforsch. 55:311-315.

PACKAGING Ready to use solution AEB190504 : 25 ml flask AEB190502 : 50 ml flask


190504 : 09/05/08 - E

KOVACS

Kovacs reagent for the detection of Indole production In Vitro use only

Store between 2 and 8C

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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 45C 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 2-8C or 2 month if frozen at 20C. 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


184002 :23/05/01 - C

D-CYCLOSERINE

Culture media supplement In Vitro use only


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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 20C and 70C. 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 micro-organism.

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

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 70C. Temperature should be at least 20C.

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.

BIBLIOGRAPHY 1. White DJ Sands R.L. 1985. Storage of Bacteria at 76C. 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 76C. Journal of Apply Bacteriology. 44:313-316.

PRESENTATION AEB400100 : box of 64 tubes containing 25 beads.

Made by AES CHEMUNEX Combourg France

400100 : 07/04/08 - D

CRYO BEADS

Preservation of reference strains In Vitro use only


20

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 45-50C 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 45-50C. Homogenize well then heat to 80C in a water bath for 15 minutes. Cool to 45C, 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 hemolytic with on horse blood plate but can be 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

STERILE DEFIBRINATED BLOOD

In Vitro use only Store between 2 and 8C

21

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 375 mg Rabbit plasma EDTA 2,5 ml Trypsine inhibitor 2,5 mg Potassium tellurite 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 48C. 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 37C 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 37C 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.

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 2-8C.

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. Mthode de routine pour le dnombrement des Staphylocoques coagulase positive par technique des colonies 37C. Partie 1 : technique avec confirmation des colonies.

3. Norme AFNOR NF V08-057-2. Mthode de routine pour le dnombrement des Staphylocoques coagulase positive par comptage des colonies 37C. Partie 2 : technique sans confirmation des colonies.

4. Norme NF EN ISO 6888-2. Mthode horizontale pour le dnombrement des Staphylocoques coagulase positive (Staphylococcus aureus et autres espces). Partie 2 : Technique de la glose au plasma de lapin et fibrinogne.

PRESENTATION Prepoured medium. Store between 2 & 8C AEB520330 : Pack of 20 dishes 90 mm AEB520329 : Pack of 120 dishes 90 mm

Ready to use Agar base: Store between 18 & 23C AEB620314 : Pack of 6 x 90 ml flasks AEB620313 : Pack of 6 x 180 ml flasks

Additive: Store between 2 & 8C 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

R.P.F AFNOR

Rabbit Plasma Fibrinogen supplement In Vitro use only

22

AAnnaallyyssiiss

23


Total flora enumeration at 30C

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+/- 1C

Enumeration of the colonies and expression of the results

according to the dilutions.

24

DESCRIPTION The standard Plate Count Agar is used for aerobic micro-organisms 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 5,00 Yeast extract 2,50 Glucose 1,00 Agar 15,00

Final pH: 7,0 +/- 0,2 to 25C

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 121C.

PROCEDURE Liquefy the agar then keep the medium at a temperature of approximately 45-50C. 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 30C for 72 hours. For thermophilic micro-organisms, incubate at 55C. Finally, for psychrophilic micro-organisms, incubate at 7C.

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

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.

BIBLIOGRAPHY 1. FIL-IDF 49 . 1970. Mthode normalise pour le

dnombrement des germes totaux dans les poudres de lait et de lactosrum (mthode de rfrence).

2. FIL-IDF 61. 1971. Crmes glaces et glaces au lait. Dnombrement des germes totaux.

3. J. O. du 27 Aot 1963. Contrle des laits concentrs sucrs et des laits secs.

4. American Public Health Association . 1960. Standard methods for the examination of dairy prducts. 11th ed. APHA Inc. , New York.

5. Mthode officielle pour le dnombrement des germes arobies msophiles. Ministre de lAgriculture. Commission XXX. Cosmtologie.

6. AFNOR V 08-011. Directives gnrales pour le dnombrement des micro-organismes. Techniques par comptage des colonies obtenues 30C.

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

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

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

Pre-poured contact dishes AEB120710C: Pack of 10 dishes 60mm

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

Made by AES CHEMUNEX Combourg France

152852:31/07/07 - G

PCA

Plate Count Agar In vitro use only

To be stored between 2 and 25C

25


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 25C1C 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.

26

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 5,00 Dextrose 10,00 Monopotassium phosphate (KH2PO4) 1,00 Magnesium Sulfate (MgSO4,H2O) 0,50 Dichloran 0,002 Rose Bengal 0,025 Chloramphenicol 0,10 Agar 15,00

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

PROCEDURE

Liquefy the medium in a boiling water bath, then cool the medium to 44-47C 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 transfer 0,1 ml of the first dilution (10-1) for fluid samples or 0,1ml of the 10-2 dilution for solid samples.

Incubate the plates in an upright position at 25C + 1C 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.

- 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 Mthode horizontale pour le dnombrement des levures et moisissures. Partie 1 : technique par comptage des colonies dans les produits activit deau suprieure 0,95.

PACKAGING

Ready-to-use medium AEB620547 : Pack of 6 flasks of 200 ml


620547 : 08/07/09 - A

D.R.B.C

Dichloran-Rose Bengal-Chloramphenicol agar

Usage In Vitro To be stored between 2 and 25C

27


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

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 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.

Incubate at 25C1C 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.

28

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.

FORMULA In grams per litre of purified water

Peptone 5,0 Glucose 10,0 Potassium dihydrogenophosphate 1,0 Sulfate de magnesium, 7H2O 0,5 Dichloran (dichloro-2,6-nitro-4-aniline) 0,002 Glycerol 220,0 Chloramphenicol 0,1 Agar 15,0 Final pH at 25C: 5,6 +/- 0,2

PROCEDURE Liquefy the medium in a boiling water bath, then cool the medium to 44-47C 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 transfer 0,1 ml of the first dilution (10-1) for fluid samples or 0,1ml of the 10-2 dilution for solid samples.

Incubate the plates in an upright position at 25C + 1C 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). Dicloran-glycerol medium for enumaration of xerophilic fungi from low moisture foods. Appl. Environm.Microbiol. 39, 488-492

2. NF ISO 21527-2 : novembre 2008 : Microbiologie des aliments Mthode horizontale pour le dnombrement des levures et moisissures. Partie 2 : technique par comptage des colonies dans les produits activit deau infrieure ou gale 0,95

PACKAGING Ready to use medium AEB611276 : Pack of 6 flasks of 100 ml


611276: 29/07/09 -C

DG18

For in vitro use only Store between 2 and 25C

29

Gram -

30


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 37C

1ml of culture + 10ml of EE broth


Subculture onto VRBG


Select the typical colonies and isolate on a nutritive agar plate


Confirm the Enterobacteriaceae with: - Oxidase reaction (-)

- Glucose fermentation (+)

31


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+/-1C

Select 5 typical colonies (pink, red or purple with or without a halo of precipitation) and inoculate on a nutritive agar


Select one colony on each plate and detect their oxidase. Subculture the colonies with negative oxidase on an agar containing glucose (AEB120129A).


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

32

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.

FORMULA In grams per liter of purified water

Enzyme digest of animal tissus 10,00 Ox Bile 20,00 Glucose 5,00 Disodium phosphate 6,45 Monopotassium phosphate 2,00 Brilliant green 0,0135

Final pH : 7,2 + 0,2 at 25C

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 30C for

RESULTS When the medium becomes cloudy or change colour (yellow / green), it reveals the presumptive presence of Enterobacteriaceae. 242 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 Mthodes horizontals pour la recherch et le dnombrement des Enterobacteriaceae partie 1 : Recherche et dnombrement laide de la technique NPP avec pr-enrichissement.

PACKAGING Ready to use medium AEB111380: Pack of 100 Tubes of 10 ml


111380 : 12/10/06 - A

EE BROTH MOSSEL

Enrichment broth for Enterobacteria according to Mossel In Vitro use only


33

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 3,00 Pepsic meat peptone 7,00 Sodium chloride 5,00 Bile salts 1,50 Dextrose 10,00 Neutral red 0,03 Violet crystal 0,002 Agar 15,00

Final pH : 7,4 + 0,2 at 25C

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 44-47C. 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 37C (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 121C could alter the properties of the VRBG medium, nevertheless, in order to keep the ready prepared media a few days autoclave 20 minutes at 110C.

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: Dcembre 2004. Mthodes horizontales pour la recherche et le dnombrement des Enterobacteriaceae. Partie 2 : Mthode par comptage des colonies 4. AFNOR NF V08-054 : Fvrier 1999. Microbiologie des aliments. Dnombrement des entrobactries 30C - Mthode de routine.

PACKAGING Dehydrated medium (To be stored between 1 and 30C) AEB153202 : 500 g

Ready to use medium (To be stored between 2 and 25C) 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 25C) AEB523210 : Pack of 20 plates 90 mm AEB123210C : Pack of 10 contact plates 65 mm AEB523209C : Pack of 120 contact plates 65 mm


153202: 26/05/08 - L

V.R.B.G.

Violet crystal, neutral Red, Bile Glucose (dextrose) agar

In Vitro use only

34

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 10,00 Ox bile 20,00 Lactose 10,00 Brilliant green 0,0133

Final pH: 7,2 +/- 0,2 at 25C

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 121C.

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 121C. 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 55C. For coliforms detection, incubate at 30C for 24 to 48 hours. For faecal coliforms detection, incubate at 44C 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.

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

Colony-count technique : Subculture 5 atypical colonies on VRBL in a BLBVB single concentration tube. Incubate at 30C or 37C (according to the validated procedure) for 242hours.

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 30C or 37C (according to the validated procedure) for 482hours.

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 aot 1963. Contrle des laits concentrs sucrs et des laits secs. 2. FIL - IDF 40 : Mthode de routine normalise pour le dnombrement des bactries coliformes dans le lait pasteuris. 3. J.O. du 21 septembre 1968. Mthode de prlvement et d'analyse bactriologique des glaces et crmes glaces. 4. FIL - IDF 62 : Crmes glaces et glaces au lait : dnombrement des coliformes. 5. FIL - IDF 65 : Poudres de lait et de lactosrums : dnombrement des coliformes. 6. FIL - IDF 64 : Laits ferments : dnombrement 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 Qualit de leau Recherche et dnombrement des coliformes, coliformes thermotolrants et des E.coli prsums - Mthode NPP. 9. NF ISO 4832- Juillet 2006 : Microbiologie des aliments - Mthode horizontale pour le dnombrement des coliformes. Mthode par comptage des colonies 10. NF ISO 4831- Octobre 2006 : Microbiologie des aliments - Mthode horizontale pour le dnombrement des coliformes. Technique du nombre le plus probable.

BRILLIANT GREEN

Lactose Brilliant Green Bile Broth In vitro use only

35

PRESENTATION Dehydrated medium To be stored between 1 and 30C AEB140502 : 500 g flask

Ready to use medium, with Durham tube (To be stored between 2 and 25C) Simple concentration AEB110509 : Pack of 100 tubes of 10 ml Double concentration AEB110520 : Pack of 100 tubes de 10 ml


140502: 05/01/09

BRILLIANT GREEN

Lactose Brilliant Green Bile Broth In vitro use only

36


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, > 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 (242) hours at (301)C or (371)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.

37

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.


Yeast extract 3,00 Pancreatic Casein peptone 7,00 Sodium chloride 5,00 Bile salts 1,50 Lactose 10,00 Neutral Red 0,03 Violet crystal 0,002 Agar 15,00

pH final : 7,4 + 0,2 25C

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 45C. 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 30C for Coliforms and 44C for faecal Coliforms.

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.

LIMITS A classic cycle of autoclave 15 minutes at 121C could alter the properties of the VRBL medium, nevertheless, in order to keep the ready prepared media a few days autoclave 20 minutes at 110C.

BIBLIOGRAPHY 1. Davis J.G. 1951. Milk Testing. Dairy Industries Ltd., London. 2. F.I.L.-I.D.F. 39. 1966. Mthode de routine normalise pour le dnombrement des bactries coliformes dans le lait cru. 3. F.I.L.-I.D.F. 62. 1971. Crmes glaces et glaces au lait. Dnombrement des coliformes. 4. NF ISO 4832. Juillet 2006. Mthode horizontale pour le dnombrement des coliformes. Mthode par comptage des colonies. 5. AFNOR NF V08-060. Mars 1996. Dnombrement des coliformes thermotolrants par comptage des colonies 44C Mthode de routine. 6. AFNOR NF V08-050. Fvrier 1999. Microbiologie des aliments. Dnombrement des coliformes par comptage des colonies obtenues 30C Mthode de routine.

PACKAGING Dehydrated medium To be stored between 1 and 30C AEB153252 : 500 g AEB153253 : 5 kg

Ready to use media To be stored between 2 and 25C,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 25C, 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


153252: 28/05/08 - H

V.R.B.L

Violet Red Bile Lactose Agar For In Vitro use only


38

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--D-Glucuronide (MUG) to the medium (100mg/l), allows to distinguish Escherichia coli. The test is based on the the -glucuronidase enzyme degrading the MUG substrate into 4-methylumbelliferone 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.


Yeast extract 3,00 Meat peptone 7,00 Sodium Chloride 5,00 Bile Salts N3 1,50 Lactose 10,00 Neutral Red 0,03 Crystal Violet 0,002 MUG 0,10 Agar 15,00

pH final : 7,4 + 0,2 25C

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.

PROCEDURE Liquefy the medium in a boiling water bath then cool the medium to 45-47C. 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 30C, and for fecal coliforms enumeration incubate at 44C

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 121C.

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 30C) AEB1532621 : 500gr

Ready to use medium (to be stored between 2 and 25C) AEB123270C: Pack of 10 contact plates


153262: 09/04/08-D

V.R.B.L MUG

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

39


Horizontal method for the enumeration of the beta-glucuronidase- positive Escherichia coli.

Colony-count technique at 44C 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+/-1C

Enumerate the blue colonies

40

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 -D-glucuronate) allows to screen for the presence of -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 20,00 Bile salts 1,50 BCIG (X-Gluc) 0,075 Agar 14,00

Final pH : 7,2 + 0,2 at 25C

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 44-47C then dispatch in tubes or flasks. Autoclave 15 minutes at 121C.

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 44C for 18 to 24 hours maximum.

RESULTS Escherichia coli will grow as blue colonies.

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 -glucuronidase negative and cannot be detected on the medium.

BIBLIOGRAPHY 1. Norme NF ISO 16649-1 aot 2001

Microbiologie des aliments Mthode horizontale pour le dnombrement des Escherichia coli -glucuronidase positive Partie 2 : Technique de comptage des colonies 44C au moyen de membranes et de 5-bromo-4-chloro-3 indolyl -D-glucuronate.

2. Norme NF ISO 16649-2 juillet 2001 Microbiologie des aliments Mthode horizontale pour le dnombrement des Escherichia coli -glucuronidase positive Partie 2 : Technique de comptage des colonies 44C au moyen de 5-bromo-4-chloro-3 indolyl -D-glucuronate

3. Norme XP ISO/TS 16649-3 dcembre 2005 Microbiologie des aliments Mthode horizontale pour le dnombrement des Escherichia coli -glucuronidase positive Partie 2 : Technique du nombre le plus probable utilisant le 5-bromo-4-chloro-3 indolyl -D-glucuronate

PACKAGING Dehydrated culture media (to be stored between 1 and 30C) AEB152812 : 500 g

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


152812 : 28/04/08-C

T.B.X. (Tryptone Bile X-Gluc)

Medium for the enumeration of Escherichia coli in foodstuffs In Vitro use only

41


Simultaneous detection

of E.coli/enterobacteria E.coli/coliforms

42

PRINCIPLE REBECCA is a chromogenic medium for the direct enumeration without confirmation in products for human and animal consumptions and in the water(1) of:

E. coli - D-glucuronidase positive, E.coli - D-glucuronidase positive and of

enterobacteria, E.coli - D-glucuronidase positive and of

coliformes(2), The enumeration of E.coli is done by the detection of - D-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 The medium REBECCA CF(2) exist in ready to use flasks for the enumeration of both E.coli and coliforms. Coliforms expressing their -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 18,00 Selective agents 5,25 Chromogenic substrate 0,25 Agar 14,00 final pH: 7,3 + 0,2 to 25C

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

FORMULA REBECCA CF In grammes per litre of purified water Peptones & Extracts 18,00 Selective agents 5,25 Chromogenic substrate 0,45 Agar 14,00 final pH: 7,3 + 0,2 to 25C

PREPARATION Dehydrated medium: See indications on the

bottle. Ready to use flasks:

Regenerate REBECCA base flasks at 100C until perfectly liquid. Cool to 44 - 47C. Application 1 : Enumeration of E. coli - D-glucuronidase positive: No supplement is necessary. Application 2: Simultaneous enumeration of E. coli - 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 - D-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 37C 1C.

REBECCA

Rapid Enterobacteriaceae Escherichia Coli Coliform Agar

Use In vitro To be stored between 2 & 8C

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

43

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 44-47C.

KEEPING & STORAGE - Ready to use medium (Flasks, supplement & plates) are stored between 2 & 8C until their expiry date. - Poured plates prepared from the dehydrated base or ready to used flasks can be kept at 2-8C for 15 days. - Flasks prepared from the dehydrated REBECCA base & REBECCA CF can be kept one month when stored at 2-8C. - Once regenerated, the supplement EB can be kept 24 hours at 2-8C, 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 44- 47C 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 44-47C. A supplemented REBECCA flask cannot undergo a melting procedure. - There exist a few strains of E. coli -D-glucuronidase negative, example: E.coli O157. - Some strains, other than E. coli have a -D-glucuronidase enzyme and therefore could grow as blue colonies. Example: Salmonella. BIBLIOGRAPHY

1- NF ISO 16649-2 : Microbiologie des aliments Mthode horizontale pour le dnombrement des Escherichia coli -D-glucuronidase positive partie 2 : Technique de comptage des colonies 44C au moyen de 5-bromo-4-chloro-3-indolyl -D-glucuronate. (2001).

2- NF ISO 21528-2 : Microbiologie des aliments Mthodes horizontales pour la recherche et le dnombrement des Entrobacteriaceae Partie 2 : mthode par comptage des colonies (2004).

3- NF ISO 4832 : Microbiologie des aliments Mthode horizontale pour le dnombrement des coliformes Mthode 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


620027 : 17/01/08 A

REBECCA

Rapid Enterobacteriaceae Escherichia Coli Coliform Agar

Use In vitro To be stored between 2 & 8C

44

Appendix 1: Procedure and interpretation guide

Enumeration of E. coli -D-glucuronidase positive

Simultaneous enumeration of E.coli -D-glucuronidase positive &

enterobacteria

Simultaneous enumeration of E.coli -D-glucuronidase positive and

coliforms (Out of validation)

MEDIUM REBECCA (Base) REBECCA Base* REBECCA CF SUPPLEMENT TO ADD TO REBECCA

(BASE)

None

REBECCA EB supplement*

None INOCULATION See technical data sheet

See technical data sheet

See technical data sheet

INCUBATION 24 hours 2 hours at 37C1C

24 hours 2 hours at 37C1C

24 hours 2 hours at 37C1C

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

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

Other enterobacteria than E. coli -D-glucuronidase +:

Pink to red colonies

E. coli -D-glucuronidase +: Dark blue colonies with or without

Coliforms other than E. coli -D-glucuronidase +:

Pink to purple

ENUMERATION (Method of calculation : confer with

current standards) E. coli -D-glucuronidase + =

Blue colonies

E. coli -D-glucuronidase + = Blue colonies

Enterobacteria = Blue colonies

+ Pink to red colonies

E. coli -D-glucuronidase + = Dark blue colonies

Coliforms = Dark blue colonies

+ Pink to purple colonies

45


Pre-enrichment

25 g sample + 225 ml of Buffered peptone water

Incubate at 371C for 16 to 20 hours

Selective enrichment

0,1 ml sample + 10 ml of RVS broth

Incubate at 41,51C for 243 hours

1 ml sample + 10 ml of MKTTn broth

Incubate at 371C for 243 hours

Isolation

Isolate on ASAP agar and on XLD agar

Isolate on ASAP agar and on XLD agar

Incubate at 371C for 243 hours Incubate at 371C for 243 hours

Salmonella spp., Salmonella H2S- non motile, lactose+ or saccharose+ (S.arizonae, S.indiana), S.typhi, S.paratyphi A, B, C

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 243 hours at 371C

Use of ASAP agar for Salmonella detection in food samples

According to ISO 6579 NF EN 12824 AFNOR V 08-013

46

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 25C

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 115C.

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 41,5C 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 35-37C for 18 to 24 hours.

RESULTS Foodstuff : After incubation subculture on to an adequate selective agar (XLD, ASAP, Hekton or XLT4 Modified).

Faeces studies : After incubation, subculture on to an adapted selective agar, (ASAP, D.C.L.S., Hekton, XLT4 Modified, S.S., etc ...).

LIMITS AND PRECAUTIONS Store the dehydrated medium below 30C. 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 procd 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, Mller-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. Dcembre 2002 - Microbiologie Mthode horizontale pour la recherche des Salmonella spp. 7. NF U 47-100 Fvrier 2005 Mthode danalyse en sant animale Isolement et identification de tout srovar ou de srovar(s) spcifi(s) de salmonelles dans lenvironnement 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

RVS (Rappaport Vassiliadis Soja)

Enrichment broth for Salmonella In Vitro use only


47

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

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