Culture Media in Food Industry

Culture media for the food industry

AES Chemunex – Rue Maryse Bastié – Ker Lann CS17219 – 35172 Bruz cedex - France Tel : +33 (0)2 23 50 12 12 – fax : +33 (0)2 23 50 12 00 – http://www.aes-lab.com – [email protected]

2

ES 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.

A

3

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).

4

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.

5

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.aeslaboratoire.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.

N.B : All our culture media follow the PCQ system excepting dehydrated versions, packs of 5 tubes and packs of 10 ready poured dishes. Your contact: Claire GARDYN – phone: 00 33(0)2 23 50 12 64 - 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

Product’s abbreviation

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

6


Buffered Peptone Water .............................................................................. p.10 Peptone salt (Maximum recovery diluent).................................................... p.11 Half Fraser broth.......................................................................................... p.12 Fraser broth ................................................................................................. p.13 Mucap test (adapted protocol for SMS method) .......................................... p.14 Test Oxidase................................................................................................ p.15 Kovacs ......................................................................................................... p.16 D-cycloserine ............................................................................................... p.17 Cryo beads. ................................................................................................. p.18 Sterile defibrinated blood ............................................................................. p.19 R.P.F AFNOR.............................................................................................. p.20

Total flora..................................................................................................... p.22 PCA................................................................................................... p.23

Yeasts and moulds ...................................................................................... p.24

Y.G.C ............................................................................................... p.25 O.G.A ................................................................................................ p.26 DG18................................................................................................. p.27

Gram-

Enterobacteria ............................................................................................ p.29 EE broth Mossel ................................................................................ p.31 V.R.B.G ............................................................................................. p.32

Coliforms...................................................................................................... p.33

V.R.B.L.............................................................................................. p.34 V.R.B.L – MUG.................................................................................. p.35

E.coli ............................................................................................................ p.36

T.B.X ................................................................................................ p.37 Salmonella ................................................................................................... p.38

ASAP................................................................................................. p.39 RVS (Rappaport Vassiliadis Soja)..................................................... p.40 MKTTn (Müller Kauffmann tetrathionate-novobiocine) ...................... p.41 X.L.D ................................................................................................. p.42 SMS .................................................................................................. p.43

1. Diluents & confirmation reagents

2. Analysis

7

X.L.T.4............................................................................................... p.46 Drigalski ........................................................................................... p.47 Wilson and Blair modified·································································· p.48 Edel – Kampelmacher (Brilliant Green Agar ISO) ····························· p.49 Kligler-Hajna······················································································ p.50

Shigella ········································································································ p.51 Mac Conkey agar ·············································································· p.52 Hektoen····························································································· p.53 T.S.I··································································································· p.54

Enterobacter sakazakii················································································· p.55 ESIA/ESSB ······················································································· p.57 mLST································································································· p.58

Other culture media ···················································································· p.59 Vibrions TCBS··················································································· p.60 Yersinia CIN······················································································ p.61

Gram+ Staphylococci······························································································· p.63

Baird-Parker ······················································································ p.67 Baird-Parker + RPF··········································································· p.69 Brain Heart Infusion Broth ································································· p.70 Lyophilised Rabbit Plasma ································································ p.71 DNA··································································································· p.72 Giolitti Cantoni ··················································································· p.73

Clostridium ·································································································· p.74

T.S.C and Tryptose sulfite································································· p.77 Thioglycollate Resazurin ··································································· p.79

Listeria ········································································································· p.80

ALOA································································································· p.83 Oxford ······························································································· p.86 Palcam ······························································································ p.87 Columbia 3 ························································································ p.88 Blood agar (base)·············································································· p.89 T.S.Y.E······························································································ p.90

Bacillus cereus····························································································· p.91

Mossel (MYP)···················································································· p.92 Campylobacter····························································································· p.93

Campylobacter according to Karmali················································· p.95 Brucella ····························································································· p.96 Preston······························································································ p.97

Lactic flora ··································································································· p.98

M.R.S ································································································ p.99

8

DDiilluueennttss

&&

ccoonnffiirrmmaattiioonn rreeaaggeennttss

9


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)

10

BUFFERED PEPTONE WATER

Dilution buffer In Vitro Use only

To be stored between 18 and 23°C

PRINCIPLE Buffered Peptone water is used for preenriching damaged Salmonella species from food specimens to increase recovery. Its formula is conform to the standards: NF V 08-011, V 08-052, V 04-501 and ISO 6579. This medium is use for the first step of the protocol when screening for Salmonella in foodstuff 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 foodstuff 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 3,56 7,12 Potassium phosphate, Monobasic 1,50 3,00 Sodium chloride 5,00 10,00

Final pH : 7,0 + 0,2 at 25°C Note: in italic characters the formula for double concentration buffered peptone water. METHOD Suspend 20,0 g of powder into 1 litre of distilled water. Bring slowly to the boil, stirring to obtain complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C PROCEDURE In sterile conditions dilute the sample (10 or 25 g) in order to get a decimal dilution. Homogenise well. Incubate round about 18 hours at 37°C, then inoculate the adequate selective enrichment broth according to the analysed product.

BIBLIOGRAPHY 1. AFNOR V 08-011. Directives générales pour le dénombrement des micro-organismes. Méthode par comptage des colonies obtenues à 30°C. 2. AFNOR V 08-013 ISO 6579 Microbiologie-Directives générales concernant les méthodes de recherche des Salmonella. 3. AFNOR V 04-501. Directives générales concernant les méthodes de recherche des Salmonella. 4. AFNOR V08-052 . Méthode de routine pour la recherche de Salmonella dans les produits alimentaires. PACKAGING Dehydrated medium AEB140301: 100 g AEB140302 : 500 g AEB140303 : 5 kg Ready measured dehydrated medium AEB240309 : qsp 9 litres AEB240312 : qsp 12 litres AEB240318 : qsp 18 litres AEB240380/15 : qsp 80 litres Ready to use media 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 AEB610306M : 6 flaks with septum of 100ml AEB110308M : 1 flask of 900ml AEB910303 : Pouch of 3 L AEB910305 : Pouch of 5 L Buffered peptone water double concentration AEB610316 : Pack of 6 flasks 100 ml Buffered peptone water with 0.5% of Tween 80 AEB1110314M : Flask of 490 ml AEB610329L : Pack of 6 flasks of 900 ml AEB910323 : Pouch of 3 L Made by AES Chemunex - Combourg - France 140302£: 02/01/2006 - O

11

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 with ISO standards 6887 and 4833. The maximum recovery diluent has no lethal action on organisms, because of 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 25°C

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. 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. 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.

PACKAGING Dehydrated medium AEB141492 : 500 g AEB141493 : 5 kg Prepared medium 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 AEB111498M : Flask with septum of 900 ml AEB611499L: 6 flasks of 900 mL AEB611501M: 6 flasks with septum of 500 mL AEB911493: Dilibag of 3L AEB911495: Dilubag of 5L Prepared medium with 0,3 % of cystein AEB111489 : 100 tubes of 9 ml Prepared medium with 0,2 % of polysorbate 80 AEB611474 : 6 flasks of 50 ml AEB611476 : 6 flasks of 100 ml Made by : AES Laboratoire - Combourg - France 141492£:16/03/05-E

PEPTONE SALT

Isotonic broth for dilution For in vitro use


12

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 increase 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 amonium ferric citrate to create a dark brown or black complex. The Fraser Demi 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 basis for Fraser Demi In grams per litre of distilled water. Peptone proteose 5.00 Tryptone 5.00 Meat extract 5.00 Yeast extract 5.00 Sodium Chloride 20.00 Disodic phosphate 9.6 Monopotassic 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 Supplement for Fraser and Fraser Demi In grams per 10ml of distilled water Ammonium ferric citrate 0.5 PREPARATION Pour 55 gram of powder into 1 litre of distilled water. If necessary, you may bring to the boil to obtain perfect dissolving. 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.

PROCEDURE Some 25g samples are enriched by homogenising in 225ml of Fraser Demi, and then incubated at 30°C for 18 to 24 hours.

• Alternative method AES 10/3 – 09/00 Inoculate a ALOA agar dish, spreading 0,1 ml of incubated broth. • Routine method AFNOR standard NFV 08 – 055 Perform a second enrichment, pouring 0.1ml of the initial enrichment broth into tubes containing 10ml of Fraser Demi broth. Incubate for 18 to 24 hours at 37°C and isolate at each step by spreading on Oxford or Palcam.

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 other 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 make 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 V 08-055. Décembre 1993. Microbiologie Alimentaire. Recherche de Listeria monocytogenes. Méthode de routine.

PRESENTATION Dehydrated medium AEB140412: 500g bottle AEB140413: 5 kg barrel AEB140414: QSP 4 litres AEB240414: QSP 10 litres AEB240412: QSP 12 litres AEB240418: QSP 18 litres

Ready to use medium AEB610416: 6 x 100ml flasks AEB610418: 6 x 225ml flasks AEB610419L: 6 x 900 ml flasks AEB6140418P: 6 x 225 ml flasks without citrate AEB910913: Dilubag 3 L AEB910915: Dilubag 5 L

Supplement for Fraser AEB110425: 5 x 10 ml tubes B1110.16 : dehydrated 1 kg barrel Made by AES Laboratoire – Combourg – France 140412£: 24/02/04 - G

Selective enrichment medium for Listeria monocytogenes In vitro use only

12

13

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 allegedly contain some esculin hydrolyzing 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. FORMULA Dehydrated basis for Fraser In grams per liter 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 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 25°C Supplement for Fraser and Fraser Demi In grams per 10ml of purified water Ferric ammonium citrate 0,5

PREPARATION Pour 55 grams of powder into 1 liter 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 0,5mg of ferric ammonium citrate. Either liquid or dehydrated ferric ammonium citrate may be added before autoclaving, leading to a final concentration of 0,5 gram per liter of broth. A slight precipitate may appear. It is not prejudicial to the analysis.

PROCEDURE ISO standard ISO 11290-1 : Add 25 grams of test material to 225 ml of Half Fraser broth and mix thoroughly. Incubate at 30°C for 24 hours. Streak the Half Fraser broth culture to Oxford and Palcam plates and transfer 0,1 ml to 10 of Fraser broth. Incubate the plates at 37°C for 24 to 48 hours at 37°C and the Fraser broth at 37°C for 48 hours. Streak the Fraser broth culture to Oxford and Palcam broth. Incubate the plates at 37°C for 24 to 48 hours. Examine plates for suspect colonies.

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 strains for species that are looked for, or they may allow the growth of other 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 make 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. ISO 11290-1 : 1996 – Microbiology of food and animal feeding stuffs – Horizontal methods for the detection and enumeration of Listeria monocytogenes – Part 1 : detection method.

PACKAGING Dehydrated medium AEB140422: 500g bottle AEB140413: 5 kg barrel AEB240414: QSP 10 litres AEB240412: QSP 12 litres AEB240418: QSP 18 litres

Ready to use medium AEB110425 : 5 tubes of 10 ml AEB110429 : Pack of 100 tubes of 10 ml AEB610428 : Pack of 6 flasks of 225 ml Supplement for Fraser AEB110425: 5 tubes of 10 ml B1110.16 : dehydrated 1 kg barrel Made by AES Laboratoire - Combourg – France 140422£ : 13/07/01-F

FRASER BROTH

Selective enrichment medium for Listeria monocytogenes For In Vitro use only


14

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 8°C

15

PRINCIPLE Oxidase is an enzyme produced by some bacteria for electron transport and nitrate metabolism. Detection of this enzyme in freshly grown cultures is useful in the identification of some species. Enterobacteria are oxidase negative Pseudomonas are oxidase positive. The test is based on the intracellular production by the bacteria of oxidase. In presence of oxygen and cytochrome c, the substrate of the enzyme is oxidised into a purple coloured metabolite. FORMULA Strips inpregnanted with N,N,N,N-tetramethyl-1,4 phenylene diamine. PROCEDURE Smear a portion of freshly cultured colony onto the strip. RESULTS A positive reaction is seen when a violet to purple coloration appears after 5 seconds. A negative results is given when an absence of coloration or a late coloration is seen. LIMITS & PRECAUTIONS 1. Freshly grown colonies on selective medium or

medium with high glucose concentrations can give false negative results.

2. Strain producing low levels of oxidase enzyme such as Pasteurella, can show a false negative test after 5 secondes. Discordent oxidase test with metabolism results should be repeated.

3. False positive reactions can be seen when microorganism cultures are not pure (ex: Pseudomonas & Neisseria).

4. A Nichrome wire to pick off the colony should not be used to proceed to the test since this metal may generate false positives.

BIBLIOGRAPHY 1. KOVACS, Nature, 178:703.1956 2. STEEL, K.J., J. Appl. Bacteriol., 25:445, 1962 PACKAGING MGNMID61G : Pack of 50 strips Distributed by : AES Laboratoire - Combourg - France MID61G: 03/11/03-A

OXIDASE TEST

Test to detect oxidase enzyme In Vitro use only

Store between 2 and 8°C

16

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 Amber flask PROCEDURE The test is carried out onto the surface of the appropriate medium such as : - peptone water without indole, - Schubert broth, - S.I.M. agar, - Urea-indole broth - Tryptophane broth. RESULTS Edwersiella, E. coli, Klebsiella oxytoca, Levinea,

Morganella morganii, Proteus rettgeri & vulgaris,

Providencia are INDOLE +. Citrobacter, Enterobacter aerogenes & cloacae,

Hafniae, 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 Laboratoire - Combourg - France 190504£ : 16/02/04-B

KOVACS

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


17

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 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 surfer 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 Laboratoire - Combourg - France 184002 :23/05/01-C

D-CYCLOSERINE

Culture media supplement In Vitro use only


18

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 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 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 –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 a medium appropriate 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 – 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.

PRESENTATION AEB400100 : box of 64 tubes containing 25 beads. Made by AES Laboratoire – Combourg –France 400100£ : 19/09/02 - C

CRYO BEADS

Preservation of reference strains In Vitro use only


19

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

STERILE DEFIBRINATED BLOOD

In Vitro use only Store between 2 and 8°C

20

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 48°C. Homogenize carefully then dispatch into sterile Petri dishes. PROCEDURE Using a glass spatula, spread 0.1 ml of the sample (or its decimals dilutions) onto the surface of a ready poured dish.

Incubate at 37°C for 24, then 48 hours if no growth is detected. RESULTS Enumerate any colonies (black or not) that are surrounded by a precipitation halo. Confirmation of the potential pathogen character of the strain is done by looking for the thermonuclease on Lachica medium.

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 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. PACKAGING Lyophilised RPF supplement Store between 2 and 8°C AEB184100 : AFNOR 12 qsp 100 ml AEB184106: AFNOR 6 qsp 100ml AEB184110: AFNOR qsp 4 l AEB184111: AFNOR qsp 1 l Ready poured medium Store between 2 and 8°C

AEB520330: Pack of 20 dishes (∅ 90 mm)

AEB520329 : Pack of 120 dishes (∅ 90 mm) Made by : AES Laboratoire - Combourg – France 184100£ : 16/01/04 –A

R.P.F AFNOR

Rabbit Plasma Fibrinogen supplement In Vitro use only


21

AAnnaallyyssiiss

22


Total flora enumeration at 30°C

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+/- 1°C

Enumeration of the colonies and expression of the results

according to the dilutions.

23

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 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. 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. 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. 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 prducts. 11

th 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.

PACKAGING Dehydrated medium 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: Pack of 10 contact dishes 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 Laboratoire – Combourg – France 152852£:26/07/04 - F

PCA

Plate Count Agar In vitro use only


24


Yeasts and moulds enumeration by colony-count technique at 25°C

NF ISO 7954 - V08-022 standard - November 1998

Prepare the mother suspension and the dilutions according to the sample.

Inoculate 1 ml of the mother suspension in a Petri dish and repeat the

operation for the dilutions chosen. Pour 15 ml of YGC agar per plate (it is also possible to proceed by spreading

0,1 ml of sample onto the surface of a poured plate)

Incubate up to 5 days at 25+/-1°C Do not turn over the plates during the incubation

Reading at 3, 4 and 5 days

Enumerate all the colonies

25

PRINCIPLE Y.G.C. agar (Yeast extract Glucose Chloramphenicol) is used for screening and enumerating yeast and molds in foodstuff. Its formula is conform to F.I.L.-I.D.F. and I.S.O. standards. It includes Chloramphenicol, a thermostable antibiotic that inhibits the growth of interfering bacteria. FORMULA In grammes per litre of purified water Yeast extract 5,00 Glucose(Dextrose) 20,00 Chloramphenicol 0,10 Agar 15,00 Final pH : 6,6 + 0,2 at 25°C

METHOD Suspend 40,1 grammes into 1 litre of purified water. Homogenize well and heat to boiling point until completely dissolved. Dispatch into flasks. Autoclave 15 minutes at 121°C. PROCEDURE Liquefy the medium then cool to 45-50°C . Place 1 ml of the test product or its decimal dilutions in sterile Petri dishes. Add 15 mL of melted Y.G.C. Homogenize well and let the medium set. Reverse the prepared dishes and incubate 3 to 5 days at 25°C.

RESULTS Enumerate dishes where yeast and molds colonies are no more than 150. Si nécessaire, effectuer un examen de confirmation morphologique au microscope pour chaque type de colonies identifiées. If necessary, carry out morphology confirmation LIMITS AND PRECAUTIONS This medium is used in exchange of OGEA that has the disadvantage that the selective agent, oxytetracycline, has to be added when the analysis is carried out. Y.G.C. has also a longer self life.

BIBLIOGRAPHY 1. International Organization for Standardization (I.S.O.). Milk and milk products - Enumeration of yeasts and moulds - Colony count technique at 25°C. Norm Entwurft ISO/DIN 6611. 2. AFNOR V 04-015. Février 1984. Laits de conserve - Microbiologie - Dénombrement des levures et moisissures. 3. AFNOR V 08-022. Août 1988. NF ISO 7954. Directives générales pour le dénombrement des levures et moisissures. Technique par comptage des colonies à 25°C. 4. AFNOR V 08-059 - Novembre 1995 – Dénombrement des levures et moisissures à 25 °C – Méthode de routine. PACKAGING Dehydrated medium AEB153502 : 500 g Ready to use dishes AEB623506 : Pack of 6 flasks of 100 ml AEB623507 : Pack of 6 flasks of 200 ml Ready poured dishes

AEB523510 : Pack of 20 dishes 90 mm ∅


AEB123501 : Pack of 10 dishes 120 mm ∅ Ready to use contact dishes

AEB123510C : Pack of 10 dishes 65 mm ∅ Made by : AES Laboratoire - Combourg - France 153502£:15/03/05-F

Y.G.C

Yeast extract Glucose Chloramphenicol agar In Vitro use only


26

DESCRIPTION Oxytetracycline Glucose Agar (O.G.A.) is used for the detection and enumeration of yeasts and moulds in food products and cosmetics. FORMULA In grams per liter of distilled water Yeast extract 5,00 Glucose 20,00 Agar 15,00

Final pH : 6,6 + 0,2 à 25°C PREPARATION Pour 40 grams of powder into 1 litre of purified water. Slowly bring to the boil, stirring until complete dissolution. Dispense into flasks..

Autoclave for 15 minutes at 121°C. PROCEDURE

Melt the medium at 45-50°C to achieve total liquefaction. Add 10 ml of sterile oxytetracycline solution to 100ml of melted medium. It is important to mix well the meted medium with its additive solution. Dispense into Petri dishes, and let to solidify. Dry in an incubator with the covers partially removed. Transfer 0,1ml of the product to be analysed and its serial tenfold dilutions to the plates, and spread on the agar surface with a sterile spreader.

Incubate at 20-25°C for 3 to 5 days. RESULTS Enumerate separately the yeasts and moulds. Carry out a microscopic confirmation test for each type of colony found.

BIBLIOGRAPHY 1. J.O. du 8 août 1972. Dénombrement des levures et des moisissures dans les produits cosmétiques. 8553-8554. 2. J.O. du 27 août 1963. Contrôle des laits concentrés sucrés et des laits secs. 3. AFNOR VO3-454. 1971. Epices et aromates. Dénombrement des levures et des moisissures. 4. Buttiaux R. et Catsaras M. 1965. L'analyse bactériologique des bières. Ann. Inst. Pasteur 16:167. 5. Mossel D.A.A., Visser M. and Mengering W.J.H. 1962. A comparison of media for the enumeration of moulds and yeasts in foods and beverages. Lab. Pract. 11:109. 6. Mossel D.A.A., Kleynen-Semmeling A.M.C., Vincentie H.M. 1970. Oxytetracycline- Glucose-Yeast Extract Agar for selective enumeration of moulds and yeasts in foods and clinical materials. J. App. Bact. 33:454-457. 7. Sainclivier M. et Roblot A.M. 1966. Choix d'un milieu de culture pour le dénombrement des levures et moisissures dans le beurre. Ann. Inst. Pasteur 17:181. PACKAGING Base for oxytetracycline glucose agar Dehydrated medium AEB152002 : 500 g flask Ready to use medium AEB622006 : Pack of 6 flasks of 100 ml AEB622007 : Pack of 6 flasks of 200 ml Oxytetracycline solution 1 mg/1 ml Ready to use AEB180307 : 10 ml Tube Lyophilised AEB184000 q.s.p. 500 ml (50 mg/tube) Made by AES Laboratoire - Combourg - France 152002£ : 30/03/04 – C

O.G.A

Base for Oxytetracycline Glucose agarrFor In Vitro use


27

PRINCIPLE DG18 agar 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 food). 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 Enzymatic Digestat of casein 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 25°C: 5,6 +/- 0,2

PROCEDURE Inoculate sterile plates with 1 ml of sample or its decimal dilutions. Pour regenerated medium cooled to 45-47°C, homogenise and let the medium to set. Incubate the plates 5 days at 25°C +/- 1°C. It is strongly recommended not to turn over the plates during incubation as to avoid inoculating the plates with the spores of the moulds. RESULTS Count yeasts and moulds present (Do not enumerate the plates where the moulds are confluent). 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. Projet de norme NF V 08-036. Microbiologie des aliments. Méthode horizontale pour le dénombrement des levures et moisissures se développant sur un milieu à faible aw.

PACKAGING Ready to use medium AEB611276 : Pack of 6 flasks of 100 ml Made by AES Laboratoire - Combourg - France 611276: 03/06/03-A

DG18

For in vitro use only Store between 18 and 23°C

28

Gram -

29


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 37°C

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 (+)

30


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+/-1°C

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.


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

31

DESCRIPTION This liquid medium whose formulation fits with Mossel recommendations, is used for the selective enrichment of Enterobacteriaceae for the bacteriological control of pharmaceutical products that may not be sterile, human and animal food. This broth is the result of a modification of Brilliant Green broth, in which lactose was replaced by glucose and whose buffer effect is reinforced to obtain an early growth and avoid microorganisms destruction by acidification. Using glucose instead of lactose makes the medium adequate for all Enterobacteriaceae. The medium also contains brilliant green and bile salts, necessary for the secondary flora inhibition. FORMULA In grams per liter of purified water Peptone 10,00 Ox Bile 20,00 Glucose 5,00 Disodium phosphate 6,45 Monopotassium phosphate 2,00 Brilliant green 0,015

Final pH : 7,2 + 0,2 at 25°C PREPARATION Pour 43,5 g of powder into 1 liter of purified water. Stir slowly until complete dissolution. Dispense into tubes or flasks. Heat up to 100°C for 30 minutes or 121°C for 5 minutes. DO NOT OVERHEAT. Once the broth has cooled down, a slightly white precipitate may appear at the bottom of the tubes or flasks. PROCEDURE 1. Perform a revivification by incubating dilutions of the product to be analyzed (1/10th in Tryptic Soy Broth), at 25°C for 2 to 8 hours. Dilution should be done so that there is at least 1 cm of liquid above the sample. Homogenize carefully. 2. After revivification mix Mossel broth with the sample (10 times the initial volume). For important volumes, mixing should be made with equal volumes with double concentration of Mossel broth. Homogenize carefully. 3. Incubate at 30°C for 18-24 hours.

RESULTS When the medium becomes cloudy or change colour (yellow / green), it reveals the presumptive presence of Enterobacteriaceae. A final diagnosis should be obtained with isolation and selective media. NOTES For an enumeration (between 1 and 100 for 1 ml or gram of samples), the “most probable number” method should be used (after a 37°C incubation for 24 hours). For enumerations that should be above 100 for 1 ml or gram of sample, an inoculation in V.R.B.D. agar should be preferred, followed by an incubation at 37°C for 24 hours. LIMITS AND PRECAUTIONS Autoclaving at 121°C for 5 minutes is ideal if the medium can be cooled down quickly. In case of a preparation with double concentration, it is advised to heat up to 100°C for 30 minutes. In any case, the medium should be green at the end of sterilization. BIBLIOGRAPHY 1. Mossel D.A.A., Visser M. and Cornelissen A.M.R. 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of all Enterobacteriaceae. J. Bact., 84:381. 2. Mossel D.A.A., Visser M. and Cornelissen A.M.R. 1963. The examination of foods for Enterobacteriaceae using a test of the type generally adopted for the detection of Salmonella. J. Appl. Bacteriol. 26:444-452. 3. AFNOR V08-021. Décembre 1985. Microbiologie alimentaire. Directives générales pour le dénombrement sans revivification des Enterobacteriaceae. 4. Pharmacopée Européenne. Milieu E. PACKAGING Dehydrated medium AEB140372 : 500 g flask Ready to use medium AEB611376 : Pack of 6 flasks of 100 ml AEB111379 : Pack of 100 Tubes of 9 ml Made by AES Laboratoire - Combourg – France 140372£: 22/01/04 - J

EE BROTH MOSSEL

Enrichment broth for Enterobacteria according to Mossel In vitro use only


32

PRINCIPLE Violet crystal, neutral red bile glucose agar is used for

screening and enumeration Enterobacteria in water,

dairy, and any foodstuff samples. Its formula corresponds

to the one described by Mossel and used in I.S.O.,

AFNOR V 08-021 and V 08-054 standards. 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 En grammes par litre d'eau distillée

Yeast ectract 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 13,00

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 and cool to 45°C. Place 1 ml of the tested product and its decimal dilutions in empty sterile Petri dishes. 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

dishes, reverse way up, 30 OR 37°C (according to

standards) for 24 hours.

RESULTS Enumerate the pink to purple colonies of at least 0,5 mm

∅ and surrounded 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 It is not necessary to autoclave this medium never the

less, in order to keep the ready prepared media a few

days autoclave 15 minutes at 121°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. International Organization for Standardization. 1977.

Meat and meat products. Detection and enumeration of

Enterobacteriaceae (Reference methods). Draft

International Standard ISO/DIS 5552.

4. AFNOR V 08-021 : Décembre 1993. Directives

générales pour le dénombrement sans revivification des

Enterobacteriaceae. 5. AFNOR V 08-054 : Octobre 1993. Dénombrement des Enterobacteriaceae par comptage des colonies. Méthode de routine.

PACKAGING Dehydrated medium

AEB153202 : 500 g

Ready to use medium

AEB623206 : Pack of 6 flasks of 100 ml

AEB623207 : Pack of 6 flasks of 200 ml

AEB123209 : Pack of 100 tubes of 15 ml

Ready poured media

AEB523210 : Pack of 20 dishes 90 mm

AEB123210C : Pack of 10 contact dishes 65 mm

AEB523209C : Pack of 120 contact dishes 65 mm

Made by :

AES Laboratoire - Combourg - France

153202£: 30/10/03-I

V.R.B.G.

Violet crystal, neutral Red, Bile Glucose (dextrose) agar For In Vitro use


33


General directives for the enumeration of coliforms

Colony-count technique

NF ISO 4832 – July 1991

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

Inoculation by incorporation on VRBL agar

(AEB623256 - 6 flasks of 100 ml) with 1 ml of each dilution + incorporation of a double layer of VRBL

agar when the first layer is dried

Incubation 24+/- 2 hours at 30°C or 35°C or 37°C

Typical colonies: Dark pink to purple, diameter ≥ 0,5mm with or without halo.

Enumeration of the typical colonies on the plates containing less than 150 colonies and expression of the results

according to the dilutions.

34

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, les 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 V 08-015

standard.

Crystal violet and bile salts inhibits 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 3,00

Pancreatic Casein peptone 7,00


Bile salts 1,50

Lactose 10,00

Neutral Red 0,03


Agar 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 dishes. 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

dishes, reverse way up, for 24 hours at 30°C for

Coliforms and 44,5°C 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 It is not necessary to autoclave this medium never the

less, in order to keep the ready prepared media a few

days autoclave 15 minutes at 121°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. AFNOR V 08-015. Juillet 1991. Directives générales

pour le dénombrement des coliformes. Méthode par

comptage des colonies obtenues à 30°C. 5. AFNOR V 08-017. Juin 1980. Directives générales pour le dénombrement des coliformes fécaux et d'Escherichia coli.

PACKAGING Dehydrated medium


AEB153252 : 500 g

AEB153253 : 5 kg

Ready to use media

To be stored between 18 and 23°C,in the dark

AEB623256 : Pack of 6 flasks 100 ml



Ready poured

To be stored between 18 and 23°C, in the dark

AEB123260C : Pack of 10 contact dishes ∅ 65 mm

AEB523259C : Pack of 120 contact dishes ∅ 65 mm

AEB523260 : Pack of 20 dishes ∅ 90 mm

Made by :


153252:26/07/04 - F

V.R.B.L

Violet Red Bile Lactose Agar For In Vitro use only


35

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. 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. When 4-Methyl-Umbelliferyl-β-D-Glucuronide (MUG) is added to the medium (100mg/l), it may be used for the direct detection of Escherichia coli.

The test is based on the the β-glucuronidase enzyme degrading the MUG substrate into 4-methylumbelliferone that is fluorescent under a 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 3,00 Peptomeat 7,00 Sodium Chloride 5,00 Bile Salts N°3 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 à 25°C

PREPARATION Pour 41,6 gr of powder into 1 litre of distilled water. While bringing to the boil, stir slowly until powder is totally dissolved. DO NOT AUTOCLAVE.

PROCEDURE Cool the medium to approximately 45°C. Place 1ml of the product to be tested (or its decimal dilutions) into sterile Petri dishes. Pour around 12ml of sterile melted medium. Mix and allow solidification on a cold horizontal surface. Pour a second 2mm layer of medium. Allow to solidify and turn the dishes over for incubation. Incubate for 24 hours. For coliforms enumeration incubate at 30°C, and for fecal coliforms enumeration incubate at 44,5°C

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 Although Proteus vulgaris presence may inhibit gaz production, Escherichia coli may be identified due to the appearance of a fluorescence after approximately 15 hours. 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 AEB1532621 : 500gr AEB123270C: Pack of 10 contact plates Made by : AES Laboratoire - Combourg - France 153262£: 14/05/02-C

V.R.B.L – MUG

V.R.B.L – MUG Agar For In Vitro diagnosis


36


Horizontal method for the enumeration of the beta-glucuronidase- positive 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

37

PRINCIPLE T.B.X. is a selective medium for the enumeration of Escherichia coli in Fodstuff. Its formula is complies to the ISO 16449-2 standard. The selectivity of the medium is due to the presence bile salts that inhibit the growth or Gram positive. The chromogenic substrat 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 20,00 Bile salts 1,50 BCIG (X-Gluc) 0,075 Agar 14,00 Final pH : 7,2 + 0,2 at 25°C METHOD Suspend 35,6 grs of powder to 1 litre of purified water. Homogenise well, and bring to the boil under continuous homogenisation in order to dissolve the Agar. Cool to 50°C then dispatch in tubes or flasks. Autoclave 15 minutes at 121°C. PROCEDURE Enumeration : Dispatch 1 ml of the sample or its dilution in then bottom of a sterile Petri plate (two essays per sample). Pour 15 ml of regenerate medium, homogenise the leave to cool on a flat surface until set. Incubate the prepared plates reverse way up at 44°C for 18 to 24 hours maximum. RESULTS Escherichia coli will show as blue colonies.

LIMITS & PRECAUTIONS High concentration of intrusive flora (> 150 CFU/plate) can trouble the enumeration. Some other strains of Entrobacteria can grow as blue colonies (Salmonella, Shigella, …) Some strains of Escherichia coli, such as

Escherichia coli 0 157, are β-glucuronidase

negative and can not be distinguished on the medium.

BIBLIOGRAPHY 1. 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. PACKAGING Dehydrated culture media AEB152812 : 500 g Ready to use medium AEB622816 : Pack of 6 flasks 100 ml AEB122019 : 100 tubes of 15ml Made by AES Laboratoire - Combourg - France 152812 : 20/01/04-B

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

Medium for the enumeration of Escherichia coli in Foodstuff

In Vitro use only To be stored between 2 and 8°C

38


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

Incubate at 41,5±1°C for 24±3 hours

1 ml sample + 10 ml of MKTTn broth

Incubate at 37±1°C for 24±3 hours

Isolation

Isolate on ASAP agar and on XLD agar

Isolate on ASAP agar and on XLD agar

Incubate at 37±1°C for 24±3 hours Incubate at 37±1°C for 24±3 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 24±3 hours at 37±1°C

Use of ASAP agar for Salmonella detection in food samples

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

39

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 coloration of their colonies.

Colonies characteristics on ASAP : Salomnella 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 molds inhibition

FORMULA In grammes per litre of distilled water: Peptones 10 Opaque agents 10 Chromogenic and inhibitor blend 13 Agar 15 Final pH: 7.2 +/- 0.2 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+/-1°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+/-1°C for 24h+/- 3 hours.

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

LIMITS When used in clinical diagnostic, some stool samples can give to the agar a pink coloration a specially 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. A prolongation of incubation could decrease the medium specificity. 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 Laboratoire – Combourg – France 520090£ : 04/11/03-D

ASAP (AES Salmonella Agar Plate)

Chromogenic medium for the isolation of Salmonella For In Vitro diagnosis


40

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. 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 1.44 Magnesium chloride 6H2O 28,6 Malachite Green Oxalate 0,036 Final pH : 5,2 + 0,2 at 25°C Bringing back the volume to 1000 ml and taking into consideration the form anhydrous of Magnesium chloride and volumetric variation the reconstitution is of 26,7 g/l.

METHOD Suspend 26,7 grammes of powder in to one litre of purified water. Homogenize slowly until complete dissolution. Dispatch 10 ml per tube. Autoclave 15 minutes at 115°C.

PROCEDURE 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. When proceeding to microbiology control of foodstuff, Rappaport-Vassiliadis 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. Homogenize well and incubate at 41,5°C for 21 to 27 hours.

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

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.

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 : 22/11/02 - A

RVS (Rappaport Vassiliadis Soja)

Enrichment broth for Salmonella In Vitro use only


41

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 extracts 4,3 Enzyme digest of casein 8,6 Sodium chloride 2,6 Calcium carbonate 38,7 Sodium Thiosulfate (5H2O) 47,8 Oxgall 4,78 Brilliant green 9,6 mg

Iodine/iodide Solution Iodine 20,00 g Potassium Iodide 25,00 g Purified sterile water 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 at 37°C +/- 1°C for 21 to 27 hours. Note : When screening for Salmonella in veterinary samples an incubation at 42°C is more appropriate.

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. Complete media prepared by the laboratory should be used immediately. Ready to use base medium can be keep up to 3 weeks at 2-8°C. This medium is use in parallel with a second selective medium, 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. PACKAGING Dehydrated medium Store between 18 and 23°C AEB140902 : 500 g Ready to use medium (The tubes provided already added with the iodine/iodide solution and novobiocine) Store between 2 and 8°C AEB121609 : Pack of 100 tubes of 10 ml Novobiocine Supplement (10 mg) AEB184150 :QSP 250 ml of medium

Made by

AES Laboratoire - Combourg – France

140902£ :11/05/04 - B

MKTTn (Müller Kauffmann tetrathionate-novobiocine)

Salmonella selective enrichment broth In Vitro use only

42

PRINCIPLE X.L.D. (Xylose Lysine Desoxycholate), as described by Taylor, was developed principally for isolating Shigella and Providencia from stools. It has been shown to be more effective than other enteric differential media. The principal assets of this medium are :

• Acid production : due to Lactose and/or saccharose and/or xylose fermentation (Medium colour change from red to yellow)

• Alkaline reversion : due to Lysine decarboxylation into cadaverin, (LDC+ colonies turn out red).

• Hydrogen sulfide (H2S): Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions.

This medium inhibits the growth gram+ micro-organisms and most unwanted coliforms. FORMULA In grammes per litre of purified water Yeast Extract 3,00 L-Lysine 5,00 Xylose 3,50 Saccharose 7,50 Lactose (monohydrated) 7,50 Sodium desoxycholate 2,50 Sodium Chloride 5,00 Sodium thiosulfate 6,80 Ferric Ammonium Citrate 0,80 Phenol Red 0,08 Agar 13,50 Final pH : 7,4 + 0,2 at 25°C

METHOD Suspend 55,0 grammes of powder in 1 litre of purified water. Heat slowly under constant agitation up to 90°C, until the agar is completely dissolved). DO NOT NE AUTOCLAVE. It is important not to boil the medium, as soon as the medium is dissolved to 50°C then pour into sterile Petri dishes.

PROCEDURE The medium must translucide and of orangey-red colour. An excess heating or prolongated periode at 50°C can cause an un wanted precipitation making the ready difficult. A filtation , of the liquefied medium allows to regain the classical aspect, never the less the efficiency of the medium is lowered. Home made medium have to be stored at 4°C in the dark and up to 15 days. Manufactured medium can de stored at room temperature up to 3 months. Inoculate directly the dishes with the sample and/or the selective enrich sample (Müller-Kauffmann broth, Selenite broth) Incubate for 18to 24 hours at 37°C.

RESULTS Enterobacteria ferment very quickly the xylose (except Edwardsellia, Proteus morganii, Proteus rettgeri, Providencia, and Salmonella paratyphi A that are xylose -) This allows to defferentiate from Shigella (Red colonies). As xylose is exhausted Salmonella then decarboxylate lysine causing reversion to alkaline conditions. Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions. Alkaline conditions reversion by other lysine-positive organisms is prevented by excess acid production from fermentation of lactose and saccharose. Moreoer these acid conditions also inhibits the H2S production. Yellow opaque colonies : Citrobacter, Enterobacter, Escherichia coli, Klebsiella, Proteus et Serratia. Red colonies : Providencia, Salmonella H2S - and Shigella. Red colonies with a black center: Arizona, Edwardsiella et Salmonella

LIMITS AND PRECAUTIONS An excess incubation 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 saccharose 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. Pharmacopée Européenne Milieu K.

PACKAGING Ready to use medium AEB623406 : Pack of 6 flasks of 100 ml Ready to use medium


AEB523409 : Pack of 120 dishes 90 mm ∅ Made by : AES Laboratoire - Combourg - France 153402£:11/06/04 – I

X.L.D

Xylose Lysine Desoxycholate Agar In Vitro use only


43


AFNOR Validation n° AES 10/04-05/04 According to NF EN ISO 16140 for all human and animal

food products and environmental samples. End of validation: 05/07/08

DAY 0 : Pre-enrichment

25 g sample +

225 ml Buffered Peptone Water

Incubation 16 to 20 h at 37°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

DAY 2 : Results

Plates presenting typical colonies within 24 h

are considered positive.

Negative: no migration or

migration are < 2 cm

Presumptive positive:

red migration area > = 2 cm

Confirmation: by any classical test from

international standards

Protocol for Salmonella detection - method

44

Selective medium for the detection of Salmonella In Vitro use only

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 halo of growth 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. PREPARATION For the preparation of the media using the dehydrated base and supplements, please refer to annex I. PROCEDURE SMS method (food and environmental samples) – Refer to annex II : • The SMS is inoculated after a pre-enrichment phase of 16 to 24 hours at (37±1)°C in buffered peptone water or any other diluent required by the screened sample (as describe 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 dish 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 plates 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 prolong 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, growth of Salmonella is visible as a red zone of at least 2 cm extending out from the inoculated drop, due to the action of lysine decarboxylase producing alkaline metabolites. A turbid migration zone precedes generally the red zone. Plates which present this profile have to be regarded as presumptively positive and will have to be subject 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 (SMS Method), all positive results have to be confirmed by one of the following methods :

1. By traditional 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 any other AFNOR 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 unmatched 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 presumptuous positive dishes by using MUCAP test (OUT OF AFNOR FRAMEWORK OF VALIDATION). Tests carried out through out the trial periods, preliminary and collaborative studies, shown 100 % specificity and sensitivity for this test. (A report is available upon request).

AFNOR VALIDATION N° AES 10/04-05/04 according to NF EN ISO 16140 standard for food and animal feeding stuffs and

environmental samples. Limit of validation (07/05/2008)

S.M.S (Simple Method for Salmonella) page1/2

45

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 dishes. It is wise to take out the dishes 15 minutes before proceeding to the analysis letting the dishes acclimatise to 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 plate 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 dish 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 in fewer numbers making it virtually impossible to have a variation of the pH indicator in 24 hours.

• As to help the reading of the dishes particularly those that have totally turned red we advise the compare then to a negative or none inoculated one. Presumptuous positive dishes are characterised by strong opacity due to the migration of Salmonella in the medium.

• After incubation, SMS dishes can be stored at refrigerated temperatures (48 hours maximum) before interpretation and possibly confirmation. • The use of the media prepared from the dehydrated base and supplements, requires some precautions (annex I)

PACKAGING Pre-poured medium AEB520069 : Pack of 120 plates (90 mm diameter) AEB520070: Pack of 20 plates (90 mm diameter) Dehydrated media and supplements: AEB141062: SMS base – 500 g (570 test) 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. Mucap test AEB191500 : Pack of 160 tests Made by AES Laboratoire - Combourg - France 520069£ : 11/01/05 – N

AFNOR VALIDATION N° AES 10/04-05/04 according to NF EN ISO 16140 standard for food and animal feeding stuffs and

environmental samples. Limit of validation (07/05/2008)

S.M.S (Simple Method for Salmonella) page1/2

Selective medium for the detection of Salmonella In Vitro use only

46

PRINCIPLE X.LT.4 modified is a highly selective medium used for the isolation of Salmonella (except Salmonella Typhi and other related species). Most 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-4-Undecyl Sulfate at 27%).

FORMULA In grammes per litre of purified water Special peptone 1,60 Yeast Extract 3,00 L-Lysine 5,00 Xylose 3,75 Saccharose 7,50 Lactose (monohydrated) 7,50 Sodium desoxycholate 2,50 Sodium Chloride 5,00 Sodium thiosulfate 6,80 Ferric Ammonium Citrate 0,80 Phenol Red 0,08 Agar 13,50 Final pH : 7,4 + 0,2 at 25°C

METHOD 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 NE 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 Enterobacteria ferment very quickly the xylose (except Edwardsellia, Proteus morganii, Proteus rettgeri, Providencia, and Salmonella paratyphi A that are xylose -) This allows to defferentiate from Shigella (Red colonies). As xylose is exhausted Salmonella then decarboxylate lysine causing reversion to alkaline conditions. Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization of hydrogen sulfide production under alkaline conditions.

Alkaline conditions reversion by other lysine-positive organisms is prevented by excess acid production from fermentation of lactose and saccharose. Moreover these acid conditions also inhibit the H2S production. Inoculate directly the dishes with the sample and/or the selective enrich sample (Müller-Kauffmann broth, Selenite broth) 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 yellow to pinkie dark red black centred colonies. 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 ET PRECAUTIONS An excess incubation end 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. 3. Norme AFNOR NF U 47-100 – juillet 2001 – Méthode d’analyse en santé animale – Isolement et identification des salmonelles ou recherche de sérovars particuliers dans

l’environnement des productions animales.

PACKAGING Dehydrated agar base Store between 18 and 23°C AEB153412: 500 g Supplement SEMUS ¼: Store between 18 and 23°C AEB180950: QSP 10 Litres Ready to use medium Store between 2 and 8°C AEB523420: Pack of 20 dishes 90 mm ∅

AEB523419 : Pack of 120 dishes 90 mm ∅ Made by : AES Laboratoire - Combourg - France 153412£:03/11/04 - E

X.L.T.4

Selective medium for screening Salmonella In Vitro use only

47

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 15,00 Meat extract 3,00 Yeast extract 3,00 Sodium Thiosulfate 1,00 Bile salt 1,00 Lactose 15,00 Violet crystal 0,005 Bromothymol blue 0,08 Agar 12,00

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 45-50°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.

RÉSULTS 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. 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

DRIGALSKI

Lactose agar for selective isolation of Enterobacteria For In Vitro use


48

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.


Casein pancreatic peptone 5,00

Meat peptone 5,00

Meat extract 5,00

Dextrose 5,00

Disodium phosphate 4,00

Bismuth sulfite 8,00

Ferrous sulfate 0,30

Brilliant green 0,025

Agar 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 dishes. Let the

dishes set on a cold horizontal surface. Dry the dishes 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 Il est conseillé d'ensemencer parallèlement des milieux

moins sélectifs tels que gélose au D.C.L. selon Hynes,

gélose au D.C.L.S., gélose Hektoën, gélose de Mac Conkey

avec cristal violet, gélose lactosée au vert brillant et au rouge

de phénol selon Kristensen ou gélose au X.L.D.

Il est conseillé d'utiliser le milieu coulé en boîtes de Pétri le

jour même de sa préparation, surtout si l'échantillon à

contrôler est suspecté d'être fortement contaminé. Après 3 à 4 jours de stockage à 4°C, le pouvoir inhibiteur est fortement atténué et il conviendra alors pour les produits faiblement contaminés.

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. 26:374-391.

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 Dehydrated medium AEB153292 : Flacon de 500 g

Ready to use medium

AEB623296 : Coffret de 6 flacons de 100 ml

Made by :


153292£ : 19/06/04 - E

WILSON AND BLAIR MODIFIED

BISMUTH SULFITE AGAR In Vitro use only


49

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 10,00 Beef extract 5,00 Lactose 10,00 Sucrose 10,00 Yeast extract 3,00 Disodium Hydrogen Phosphate 1,00 Sodium Dihydrogen Phosphate 0,60 Phenol red 0,09 Brilliant green 0,0047 Agar 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. PROCEDURE Liquefy the medium then cool down to 45-50°C. Dispatch

20 ml by sterile Petri dishes. Let the dishes set on a cold

horizontal surface. Dry the dishes in the incubator with

the lid slightly open. Using an inoculating loop inoculate the dish by isolation streaks with a sample from a selective enrichment broth such as Rappaport-Vassiliadis. Incubate at 35-37°C for 48 hours.

RESULTS Characterise 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 confirmation tests.

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 AEB151492 : 500 g Ready to use medium AEB621497 : Pack of 6 flasks of 200 ml Ready poured medium AEB521500 : Pack of 20 dishes 90 mm ∅ Made by : AES Laboratoire - Combourg - France 151492£:01/10/04 –E

EDEL-KAMPELMACHER

BRILLIANT GREEN AGAR ISO In Vitro use only


50

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 20,00 Yeast extract 3,00 Meat extract 3,00 Dextrose 1,00 Lactose 10,00 Sodium chloride 5,00 Sodium thiosulfate 0,50 Ferrous citrate 0,50 Phenol red 0,025 Agar 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 well-isolated 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 (non-fermentation). Cracks, plits, or bubbles in the medium indicate gas production. A black precipitate in the butt indicates hydrogen sulfide production.

GLU. LAC. H2S GAS

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 + - - -

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 Kligler-Hajna 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.

KLIGLER-HAJNA

Kligler-Hajna Agar In Vitro use only

To be stored between 18 & 23°C

51


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 )

Isolate on XLD agar

Incubate 20 -24 hours at 37+/-1°C

Incubate an additional 24 h if the result is

negative

Isolate on Hektoen 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

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

52

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 clinical, dairy, foodstuff, waters, pharmaceutical and industrial sources. In clinical samples this medium is adapted for the screening of Salmonella, Shigella et des Escherichia coli in babies stools. 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 17,00 Proteose Peptone 3,00 Monohydrated Lactose 10,00 Bile salts 1,50 Sodium chloride 5,00 Neutral red 0,03 Crystal violet 0,001 Agar 13,50 Final pH : 7,1 + 0,2 at 25°C

METHOD Pour 50,0 g of powder into 1 litre purified water. Bring slowly to the boil, homogenise until complete dissolution. Dispense into tubes or flasks. Sterilize in autoclave for 15 minutes at 121°C.

PROCEDURE Liquefy the medium then cool to 45-50°C and dispense into sterile Petri dishes. Inoculate the surface in order to obtain isolated colonies. When analysing, water, dairy, foodstuff, dispense 1 ml of the sample or its decimal dilutions into a sterile Petri dish then pour about 15 ml of medium (45-57°C), homogenise and let the medium set. When screening for pathogen Enterobacteria in babies stools or urine use in parallel a second selective medium such as D.C.L.S for Salmonella and Shigella. Incubate 18 to 24 hours at 37°C.

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 AEB151602 : Flacon de 500 g* Ready to use medium AEB621606 : Pack of 6 Flasks of 100 ml* AEB621607 : Pack of 6 flasks of 200 ml* Ready poured medium

AEB521610 : Pack of 20 dishes 90 mm ∅ Made by : AES Laboratoire - Combourg - France 151602: 22/01/04-G * : Products not stamped.

MAC CONKEY AGAR

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


53

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 12,00 Yeast extract 3,00 Bile salts 9,00 Lactose 12,00 Saccharose 12,00 Salicine 2,00 Sodium chloride 5,00 Sodium thiosulfate 5,00 Ferric citrate 1,50 Bromothymol blue 0,064 Acid Fuchsine 0,10 Agar 13,50

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.

RÉSULTS 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.

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. 21:32-37. 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 : Flask of 200 ml Ready to use plates AEB521160 : Pack of 20 plates 90 mm AEB52159 : Pack of 120 plates 90 mm

Made by : AES Laboratoire - Combourg - France 151152: 30/05/05 - G

HEKTOEN

Hektoen Agar In Vitro use only


54

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 In grammes per liter of purified water Mix of peptone 20,00 Dextrose 1,00 Lactose 10,00 Sucrose 10,00 Sodium chloride 5,00 Sodium thiosulfate 0,20 Ferrous sulfate 0,20 Phenol red 0,025 Agar 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 well-isolated 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 (non-fermentation). 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.

Butt Slant H2S Citrobacter AG A + Edwardsellia A/AG A + Enterobacter aerogenes AG A - Enterobacter cloacae AG A - Escherichia coli AG A - Klebsiella AG A - Proteus mirabilis AG A + Proteus vulgaris AG A + Proteus morganii A/AG NC/ALC - Proteus rettgeri A NC - Shigella dysenteriae A NC/ALC - Shigella sonnei A NC/ALC - Salmonella typhi A NC/ALC + Salmonella paratyphi A AG NC/ALC - Salmonella cholereasuis AG NC/ALC - Salmonella enteritidis AG NC/ALC + Salmonella typhimurium AG NC/ALC + AG : acid production (yellow) and gas A : acid production (yellow) NC : No colour change (red) ALk : 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 AEB152902 : 500 g Prepared medium in flask AEB122909 : Pack of 100 slanted tubes Made by AES Laboratoire - Combourg - France 152902:09/01/03-F£

T.S.I

Triple Iron Sugar Agar In Vitro use only


55

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 (3L) & AEB910305 (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 (45 ± 0,5)°C(1) for (24 ± 2) hours.

(1) : It is recommended to use a water bath as incubation temperature should never go over 45,5°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)

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.

Typical colonies None typical colonies: Purple to mauve

colonies

56


Homogenize X g of sample In 9 X ml of ESSB broth


Isolate on ESIA agar


Presence of typical colonies (blue colonies)

Confirmation on microwell or

automated identification system

Absence of typical colonies (purple colonies)

Absence of Enterobacter

sakazakii in X gram of sample

How to use ESIA agar for the detection of Enterobacter sakazakii

57

PRINCIPLE Enterobacter sakazakii is an enterobacteria which was blamed in cases of neonatals infections convereyed 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 specie 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 which produces typical blue colonies. FORMULA In grammes per litre of purified water Peptone 7 Yeast extract 3 Sodium chloride 5 Sodium desoxycholate 0.6 Crystal violet 0.002 X-alpha-glucopyranoside 0.15 Agar 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 45-50°C then pour in Petri plates. PROCEDURE Detection of Enterobacter sakazakii according to standarded procedure: Refer to ISO/TS 22964 standard. 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 foodstuff 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 sakazakii spp. appear blue. Using well isolated typical colonies Enterobacter sakazakii procedure to confirmation tests. LIMITS & PRECAUTIONS Some coliform can grow on ESIA inolation 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. 10:398-401.

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. 39:293-297.

3. ISO/TS 22964 : Milk and milk products – Detection of Enterobacter sakazakii.

PACKAGING Dehydrated AEB150002 : 500 grs Ready to use ESIA AEB520010 : Pack of 20 plates 90 mm Ready to use ESSB AEB611448 : Pack of 6 flasks 225 ml Made by AES Laboratoire - Combourg - France 520010£ : 05/09/05 –B

ESIA

Enterobacter sakazakii isolation agar In Vitro use


58

PRINCIPLE Modified Lauryl sulfate with Vancomycin (m LST) was specially formulated for the detection of Enterobacter sakazakii in the frame work of the technical specification project : ISO/PRF 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 vancomicin on the other hand associated to an incubation at 45°C, make this medium an ideal enrichment broth for Enterobacter sakasakii while inhibiting the growth of interfering germs present in this category of sample. FORMULA En grammes par litre d'eau distillée Tryptose 20,00 Lactose 5,00 Sodium chloride 34,00 Potassium dihydrogen phosphate 2,75 Dipotassium hydrogen phosphate 2,75 Sodium lauryl sulfate 0,10 Vancomycin 0.01

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 45°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 45,5°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.

LIMITS & PRECAUTIONS In the refrigerator (2-8°C), the sodium laureth sulphate could precipitate. The reaction is reversible with increasing of temperature. This will not affect the properties of the broth. BIBLIOGRAPHY ISO/PRF TS 22964: Milk and milk products – Detection of Enterobacter sakazakii (2005). PACKAGING Ready to use medium AEB110549 : Pack of 100 tubes of 10 ml Made by AES Chemunex - Combourg - France 110459£ : 03/11/05 - B

m LST

Modified Lauryl sulfate with Vancomycine In Vitro use only

To be stored beteen 2 and 8

59


Other culture media

• Vibrions TCBS

• Yersinia CIN

60

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 salt

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 distilled water

Yeast Extract 5,00

Peptone 10,00

Sodium Thiosulfate 10,00

Sodium Citrate 10,00

Biliary salt 8,00

Saccharose 20,00

Sodium Chloridea 10,00

Ferric Citrate 1,00

Bromothymol Blue 0,04

Thymol Blue 0,04

Agar 16,00

Final pH : 8,4 + 0,2 à 25°C

PREPARATION Suspend 90,0 grammes of powder in 1 litre of distilled

water.

Bring slowly to the boil, shaking until complete

dissolution DO NOT AUTOCLAVE

PROCEDURE Liquefy the medium 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, every

other 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-Sucrose-

Agar 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 Laboratoire - Combourg – France 153182:20/02/02-D

VIBRIONS TCBS

Selective medium for Vibrio cholerae For in vitro use


61

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.


Peptone 20,00

Yeast extract 2,00

Mannitol 20,00

Sodium Pyruvate 2,00


Magnesium sulfate 0,01

Sodium desoxycholate 0,50

Irgasan 0,004

Neutral red 0,03


Agar 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 4°C for 21 days)

Incubate the plates at 22-32°C for 24 to 48 hours.

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

AEB153452 : 500 g

CN. Selective supplement AEB184011 : q.s.p. 500 ml, lyophilised*

Ready to use medium

AEB123460 : Coffret 10 boîtes de 90 mm

Fabriqué par


153452:30/05/05 -E * : Product none CE stamped.

YERSINIA C.I.N

Yersinia Selective Agar In vitro use only


62

Gram+

63


Enumeration of Staphylococci aureus

NF EN ISO 6888-1 standard ; NF EN ISO 6888-2 standard - 2003

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

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

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

6888-1 standard

6888-2 standard

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.

64


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

65


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 Enumeration method (NPP)

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, …)

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, …)

66


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.

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

OR

67

DESCRIPTION Baird Parker Agar is a highly specific and selective medium for isolation and enumeration of coagulase-positive 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 10,00 Meat extract 5,00 Yeast extract 1,00 Sodium pyruvate 10,00 Glycine 12,00 Lithium chloride 5,00 Agar 15,00 Final pH : 7,0 + 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 distilled 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. 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 dishes.

PROCEDURE Samples are diluted as desired and the dilutions spread-inoculated onto the agar surface. Incubate plates at 37°C for 24 hours and 48 hours if no typical colony grow 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 probably to the action of a lipoproteinase and 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 sulfamethazine is recommanded in order to inhibit the growth of Proteus. Bacillus give brown colonies, yeasts 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 opac halo and, very rarely, a clear zone. 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. Europeen Pharmacopa. O medium.

PACKAGING Dehydrated medium. To be stored between 18 and 23°C AEB150302 : 500 g Ready to use base. To be stored between 18 and 23°C AEB620316 : 6 bottles of 100 ml AEB620317 : 6 bottles of 200 ml AEB620314 : 6 bottles of 90 ml AEB620313 : 6 bottles of 180 ml

BAIRD –PARKER (page 1/2)

Base for Baird-Parker Agar In Vitro use only

68

Ready to use Baird Parker (Pharma.) base. To be stored between 18 and 23°C AEB620316P: pack of 6 flasks of 100 ml Ready poured Baird Parker with Sulfamethazine To be stored between 2 and 8°C

AEB520320S: Pack of 20 dishes 90 mm ∅ AEB520319S: Pack of 120 dishes 90 mm ∅ Ready poured medium. To be stored between 2 and 8°C


AEB520319 : Pack of 120 dishes 90 mm ∅ Ready poured Baird Parker RPF To be stored between 2 and 8°C

AEB520330 : Pack of 20 dishes 90 mm ∅ AEB520329 : Pack of 120 dishes 90 mm ∅ Ready to use sterile tellurite egg yolk. To be stored between 2 and 8°C AEB180053 : 5 ml tube AEB180054 : 25 ml bottle AEB180052 : 50 ml bottle AEB180056 : 100 ml bottle Ready to use sterile solution of sulfamethazine at 0,2 %. To be stored between 2 to 8°C. AEB180042 : 2,5 ml tube AEB180408 : 12,5 ml tube RPF supplement To be stored between 2 and 8°C AEB184100: 12 qsp 100ml AEB184106: 6 qsp 100 ml AEB184107: 6 qsp 200 ml Made by : AES Laboratoire - Combourg - France 150302£ : 07/01/05 - K

BAIRD –PARKER (page 2/2)

Base for Baird-Parker Agar In Vitro use only

69

DESCRIPTION Baird Parker Agar with Rabbit Plasma Fibrinogen (RPF) is a higly selectif and specific for isolation and enumaration of coagulase-positive Staphylococcus from foodstuffs and pharmaceutical products. The direct detection of a coagulase on the medium is possible due to the presence of R.P.F. FORMULA In grammes per liter of distilled water Tryptone 10,00 Meat extract 5,00 Yeast extract 1,00 Sodium pyruvate 10,00 Glycine 12,00 Lithium chloride 5,00 Rabbit plasma 25,00 ml Trypsine inhibitor 0,025 Potassium tellurite 0,025 Agar 17,00 pH final : 7,2 + 0,2 à 25°C METHOD 1. Bring a bottle of BP agar base to boiling and cool to 45-47°C. 2. Reconstitute a vial of RPF lyophilized supplement with 20 ml sterile purified water and mix gently until complete dissolution. Pre-heat the regenerated supplement at 37°C. 3. Poor the reconstituted supplement in the bottle of BP agar base. Mix well. 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.

RESULTS Enumerate colonies (with black coloration or not) with 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. 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 520330 : 03/10/05- F£

BAIRD-PARKER + RPF

Baird-Parker Agar with R.P.F For In Vitro diagnosis

70

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 NF V08-014. 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.

FORMULA In grammes per liter of distilled water 200 grs of infusion from calf brains 12,50 250 grs of infusion from beef heart 5,00 Proteose peptone 10,0 Dextrose 2,0 Sodium chloride 5,0 Disodium phosphate 2,50

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 V08-014. 1983. Microbiologie alimentaire - Directives générales pour la recherche et le dénombrement de Staphylococcus aureus. Méthode par comptage des colonies. 4. AFNOR V59-105 Octobre 1982. Gélatine alimentaire -

Recherche de Staphylococcus aureus.

PACKAGING Dehydrated medium 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* Made by : AES Laboratoire - Combourg - France 140102£ : 31/05/06 -H * : Product not stamped.

BRAIN HEART INFUSION BROTH

B.H.I BROTH For In Vitro diagnosis


71

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.

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.

PACKAGING Lyophilised rabbit plasma

AEB184088 : Flask of 7,5 ml for 15 tests

Ready to use BHI broth (to be stored between 18-23°C) AEB110110 : Coffret de 100 tubes de 10 ml

Made by :


184087£: 07/04/05 – D

LYOPHILISED RABBIT PLASMA

Detection of free coagulase In Vitro use only


72

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 Pancreatic Digest of Casein 15,00 Papaic Digest of Soybean Meal 5,00 Deoxyribonucleic Acid 2,00 Sodium Chloride 5,00 Agar 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 AEB150052 : 500 g Ready to use medium AEB120059 : 100 tubes of 20 ml Prepared medium AEB121061 : Pack of 10 plates 55 mm Made by AES Laboratoire - Combourg - France 150052:29/10/04 - D

D.N.A

DeoxyriboNucleic Acid agar For In Vitro use

To be stored between 18 and 23°°°°C

73

PRINCIPLE

Giolliti-Cantoni broth is an enrichment medium used to enrich small quantities of Staphylococcus

aureus in foods. The concentrations of lithium chloride and tellurite potassium prevent the growth of interfering Gram negative and negative 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 gramme 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. 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. PROCEDURE Regenerate prepared tubes at 100°C for 15 minutes. Cool then add 0.1 ml of a 1% tellurite solution of each tube (single concentration). Add 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 Agar-Agar. When using double concentration broth, proceed

as with single concentration broth but add 0,2 ml of

tellurite solution and 10 ml of the tested product.

Incubate at 37°C for 24 to 48 hours

RESULTS Subculture on to Baird Parker plates all tubes that show a dark precipitate or blackening of the medium. LIMITS & 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 AEB140392 : 500 g Preapred medium base (with polysorbate and

without potassium tellurite) – single concentration (SC) AEB110399 : Pack of 100 tubes 10 ml Preapred medium base (with polysorbate and

without potassium tellurite) –double concentration (DC) AEB110400 : Pack of 100 tubes 10 ml Sterile tellurite solution (1 %) AEB180453 : Tube 5 ml Made by : AES Laboratoire - Combourg - France 140392:14/02/04-E

GIOLITTI-CANTONI

Giolitti-Cantoni broth (base) In vitro use only


74


Horizontal method for the Clostridium perfringens enumeration

NF EN ISO 7937 standard - 2005

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

on 5 colonies by plate.

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 37°C

Confirmation with biochemical

identification test

Subculture the selected colonies in a Thioglycollate broth.

Incubate in anaerobic

atmosphere 18 - 24 hours at 37°C

If the black colonies are not well isolated, inoculate 5 typical colonies

in a Thioglycollate broth.

Incubate in anaerobic atmosphere

18 - 24 hours at 37°C

75


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 nitrate-motility medium.

Incubate in anaerobic atmosphere 24 hours at

37°C

Inoculate the gelatine

lactose broth.

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.

Note the gas production and the lactose fermentation (yellow colouring).

Place the tubes 1h at

5°C. If the medium

solidifies (gelatine liquefaction), re-

incubate 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.

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

Incubate 18-24 hours at 46°C

Isolate on TSC agar plates

Incubate in anaerobic atmosphere

18-24 hours at 37°C

76


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.

77

DESCRIPTION Tryptone Sulfite agar with or without cycloserine, as described by Harmon, Kautter and Peeler, is used for selective isolation and enumeration of anaerobe sulfito-reducing germs in particular Clostridium perfringens in animal and human foods. Tryptose sulfite agar (without egg yolk) has the advantage of producing small colonies that are easy to count (especially dishes 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 polymyxine or kanamycine. 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 liter of distilled water Tryptone 15,00 Papaic digest of soybeam meal 5,00 Yeast extract 5,00 Sodium metabisulfite 1,00 Ferric ammonium Citrate 1,00 Agar 15,00 Final pH : 7,6 + 0,1 at 25°C

PREPARATION Pour 42 grams of powder in 1 litre of distilled 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.

PROCEDURE � Food stuff analysis with T.S.C. agar:

Liquefy the medium around 45-50°C. Reconstitute a tube of D-cyclosérine supplement with 5 ml of distilled water, in order to obtain a 4% solution (1 tube of reconstituted supplement gives 500ml of medium). 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 Dish: Place 1 mL of each decimal dilution into a sterile Petri dish. 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 dishes are set, incubate the dishes, 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 dish of Tryptose sulfite. Incubate under anaerobic atmosphere for 24 to 48 hours at 37°C.

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

LIMITS AND PRECAUTIONS Avoid heating inoculated tubes. The base medium (without cycloserine) prepared in laboratory may be kept for 2 weeks at 4°C in compliance with the V08-019 and V08-056 standards. Either ready to use or prepared in laboratory, the base medium should be regenerated at 100°C for 20 minutes before use.

T.S.C and Tryptose sulfite (page1/2)

Base for Tryptone Sulfite Cycloserine agar In Vitro use


78

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. AFNOR T90-417. 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 : Microbiology of food and animal feeding stuffs – Horizontal method for enumaration of Clostridium perfringens – Colony-count technique. 4. AFNOR V08-056 : Microbiologie des aliments - Dénombrement des Clostridium perfringens à 37°C - Méthode de routine. 5. AFNOR V08-061 : Microbiologie des aliments - Dénombrement en anaérobiose des bactéries sulfito-réductrices par comptage des colonies - Méthode de routine. PACKAGING Dehydrated medium AEB152892 : 500 g bottle Ready to use medium (without D-cycloserine) for food stuff analysis AEB122895 : 5 tubes of 20 ml 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 (Controled by NF T 90-461 standards) AEB122895E: Pack of 5 tubes of 20 mL AEB122899E: Pack of 100 tibes of 20 mL AEB622896E: Pack of 6 flasks of 100 mL Lyophilised D-Cycloserine - 200 mg/tube AEB184002 : q.s.p. 500 ml Made by AES Chemunex - Combourg - France 152892£: 21/12/05 - I

T.S.C and Tryptose sulfite (page 2/2)

Base for Tryptone Sulfite Cycloserine agar In Vitro use


79

PRINCIPLE

The fluid thioglycollate medium is used for detecting microorganisms in normally sterile materials. It support the growth of aerobic and anaerobic microorganisms. It’s prepared according to the formula of NF V 08-019 standard, European and US Pharmacopoeia. Sodium thioglycollate and L-cystine lower the oxidation-reduction potential of the medium by removing oxygen and preventing the accumulation of peroxides which can be toxic to 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 liter of purified water Pastone 15,00 Yeast extract 5,00 Dextrose 5,50 Sodium chloride 2,50 L-Cystine 0,50 Sodium Thioglycollate 0,50 Resazurin 0,001 Agar 0,75

pH final: 7,0 + 0,2 à 25°C PREPARATION Suspend 14,6 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. PROCEDURE Homogenize the sample to be analysed or its dilutions in the diluent. REFERENCES 1. United States Pharmacopeia. XXIX edition. Sterility tests. 2. J.O. du 25 Octobre 1978. Essai de stérilité. 8233-8237. 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. AFNOR V 08-019. Directives générales pour le dénombrement de Clostridium perfringens.

PACKAGING Dehydrated medium AEB141292 : 500g AEB141292RSTE : 500g* Prepared medium AEB111404: Pack of 100 tubes of 10 ml AEB111403: Pack of 5 tubes of 10ml* AEB111409: Pack of 100 tubes of 20 ml AEB611404 : Pack of 6 flasks of 90 ml* Prepared medium in flask with septum AEB611406M: Pack of 6 flask of 100 ml* AEB611403MAF: Pack of 6 flasks of 50 ml* AEB111410M: Flasks of 750 ml* Ready to use medium doubled sealed 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 141292£ : 23/12/2005 - N * : Product no stamped.

THIOGLYCOLLATE RESAZURIN

Fluid Thioglycollate medium For In Vitro use


80


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.

Incubation 24 h±3h at 37±1°.

Incubation de 24 h ± 3h if necessary

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

81

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 -

Confirmation :

1 – Catalase test 2 – Gram colouring

3 – Motility examination (optional test)

4 – Research of haemolysis

5 – Carbohydrates use

6 – CAMP test


82


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)

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.

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

83

ALOA (Agar Listeria selon Ottaviani & Agosti) page1/2

A selective medium for the isolation of Listeria monocytogenes In Vitro use diagnostic only


PRINCIPLE ALOA is a medium used for the screening of L .monocytogenes in foodstuffs samples (AFNOR validated method – see enclosed protocol). This medium is also used to carry out screening and enumeration of L.monocytogenes & L.spp in foodstuffs and any kind of samples. 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 in obtained the combination of lithium chloride, anti microbial and antifungical components.

FORMULA (IN G/L OF MEDIUM) Animal tissu enzyme digest 18 g Caseine enzyme digest 6 g Yeast extract 10 g Sodium pyruvate 2 g Glucose 2 g Magnesium glycerophospahte 1 g Magnesium Sulphate 0,5 g Sodium chloride 5 g Lithium chloride 10 g Disodium hydrogen phosphate anhydrous 2,5 g X-glucoside 0,05 g Nalidixic acid 0,02 g Ceftazidime 0,02 g Polymyxin B 76700 U Amphotericin B 0,01 g Phosphatidylinostol 2 g Agar 13,5 g Final pH 7,2 +/- 0,2 at 25°C

PROCEDURE Detection and enumeration of Listeria monocytogenes according to standard method : Refer to standards ISO 11290-1 & -2 amendment 1 (2004). Detection of Listeria monocytogenes according to ALOA ONE DAY method ( AFNOR validated) : Inoculate the plates directly from the sample or from an appropriate enrichment culture medium (to perform the isolation, see the enclosed procedure AFNOR validated ALOA ONE DAY method). Incubate at 37°C and read plates after 24 and 48 hours.

RESULTS Note colonies characteristics : Listeria spp.: blue to blue-green colonies, round, regular, without any opaque halo, diameter from 1 to 2 mm. Listeria monocytogenes: colonies with Listeria spp. characteristics and surrounded by an opaque halo. Listeria monocytogenes strains grow as typical colonies in 24 hours. Within the framework of the AFNOR validation (ALOA ONE DAY Method), all positive results have to be confirmed by one of the following methods : 1 – By traditional tests described in the standardized methods ISO, CEN or AFNOR on colonies isolated from ALOA plates. An initial purification of isolated colonies is necessary. 2 – By any other AFNOR validated 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). Conservation of incubated Half Fraser broth cannot exceed 48 hours. In the event of unmatched results (positive result with ALOA ONE DAY Method not confirmed by the selected confirmation method), the laboratory must carry out sufficient means to be ensured of the validity of the result.

PROCEDURES LIMITATIONS When in presence of plates highly contaminated, the reading can be helped by comparing the opacity of the Agar between the centre and the sides of the dish (confer to the protocol) or by comparing to a none inoculated ALOA plate. L.monocytogenes presence, even in great numbers, is characterised by an intense opacification of the medium. This allows to easily differentiate plates where L.monocytogenes are present (opaque medium) from those where they are absent (clear medium). When in doub, subculture on to a second ALOA plate. After 24 hours of incubation, some strains Listeria ivanovii can show a fine 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 allows to differentiate the two species without

hesitation.

ALOA ONE DAY AFNOR VALIDATION N° AES 10/3-09/00 for food and animal feeding stuffs and environmental samples.

84

ALOA (Agar Listeria selon Ottaviani & Agosti) page2/2

A selective medium for the isolation of Listeria monocytogenes In Vitro use diagnostic only


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 none homogeneous colour and a very intense halo.

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 medium : AEB520080 : Pack of 20 plates ∅ 90 mm

AEB520079 : Pack of 120 plates ∅ 90 mm

AEB120082 : pack of 10 plates ∅ 140 mm Dehydrated medium + supplements : Please contact us. Made by AES Laboratoire – Combourg – France 520080: 08/03/05 - Q

ALOA ONE DAY AFNOR VALIDATION N° AES 10/3-09/00 for food and animal feeding stuffs and environmental samples.

85

Listeria monocytogenes detection in food samples

Protocol (AFNOR validated n°10/3-09/00)

Enrichment

25 g sample + 225 ml ½ Fraser

Incubate at 30±1°C for 24±2 hours

Inoculation Spread 0,1 ml on ALOA®

Incubate at 37±1°C for 24 to 48 hours

Advice : A 0,1 ml inoculum spread on the plate with a 0,5 com uninoculated edge is the ideal method

Results

Absence of typical colonies

Absence of Listeria monocytogenes

Typical colonies

Presence* of Listeria monocytogenes

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

Confirmation

Biochemical Listeria identification

Method

86

OXFORD

Isolation of Listeria monocytogenes For in vitro diagnosis

To be stored between 2 and 8 °C

DESCRIPTION The OXFORD medium, which is based on Curtis's formulation, is recommanded for the isolation of Listeria monocytogenes in biological samples and foods. It contains a nutritif base and inhibitors : lithium chloride inhibits Enterococcus growth, acriflavin inhibits Gram - 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 liter of distilled 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 Cycloheximide 400,00 mg Colistin sulphate 20,00 mg Cefotetan 2,00 mg Fosfomycin 10,00 mg Acriflavin 5,00 mg pH: 7,0 +/- 0,2 at 25°C

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.

LIMITS AND PRECAUTIONS THE GROWTH OF MOST OF THE GRAM + STRAINS IS INHIBED, NEVERTHELESS, A FEW ENTEROCOCCUS STRAINS ARE PARTIALLY INHIBED AND GIVE COLONIES WITH SLIGHT HALO AFTER A 40 HOURS INCUBATION. Some of Staphylococcus strains growth without halo.

BIBLIOGRAPHY 1. Curtis G.D.W., Mitchell M.G., King A.F. and Griffin E.J. 1988. Personnal Communication. John Radcliffe Hospital, Oxford, U.K.

PACKAGING Dehydrated medium AEB151992N: 500g AEB151993N 5 Kg Prepoured medium AEB522000: pack of 20 dishes 90mm AEB521999: pack of 120 dishes 90mm AEB122002: Pack of 10 dishes 140 mm Made by: AES Laboratoire – Combourg – France 151992N: 20/02/02 - G

87

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.

FORMULA In grammes per litre of purified water Peptones 23,00 Corn starch 1,00 Yeast extract 3,00 Sodium chloride 5,00 D- Glucose 0,50 Mannitol 10,00 Esculin 0,80 Ferric ammonium citrate 0,50 Lithium chloride 15,00 Phenol red 0,08 Agar 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.

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.

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 : 1,5-3 mm, surrounded by a white halo. Identification of Listeria monocytogenes typical colonies will be carried out according to ISO standard specifications.

BIBLIOGRAPHY 1. 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.

2. 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.

3. 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.

4. 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

PALCAM

Listeria monocytogenes selective medium In Vitro use only

88

PRINCIPLE Columbia 3 agar is a riche peptone media used for the growth of fastidious microorganisms with or without the addition of blood, for example Streptococcus or Pneumococcus. Columbia 3 agar has a similar formula to the previous one, but a more accurate chose of peptone helps to improve the haemolyse reading.

FORMULA In grammes per litre of distilled water Special mix of peptone 23,00 Strach 1,00 Sodium Chloride 5,00 Agar 13,00 Final pH : 7,3 + 0,2 at 25°C

METHOD Suspend 42,0 grammes of powder in 1 litre of purified water. Heat with frequent agitation up to boiling point to completely dissolve the agar. Dispense and sterilize by autoclaving at 121°C for 15 minutes.

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

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

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

PACKAGING Dehydrated medium To be stored between 18 et 23°C AEB151002N : 500 g Flask * Ready to use medium base To be stored between 18 and 23°C AEB620657 : Pack of 6 flasks of 200 ml Ready poured medium with Sheep’s blood To be stored between 2 and 8°C

AEB520680 : Pack of 20 dishes 90 mm ∅ Dehydrated medium + CNA To be stored between 18 and 23°C AEB150662 : 500g Flask* Ready to use medium base with CNA To be stored between 18 and 23°C AEB620666 : Pack of 6 flasks of 100 ml Ready poured medium with Sheep’s blood and CNA To be stored between 2 and 8°C AEB520690 : Pack of 20 dishes 90 mm ∅ AEB125970 : 10 half dishes (Drigalsky/Columb. CNA SB) Made by AES Laboratoire - Combourg – France Réactif enregistré à l'Agence du Médicament 151002N£: 30/05/05 -E * : Product not stamped.

COLUMBIA 3

Columbia 3 Agar base In Vitro use only

89

PRINCIPLE

The medium favours the growth of fastidious germs such Streptococci, pneumococci and Haemophilus. The pigment production of germs is facilitated. FORMULA In grammes per litre of purified water Proteose peptone 15,00 Liver digestion 2,50 Yeast extract 5,00 Sodium chloride 5,00 Agar 13,00

Final pH : 7,4 + 0,2 at 25°C PREPARATION Suspend 40,5 grammes of powder in one litre of purified water. Bring slowly to the boil under constant homogenisation. Dispatch in appropriate containers.

Autoclave 15 minutes at 121°C. PROCEDURE Fresh blood Agar Add to the liquefied cooled to 42+/-1°C Agar base 5 % of fresh blood homogenise well the pour into sterile Petri plates. After inoculation incubate the plates at 37°C for 18 to 24 hors. Note: when screening for Haemophilus it is wise to star the isolation with and Horse blood Agar plate.

Cooker blood Agar To prepare chocolate agar or « Cooked blood agar », add 10% of defibrinated sterile Horse blood to liquefied Columbia 3 at 42° C + 1°C. Under frequent agitation raise the temperature 80°C and cook until the medium takes a chocolate colour, then dispense in sterile dishes. Mycoplasme : Blood Agar base can be added before sterilisation with 5 grammes of dextrose, 0.125 grammes of thallium acetate. And after sterilisation with (medium cooled to 45°C) 100.000 IU of penicillin and 200 ml of sterile horse serum.

BIBLIOGRAPHY 1. Waterworth P.M. 1955. Brit. J. Exp. Path. 36(2):186-194. PACKAGING Dehydrated base AEB152452 : 500 g Made by : AES Laboratoire - Combourg - France 152452£ : 31/05/05 - D

BLOOD AGAR (BASE)

Base for Blood Agar In Vitro use only


90

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 AFNOR NF V 08-055 and ISO 11290. FORMULA In grammes per liter of purified water Tryptone 17,0 Soytone 3,0 Sodium chloride 5,0 Dipotassium Phosphate 2,5 Dextrose 2,5 Yeast extract 6,00 Agar 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 NF V08-055. Décembre 1993.

Microbiologie alimentaire. Recherche de L. monocytogenes. Méthode de routine.

2. Norme ISO 11290. Microbiologie des aliments. Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes.

PACKAGING Pre-poured dishes AEB522865 : Pack of 20 dishes (90 mm) AEB522864 : Pack of 120 dishes (90 mm) Made by AES Laboratoire - Combourg - France 122865£ : 17/01/03-C

T.S.Y.E

Listeria monocytogenes isolation medium In Vitro use only


91


Enumeration of Bacillus Cereus by colony-count technique at 30°C

PR NF EN ISO 7932 - 2003

Decimal dilution of a sample in Peptone water

(AEB111499 - pack of 100 tubes of 9 ml)

Inoculation by spreading on surface a Mossel agar (AEB521750 - plates 90 mm)

with 0,1 ml of each dilution (2 plates per dilution)

Incubation 18 - 48 hours at 30+/-1°C

Enumeration of the suspected colonies on the plates containing less than 150 colonies and expression of the results according to the dilutions,

then selection of 5 suspected colonies

Confirmation

- Agar with glucose: glucose +

- Motility nitrate medium: nitrate reductase +

- VP test: VP+

92

PRINCIPLE Bacillus cereus Agar is used for the detection and enumeration of spores and vegetative cells of Bacillus cereus in foodstuff. 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.

FORMULA Bacillus cereus agar base In grammes per litre of purified water Peptone 10,00 Beef extract 1,00 Sodium chloride 10,00 Mannitol 10,00 Phenol red 0,025 Agar 15,00

Final pH : 7,2 + 0,2 at 25°C 50% egg yolk solution Sterile egg yolk diluted at 50% with physiological water (9g/L). Polymixine B sulfate 50.000 UI/qsp 500 ml

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 47-50°C. As to prepare 1 litre of medium add 50 ml of the 50 % egg yolk solution, and if necessary 2 doses of Polymyxin B (50 000 UI/ml) previously hydrated with 5 ml of purified water. It is important to homogenize well before dispensing in dishes. Spread 0,1 ml of the prepared sample or its decimal dilutions at the surface of a prepared dish.

Incubate at 30°C for 48 hours with a daily observation.

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 also enclin to be intrusive, irrégulières, rugueuses et ont tendance à l'envahissement.

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 bacterim morphology, testing dextrose fermentation, nitrate reduction and the production of acetylmethylcarbinol (VP).

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 V08-023 ISO 7932 Octobre 1993. Microbiologie-Directives générales pour le dénombrement de Bacillus cereus. Méthode par

comptage des colonies à 30°C. 4. AFNOR V08-058. 1995. Microbiologie des aliments. Méthode de routine. Dénombrement de Bacillus cereus

par comptage des colonies à 30°C.

PACAKGING Deshydrated medium

To be stored between 18 and 23°C AEB151732 : 500 g 50% egg yolk solution ready to use To be stored between 2 and 8°C AEB180103 : 5 ml tube AEB180104 : 25 ml flask AEB180102 : 50 ml flask AEB180106 : 100 ml flask Polymyxin B sulfate supplement To be stored between 2 and 8°C AEB184001 : q.s.p. 500 ml Ready to use medium base without supplements

To be stored between 18 and 23°C AEB621736 : 6 Flasks of 100 ml Ready poured medium



AEB121739 : Pack of 10 dishes 140 mm ∅ Made by : AES Laboratoire - Combourg - France 151732£ : 28/12/00-F

MOSSEL

Bacillus cereus Agar according to Mossel specifications In Vitro use only

93


Horizontal method for the detection of thermotolerant Campylobacter

NF EN ISO 10272 (V08-26) - June 1996

Sample preparation of X gramme of product in 9x ml of Preston broth Preston broth (500 ml) = 12,5 grammes of nutrient broth at 2,5% (AEB140852) + 25 ml of defibrinated horse blood (AEB300025) + 1 supplement of Preston

(AEB184017)

Incubate 18 hours at 42°C in microaerophilic atmosphere

(Campypack H2CO2 - BBL71034 - 10 envelopes without catalyst)

Inoculate by isolation a Karmali agar plate and at the same time another selective medium, like the Preston agar (see the technical date sheet of the Preston

supplement)


(Campypouch System - BBL60656 - 25 pouches)

Select 5 typical colonies on the two incubated plates From each colony, carry out:

- a Gram colouring - an inoculation of one tube of 1 ml of Brucella broth (AEB140072 - 500g)

From the Brucella broths, carry out fresh microscopy observation

Retain the curved negative bacilli spiral motility

From each tube of selected Brucella broth, carry out an inoculation of Blood Columbia agar (AEB520679 - 120 plates 90 mm)


J0: Enrichment

J0: Isolation

J+2: Confirmation

94


From the colonies isolated on Blood Columbia agar, carry out: - an oxidase detection (Oxidase test – MGNMID61G) - the inoculation of a TSI (Triple Sugar Iron) agar - 100 tubes in slopes

Incubate 1 - 5 days at 42°C in microaerophilic atmosphere

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.

J+3 - J+8: Confirmation

Interpretation of the results

95

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 23,00


Corn starch 1,00

Activated charcoal 4,00

Haematin 0,032

Sodium pyruvate 0,10

Cycloheximide 0,10

Tris Buffer 1,00

Agar 14,00

Final pH : 7,4 + 0,2 at 25°C

Selective supplement CV for Campylobacter

(for 500 ml of medium base)

Cefoperazon 0,016

Vancomycin 0,010

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.

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 :07/12/03-F

CAMPYLOBACTER according to KARMALI

Selective medium for Campylobacter according to Karmali In Vitro use only


96

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. 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) 10,00

Peptic digest of animal tissues 10,00

Dextrose 1,00

Yeast extract 2,00


Sodium bisulfite 0,10

Final pH : 7,0 + 0,2 at 25°C METHOD Suspend 28,1 grammes of powder in one litre of purified water, homogenise 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 Laboratoire - Combourg - France 140072£ : 31/05/05 - E

BRUCELLA

Brucella Broth In Vitro use only


97

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 50,00 mg

Polymyxin B 2500 UI

Rifampicin 5,00 mg

Trimethoprim 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

PRESTON

Preston selective supplement In Vitro use only


98


Lactic flora enumeration

NF ISO 15214 standard — 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 +/- 1°c

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

99

PRINCIPLE Man, Rogosa et Sharpe agar (M.R.S.) is recommended for isolation and enumeration of Lactobacillus species in milk, milk products and any foodstuff. Its formula is conform to the one described in the NF V 04-503 standard. 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 liter of purified water. Proteose petone 10,00 Yeast extract 5,00 Dextrose 20,00 Potassium phosphate, dibasic 2,00 Beef extract 10,00 Sodium acetate 5,00 Ammonium citrate 2,00 Magnesium sulfate 0,20 Manganese sulfate 0,05 Polysorbate 80 1,00 Agar 15,00

METHOD OF PREPARATION Suspend 70,0 grammes in 1 liter 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 poor 15 ml of the liquefied medium (45-47°C), homogenise well. Let the medium set then put the prepared dishes under anaerobic atmosphere. Incubate at 37 °C for 2 to 3 days or at 30°C for at least 5 days. 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. PACKAGING Dehydrated medium 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 Laboratoire - Combourg - France 151752£:15/03/02 –I

M.R.S

Man, Rogosa and Sharpe agar In Vitro use only


100

Alphabetical Index

Buffered Peptone Water .............................................................................. p.10

Cryo beads .................................................................................................. p.18

D-cycloserine ............................................................................................... p.17

Fraser broth ................................................................................................. p.13

Half Fraser broth.......................................................................................... p.12

Kovacs ......................................................................................................... p.16

Mucap test (adapted protocol for SMS method) .......................................... p.14 Oxidase test................................................................................................. p.15 Peptone salt (Maximum recovery diluent).................................................... p.11

RPF AFNOR................................................................................................ p.20

Sterile defibrinated blood ............................................................................. p.19

ALOA .......................................................................................................... p.83 ASAP .......................................................................................................... p.39 Baird Parker................................................................................................. p.67 Baird Parker + RPF...................................................................................... p.69 BHI (Brain Heart Infusion)............................................................................ p.70 Blood agar base........................................................................................... p.89 Brucella........................................................................................................ p.96 Campylobacter according to Karmali ........................................................... p.95 Columbia 3................................................................................................... p.88 DG18 .......................................................................................................... p.27 DNA .......................................................................................................... p.72 Drigalski ....................................................................................................... p.47 Edel Kampelmacher (Brilliant Green Agar ISO)........................................... p.49 EE broth Mossel .......................................................................................... p.31 ESIA / ESSB................................................................................................ p.57

1. Diluents

2. Analysis

101

Giolitti Cantoni ............................................................................................. p.73 Hektoen ....................................................................................................... p.53 Klingler-Hajna .............................................................................................. p.50 Lyophilised Rabbit plasma........................................................................... p.71 Mac Conkey................................................................................................. p.52 mLST .......................................................................................................... p.58 MKTTn......................................................................................................... p.41 Mossel (MYP) .............................................................................................. p.92 MRS .......................................................................................................... p.99 OGA .......................................................................................................... p.26 Oxford.......................................................................................................... p.83 Palcam......................................................................................................... p.87 PCA .......................................................................................................... p.23 Preston ........................................................................................................ p.97 SMS .......................................................................................................... p.43 TBX .......................................................................................................... p.37 Thioglycollate resazurin ............................................................................... p.79 TSC and Tryptose sulfite ............................................................................ p.77 TSI .......................................................................................................... p.54 TSYE .......................................................................................................... p.90 RVS (Rappaport Vassiliadis Soja) ............................................................... p.40 Vibrions TCBS ............................................................................................. p.60 VRBG .......................................................................................................... p.32 VRBL .......................................................................................................... p.33 VRBL + MUG............................................................................................... p.35 Wilson and Blair........................................................................................... p.48 XLD .......................................................................................................... p.42 Yersinia CIN................................................................................................. p.61 YGC .......................................................................................................... p.25

102

YYoouurr ccoonnttaaccttss

Technical support

Name Job title Phone number E-mail

Claire Gardyn Microbiology Engineer + 33 (0)2 23 50 12 64

[email protected]

Export department

Name Job title Phone number E-mail

Jean-Philippe Aurel Export Manager + 33 (0)2 23 50 12 26

[email protected]

Yveline Jehannes Export Sales Assistant + 33 (0)2 23 50 12 24

[email protected]

Emmanuelle Hubert Export Sales Assistant + 33 (0)2 23 50 12 25

[email protected]

Laëtitia Pinel

Export Sales Assistant +33 (0)2 23 50 12 55

[email protected]

Aurélie Bossard Export Assistant + 33 (0)2 23 50 12 27

[email protected]

Louis Labriffe

Sales representative

(Europe, Turkey, Latin America)

+ 33 (0)2 23 50 12 86 [email protected]

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

Yveline Emmanuelle Laëtitia

- Subsidiary: Germany

- Europe: Belgium, Denmark, Norway, Sweden, Finland, Germany, the countries of Central and Western Europe

- Asia: China, Korea, Japan

- Africa

- Europe: UK, Ireland, The Netherlands,

Portugal, Spain, Switzerland, Italy, Greece, Turkey, Cyprus

- Middle East: Israel

- India, South-East Asia

- America: Canada, USA, Latin America

- Oceania: Australia, New Zealand

- Subsidiaries: Spain, Italy,

United States

Culture Media in Food Industry

Documents

Transcript of Culture Media in Food Industry