Anda di halaman 1dari 3

BACILLUS CEREUS RAPID AGAR (BACARA)

Selective medium for presumptive Bacillus of cereus group

For In Vitro use only To store between 2 and 8C Page 1/2


PREPARATION
AES 10/10-07/10 BACARA Method ALTERNATIVE METHODS FOR AGRIBUSINESS ANALYSIS Certified by AFNOR Certification www. afnor-validation.org For human food and animal feeding stuffs. End of validity : 2014/07/02

For the medium in flask, liquefy the medium in a water-bath at 100C and cool to 44-47C. Per flask of 100 ml of BACARA medium, add aseptically 4 ml of BACARA enrichment supplement and 0.5 ml of BACARA selective supplement (chromogenic selective mix). Homogeneize well the agar base and the supplements, avoiding the incorporation of air bubbles.

PRINCIPLE
Bacillus cereus is responsible for food-borne outbreaks. It produces thermoresistant spores that make it particularly adapted to foodstuffs submitted to a thermal treatment. Some strains of B. cereus can grow at refrigeration temperature, which is an emerging risk for ready-to-use products. They represent by themselves 5% of the collective food poisoning in France and are also involved in a lot of opportunistic infections on in-patients. Bacillus cereus belongs to the Bacillus cereus group within which can be found B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides et B. anthracis. Except the last-mentioned, the phenotypic differentiation between species of Bacillus cereus is impossible with actual culture methods. BACARA agar is a selective chromogenic medium that allows the enumeration of Bacillus of the cereus group without confirmation. On this medium, typical colonies of B. cereus show a pink / orangey colour due to the metabolism of the chromogen substrate and are surrounded with an opaque halo due to the phospholipase activity. The selectivity of BACARA agar has been especially optimized to prevent growth of interfering flora and thus to allow an easy interpretation of plates even when matrix highly contaminated with competitive flora are analysed.

PROCEDURE (See also Annex I)


Surface method : After preparation and homogeneization of the primary solution in the suitable diluent, spread 0.1ml of the solution obtained onto the surface of a 90mm BACARA plate (to increase the accuracy, spread 1ml onto a 140mm BACARA plate or split onto 3 90mm BACARA plates). If necessary, repeat the step with the decimal dilutions of the primary solution. Incubate the BACARA plates for 24 2h at 30C 1C. Pour-plate method : After preparation and homogeneization of the primary solution in the suitable diluent, inoculate 1ml of the solution obtained in a sterile Petri plate. Pour about 18 ml of BACARA medium. Homogeneize well and let it solidify. If necessary, repeat the step with the decimal dilutions of the primary solution. Incubate the BACARA plates for 24 2h at 30C 1C.

RESULTS
After incubation, Bacillus from cereus group grow as large pink/orangey colonies surrounded with an opaque halo. Refer to the standard NF EN ISO 7218 for calculation and expression of results.

FORMULA
In grams per litre of purified water Special mix of peptones Yeast extract Sodium chloride Phosphate buffer Agar Mix of antibiotics Chromogen substrate Phospholipids Final pH : 7.2 + 0.2 at 25C 10.00 4.00 4.00 10.00 18.00 0.26 0.05
Pink orangey colour

Opaque halo : Phospholipase activity

AES CHEMUNEX Rue Maryse Basti Ker Lann CS 17219 35172 Bruz Cedex Tl. 02 23 50 12 12 Fax. 02 23 50 12 00 http://www.aeschemunex.com - contact@aeschemunex.com

BACILLUS CEREUS RAPID AGAR (BACARA)


Selective medium for presumptive Bacillus of cereus group

For In Vitro use only Page 2/2


PACKAGING : LIMITS AND PRECAUTIONS
Comply with Good Laboratory Practice (refer to the standard NF EN ISO 7218). Thanks to the high specificity and selectivity of BACARA, it is not necessary to carry out confirmation tests on typical colonies. All the strains belonging to the Bacillus cereus group will give characteristic colonies. Some bacteria can also grow as orangey coloured colonies on BACARA but without expression of the phospholipase activity. The absence of the opaque halo will make them easily distinguishable from the Bacillus cereus. After opening, the BACARA enrichment supplement can be used up to its expiry date if it was manipulated according to good laboratory practice (ie: Use in aseptic conditions and storage between 2 and 8C). After opening, the BACARA selective supplement can be used up to 15 days maximum if it was manipulated according to good laboratory practice. Warning: DO NOT FLAME THE TUBE before closing it. This supplement contains a flammable solvent. Supplements have to be stored away from light. The supplemented agar base has to be used immediately or poured into sterile Petri plates. The plates poured from flasks can be kept up to 2 weeks at 2-8C in a hermetically sealed package. For the modes of storage, refer to the standard NF EN ISO 7218. BACARA plates can be placed in the refrigerator after incubation up to 48 hours. The halo and the colour of the colonies will not be altered at those low temperatures.

Ready to use base medium To store between 2 and 8C AEB620106 : Kit BACARA base + supplements for 5x100 ml Pre-poured complete medium To store between 2 and 8C AEB520100 : Pack of 20 plates of 90 mm AEB120102: Pack of 10 plates of 140 mm Manufactured by AES CHEMUNEX - Combourg France 520100 : 02/07/10- B

BIBLIOGRAPHY
1- AFNOR NF EN ISO 7932 Juillet 2005. Microbiologie des aliments Mthode horizontale pour le dnombrement de Bacillus cereus prsomptifs. Technique par comptage des colonies 30C. 2- N.A LOGAN, P.C.B. TURNBULL : Actualit permanente en Bactriologie Clinique Bactries arobies sporules 3- F.A DROBNIEWSKI : Bacillus cereus and related species Clin Microbiol Rev 1993 October Species ; 6 (4): 324-338. 4- NF EN ISO 7218. : Microbiologie des aliments - Exigences gnrales et recommandations.

AES CHEMUNEX Rue Maryse Basti Ker Lann CS 17219 35172 Bruz Cedex Tl. 02 23 50 12 12 Fax. 02 23 50 12 00 http://www.aeschemunex.com - contact@aeschemunex.com

Culture media for food industry

Annex I Enumeration of presumptive Bacillus cereus


BACARA METHOD
Prepare and dilute the sample according to the specifications of NF EN ISO 6887, NF EN ISO 8261 standards and the specific standard of the product.

SURFACE METHOD

POUR-PLATE METHOD

Spreading of 0.1 ml* onto 1 90 mm plate of BACARA medium (AEB520100 Pack of 20 plates 90 mm) previously dried in an incubator. To increase the accuracy, 1 ml* onto 1 140 mm plate (AEB120102 Pack of 10 plates 140 mm) or 1 ml* onto 3 90 mm plates. Incubation 24 2 hours at 30 1C

Inoculate 1 ml* in 1 sterile Petri plate, pour about 18 ml of BACARA medium (AEB620106 Kit base + supplements for 5x100ml) cooled at 44-47C. Incubation 24 2 hours at 30 1C

Enumerate the plates containing less than 150 characteristic colonies. Characteristic colonies of presumptive Bacillus of cereus group : large pink / orangey surrounded with an opaque halo.

520100 : 02/07/10 B
AES 10/10-07/10 ALTERNATIVE METHODS FOR AGRIBUSINESS ANALYSIS Certified by AFNOR Certification www. afnor-validation.org

* : repeat the step with the decimal dilutions of the primary solution if necessary.

AES CHEMUNEX Rue Maryse Basti Ker Lann CS17219 35172 Bruz cedex Tel : 02 23 50 12 12 fax : 02 23 50 12 00 - http://www.aeschemunex.com contact@aeschemunex.com