Bacillus Cereus Selective Agar Base (PEMBA) ISO

Casein peptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Sodium pyruvate reduces the colony size of the organisms. Bromothymol blue acts as pH indicator to detect mannitol fermentation. Mannitol is the fermentable carbohydrate providing carbon and energy; Bacillus cereus is mannitol-negative. The mannitol content allows the identification of the accompanying mannitol-positive flora. Sodium chloride maintains the osmotic balance of the medium. Bacteriological agar is the solidifying agent.

Bacillus cereus produces lecithinases. The insoluble degradation products from the lecithin of egg yolk accumulate around the Bacillus cereus colonies, forming a white precipitate. The colonies of Bacillus cereus will appear as blue colonies surrounded by a zone of opacity in the medium.

Preparation

Suspend 40 grams of the medium in 950 mL of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 °C for 15 minutes. Cool to 45-50 °C and aseptically add 50 mL of Egg Yolk Emulsion (LC-5152) and, if desired, aseptically add 2 vials of Bacillus Cereus Supplement (LC-6021). Homogenize gently and dispense into appropriate containers.

According to ISO 21871

For the enumeration of low numbers of Bacillus cereus according to ISO 21871:

1. Enrichment in selective liquid medium TSPB: 3 tubes with 10 mL of medium at double concentration and 3 tubes with 9 mL of the medium at single concentration.
2. Inoculate the double concentration tubes with 10 mL of the initial suspension of the sample to be analyzed, and inoculate the single concentration tubes with 1 mL of the initial suspension or subsequent dilutions.
3. Incubate at 30 °C for 48 ± 4 hours.
4. Shake the tubes gently and inoculate the Selective Agar for Bacillus Cereus (PEMBA) with an inoculating loop (10 µL).
5. Incubate the plates in inverted position at 37 °C for 18-24 hours. If the colonies have not been identified correctly, continue the incubation for 24 more hours.
6. Examine the plates to detect the presence of colonies.

• Donovan, K.O.: A Selective Medium for Bacillus cereus in Milk, J. appl. Bact., 21; 100:103 (1958)
• Mossel, D.A.A., Koopman, M.J. and Jongerius, E.: Enumeration of Bacillus cereus in Foods. Appl. Microbiol., 15; 650:653 (1967)
• ISO 21871 Horizontal method for the determination of low numbers of presumptive Bacillus cereus.