Bacillus cereus selective agar

Cat. no. G156 Bacillus cereus Selective Agar, 15x100mm plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Bacillus cereus Selective Agar is a selective medium for the isolation and differentiation of Bacillus cereus in foods.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Holbrook and Anderson described a highly selective and diagnostic medium (PEMBA) for the isolation and determination of Bacillus cereus from food. (1) The medium is formulated to detect small numbers of B. cereus in the presence of a large number of other contaminants. Bacillus cereus Selective Agar has the ability to differentiate B. cereus from other species of Bacillus based on its resistance to polymyxin, lack of mannitol fermentation, and the presence of lecithinase. B. cereus can be present in a variety of produce and processed foods and the organism can cause gastointestinal illness if allowed to proliferate. Past outbreaks have been associated with the ingestion of rice, cooked meats, and cooked produce contaminated with B. cereus.

Hardy Diagnostics Bacillus cereus Selective Agar contains nitrogen, carbon, and meat peptone, which provide essential nutrients. Sodium chloride maintains osmotic equilibrium. The addition of sodium pyruvate enhances sporulation and egg yolk precipitation. Magnesium sulfate provides divalent cations which act as special growth factors for Bacillus spp. Mannitol fermentation is detected by the pH indicator bromothymol blue, resulting in Bacillus cereus colonies having a distinctive turquoise blue color. Egg yolk suspension is added as a source of lecithin to demonstrate the lecithinase reaction. The addition of polymyxin B inhibits gram-negative bacteria.

Typical colonies of B. cereus are crenated, about 5mm in diameter, and have a distinctive turquoise to peacock blue color surrounded by a zone of precipitate of the same color. These features distinguish B. cereus from other Bacillus species, except B. thuringiensis . Other egg yolk-reacting organisms which can grow on this medium, including Staphylococcus aureus , Serratia marcescens and Proteus vulgaris , are distinguished from B. cereus by colony morphology and color. These organisms produce an egg yolk clearing reaction in contrast to the egg yolk precipitate produced by B. cereus.

Microscopic examination for the presence of lipid globules in the vegetative cells is recommended as a rapid and confirmatory test for B. cereus and replaces the need for biochemical testing. Holbrook and Anderson have confirmed that only B. cereus in this genus is capable of possessing lipid globules in its vegetative cells when grown on the selective medium. (1) One further advantage of this test is that strains of B. cereus that react only weakly or not at all with egg yolk can be detected and confirmed.

FORMULA

Ingredients per 900ml of deionized water:*

Mannitol 10.0gm
Sodium Pyruvate 10.0gm
Disodium Phosphate 2.5gm
Sodium Chloride 2.0gm
Peptic Digest of Animal Tissue 1.0gm
Dipotassium Phosphate 0.25gm
Bromothymol Blue 0.12gm
Magnesium Sulfate 0.1gm
Sterile Egg Yolk Suspension 100ml
Polymyxin B 100,000IU
Agar 15.0gm

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample collection: Consult listed references for appropriate sampling plans. (2-5)

1. Samples should be processed in a manner suitable for the source material: liquid, solid, semi-solid, or frozen.

2. Prepare appropriate decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

3. Aseptically inoculate the agar surface with 0.1ml of a well mixed diluted sample.

4. Using a sterile spreader device, spread the inoculum evenly over the surface of the agar to achieve isolated colonies.

INTERPRETATION OF RESULTS

Typical colonies of B. cereus are crenated, about 5mm in diameter and have a distinctive turquoise to peacock blue color surrounded by a zone of precipitate of the same color. These features distinguish B. cereus from other Bacillus species, except B. thuringiensis . Other egg yolk-reacting organisms which can grow on the medium, including Staphylococcus aureus , Serratia marcescens and Proteus vulgaris , are distinguished from B. cereus by colony morphology and color. These organisms produce an egg yolk clearing reaction in contrast to an egg yolk precipitate produced by B. cereus .

Microscopic examination for the presence of lipid globules in the vegetative cells is recommended as a rapid and confirmatory test for B. cereus and replaces the need for biochemical testing. Holbrook and Anderson have confirmed that only B. cereus in this genus is capable of possessing lipid globules in its vegetative cells when grown on the selective medium. (1) One further advantage of this test is that strains of B. cereus that react only weakly or not at all with egg yolk can be detected and confirmed.

LIMITATIONS

On this medium, B. cereus is indistinguishable from B. thuringiensis . B. thuringiensis can be distinguished from B. cereus using molecular methods or the protein toxin crystal stain.

Occasional strains of B. cereus shows weak or negative egg yolk reactions.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacillus cereus
ATCC ® 13061**
A 24-48hr 35°C Aerobic Growth; blue colonies with opaque halo
Bacillus subtilis
ATCC ® 6633**
A 24-48hr 35°C Aerobic Growth; white colonies without halo
Pseudomonas aeruginosa
ATCC ® 27853**
B 24-48hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

** Recommended QC strains for User Quality Control.

Physical Appearance

Bacillus cereus Selective Agar should appear translucent, and yellow in color.

REFERENCES

1. Can. 1980. J. Microbiol. ; 26:753-7590.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Association of Official Analytical Chemists. Official Methods of Analysis , AOAC, Washington, D.C.

4. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

5. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm


ATCC is a registered trademark of the American Type Culture Collection.

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