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Bacillus Cereus Agar Base

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M833
Used as a selective medium for isolation, detection and enumeration of Bacillus cereus.

Intended use

Recommended as a selective medium for the isolation and enumeration of Bacillus cereus from food and clinical samples.

Composition**

Ingredients g/L
Peptone1.000
Mannitol10.000
Sodium chloride2.000
Magnesium sulphate0.100
Disodium hydrogen phosphate2.500
Potassium dihydrogen phosphate0.250
Sodium pyruvate10.000
Bromo thymol blue0.120
Agar15.000
Final pH (at 25°C)7.2±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 20.5 grams in 475 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add rehydrated contents of 1 vial of PolyB Selective Supplement (FD003) and 25 ml of sterile Egg Yolk Emulsion (FD045). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Bacillus cereus causes food poisoning due to the consumption of contaminated rice (1,2), eye infections (3) and a wide range of other clinical conditions like abscess formation, meningitis, septicemia and wound infection. Bacillus cereus is a known cause of disease mastitis, especially in ewes and heifers among the veterinarians (4). Holbrook and Anderson (5) developed Bacillus Cereus Agar, which is a highly specific and selective medium for the isolation and enumeration of Bacillus cereus from foods. It supports the growth of even a small number of Bacillus cereus cells and spores in the presence of large number of other food contaminants. The typical colonies of Bacillus cereus are crenate, about 5 mm in diameter and have a distinctive turquoise to peacock blue colour surrounded by a good egg yolk precipitate of the same colour. The bacteria do not ferment mannitol and thus there is no change in colour of the indicator dye around the colonies. Addition of polymyxin-B sulphate (6,7) at a final concentration of 100 units per ml of medium is sufficient to make the medium selective for the isolation of Bacillus cereus. It suppresses the growth of accompanying bacterial flora. If moulds are suspected in the inoculum, 40 mcg per ml filter-sterilized cycloheximide may be incorporated to suppress the moulds contamination. Some strains of Bacillus cereus have very weak egg yolk reaction. Moreover, on this medium Bacillus cereus is indistinguishable from Bacillus thuringiensis

Peptone provides and sodium pyruvate improve egg yolk precipitation and enhance sporulation. Bromothymol blue acts as pH indicator to detect mannitol fermentation. For the isolation and enumeration of Bacillus cereus in foodstuffs the following method is recommended. Distribute 0.1ml of the homogenized specimen diluted in Peptone Water (M028) onto the surface of the medium. Incubate at 37°C under aerobic conditions for 24-48 hours. Possible growth of contaminants is greatly reduced by incubation for 24 hours. Report the results as the number of Bacillus cereus colonies per gram weight of the food sample. Confirmatory tests should be carried out before interpretation.

Type of specimen

Food and dairy samples, Clinical sample : abscess and wound samples.

Specimen Collection and Handling:

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (8,9). For clinical samples, follow appropriate techniques for sample collection and processing as per guidelines (10,11).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

  • Bacillus cereus and Bacillus thuringiensis shows identical characteristics and hence difficult to identify.
  • Identification of Bacillus cereus is done by colony characteristics and reaction, however further biochemical characteristics should be carried out for confirmation.
  • Some strains of Bacillus cereus may show poor growth due to nutritional variations.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance
Cream to greenish yellow homogeneous free flowing powder

Gelling
Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium
Basal medium :Green coloured clear to slightly opalescent gel. After addition of egg yolk emulsion : Yellowish green coloured opaque gel forms in Petri plates

Reaction
Reaction of 4.1% w/v aqueous solution (basal medium) at 25°C. pH: 7.2±0.2

pH
7.00-7.40

Cultural Response

Cultural characteristics observed with added PolyB Selective Supplement (FD003) and Egg Yolk Emulsion (FD045) after an incubation at 35-37°C for 24-48 hours.

Organism Inoculum
(CFU)		 Growth Recovery Colour of
colony		 Egg Yolk
Reaction
Bacillus cereus ATCC 1087650-100good-luxuriant>=50%bluepositive precipitation
Escherichia coli ATCC 25922 (00013*)>=104inhibited0%
## Proteus hauseri ATCC 1331550-100good-luxuriant>=50%greennegative
Serratia marcescens ATCC 810050-100good-luxuriant>=50%yellow-light pink
(pigment production is
enhanced by
incubation at 25-30°C)		negative
Staphylococcus aureus subsp. aureus ATCC 25923 (00034*)50-100good-luxuriant>=50%yellowpositive, clearing

Key: *Corresponding WDCM numbers, ## Formerly known as Proteus vulgaris.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (10,11).

References

  1. Mortimer P.R. and McCann G., 1974, Lancet, 1043-1045.
  2. Wohlgemuth K., Kirkbride, C.A., Bicknell, E. J. and Ellis, R.P., 1972, J. Am. Vet. Med. Ass. 161:1691-1695.
  3. Bouza E., Grant S., Jordan C. et al, 1979, Arch. Ophthalmol. 97:498
  4. Kirnbull P.C., J. Clin. Pathol. 32:289
  5. Holbrook R. and Anderson J., 1980, Can. J. Microbiol. 26(7):753-759
  6. Donovan K.O., 1958, J. Appl. Bacteriol, 21(1): 100.
  7. Mossel D.A.A., Koopman J. and Jongerius E., 1967, J. Appl. Microbiol. 15(3):650-653.
  8. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
  9. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
  10. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
  11. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
More Information
Product Name Bacillus Cereus Agar Base
SKU M833
Product Type Regular
Physical Form Powder
Origin Animal
Packaging type HDPE
References 1. Holbrook R. and Anderson J., 1980, Can. J. Microbiol. 26(7):753-7592.Donovan K.O., 1958, J. Appl. Bacteriol, 21(1): 100.3.Mossel D.A.A., Koopman J. and Jongerius E., 1967, J. Appl. Microbiol. 15(3):650-653.4.Mortimer P.R. and McCann G., 1974, Lancet, 1043-1045.5.Bouza E., Grant S., Jordan C. et al, 1979, Arch. Ophthalmol. 97:4986.Wohlgemuth K., Kirkbride, C.A., Bicknell, E. J. and Ellis, R.P., 1972 , J. Am. Vet. Med. Ass. 161:1691-1695.7.Kirnbull P.C., J. Clin. Pathol. 32:2898.American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., WashingtonD.C.9.Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination ofFoods, 5th Ed., American Public Health Association, Washington, D.C.10.Isenberg, H.D. Clinical Microbiology Procedures Handb0ook. 2nd Edition.11. Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015)Manual of Clinical Microbiology, 11th Edition. Vol. 1.12.Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed.,APHA Inc., Washington, D.C.
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