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Dehydrated Culture Media

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Code: CM0617

A selective and diagnostic medium for the isolation and enumeration of Bacillus cereus

Typical Formula*






Sodium chloride


Magnesium sulphate


Disodium hydrogen phosphate


Potassium dihydrogen phosphate


Bromothymol blue


Sodium pyruvate




pH 7.2 ± 0.2 @ 25°C


* Adjusted as required to meet performance standards


Code: SR0099

Vial contents

(I vial per 500ml

per litre

Polymyxin B



Suspend 20.5g in 475ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and aseptically add the contents of one vial of Polymyxin B Supplement (SR0099) reconstituted as directed, then add 25ml of sterile Egg Yolk Emulsion (SR0047). Mix well and pour into sterile Petri dishes.

Bacillus Cereus Selective Agar, is based on the highly specific diagnostic and selective PEMBA medium, developed by Holbrook and Anderson1 for the isolation and enumeration of Bacillus cereus in foods. It meets the requirements for a medium that is sufficiently selective to be able to detect small numbers of Bacillus cereus cells and spores in the presence of large numbers of other food contaminants. The medium is also sufficiently diagnostic that colonies of Bacillus cereus are readily identified and confirmed by microscopic examination.

The role of Bacillus cereus in food poisoning, particularly from the consumption of contaminated rice, is now well documented2,3,4. The organism has also been implicated in eye infections5,6 and a wide range of other conditions, including abscess formation, meningitis, septicaemia and wound infection. Bacillus cereus is recognised as a significant pathogen in post-operative and post-traumatic wounds of orthopaedic patients7. Amongst veterinarians, Bacillus cereusis a known cause of disease, especially mastitis, in ewes and heifers8.

In the formulation of Bacillus cereus Selective Agar, a peptone level of 0.1% and the addition of sodium pyruvate improve egg yolk precipitation and enhance sporulation. Bromothymol blue is added as a pH indicator to detect mannitol utilisation. The medium is made selective by addition of Polymyxin B Supplement (SR0099) which gives a final concentration of 100IU of polymyxin B per ml of medium. Polymyxin B, as a selective agent for the isolation of B. cereus has been previously suggested by Donovan9 and found to be satisfactory by Mossel10. It is recommended that, where large numbers of fungi are expected in the inoculum, cycloheximide (SR0222) is added to the medium at a final concentration of 40mg/l.

The primary diagnostic features of the medium are the colonial appearance, precipitation of hydrolysed lecithin and the failure of Bacillus cereus to utilise mannitol. The typical colonies of Bacillus cereus are crenated, about 5mm in diameter and have a distinctive turquoise to peacock blue colour surrounded by a good egg yolk precipitate of the same colour. These features distinguish Bacillus cereus from other Bacillus spp. except Bacillus thuringiensis.

Other egg yolk reacting organisms which can grow on the medium, including Staphylococcus aureus, Serratia marcescens and Proteus vulgaris, are distinguished from Bacillus cereus by colony form and colour. These organisms also produce an egg yolk-clearing reaction in contrast to egg yolk precipitate produced by Bacillus cereus.

Microscope examination for presence of lipid globules in the vegetative cells is recommended as a rapid and confirmatory test for Bacillus cereus and replaces the need for biochemical testing. Holbrook and Anderson1 have confirmed that only Bacillus cereus of the Bacillus spp. are capable of possessing lipid globules in their vegetative cells when grown on the selective medium. One further advantage of this test is that strains of Bacillus cereus that react only weakly or not at all with egg yolk can be detected and confirmed.


  1. Homogenise 10g of the food sample for 30 seconds in 90ml of 0.1% Peptone Water CM0009 using a Stomacher 40011. Dried foods should first be rehydrated by soaking 20g in 90ml of tryptone salt solution for 50 minutes at room temperature. Add a further 90ml of 0.1% peptone water to give a final dilution of 10-1. Homogenise for 30 seconds using the Stomacher 400.
  2. Further dilutions of the homogenate should be made in 0.1% peptone water.
  3. Inoculate 0.1ml amounts of the 10-1 and higher dilutions on to the surface of the medium.
  4. Incubate the plates at 37°C for 24 hours.
  5. Examine for typical colonies of Bacillus cereus.
  6. If no initial colonies were seen at 24 hours, re-incubate the plates for a further 24 hours at 37°C.
  7. Confirm the presumptive identification of Bacillus cereus by the Rapid Confirmatory Staining Procedure.
  8. Report the results as the number of Bacillus cereus colonies per gram weight of the food sample.
Tryptone (LP0042) 0.3%, sodium chloride 0.8%, pH 7.3

The medium may also be used for detecting Bacillus cereus in milk. When necessary, decimal dilutions of the samples should be made in 0.1% peptone water. Undiluted and diluted samples are inoculated directly on to agar plates and incubated. An incubation temperature of 30°C for 18 hours is recommended as optimal for promoting the growth of Bacillus cereus relative to that of other organisms9. For examining clinical specimens plates may be inoculated in the usual way.

Rapid Confirmatory Staining Procedure
This staining method was developed by Holbrook and Anderson1 combining the spore stain of Ashby12 and the intracellular lipid stain of Burdon13. For reasons of safety, Citroclear‡ replaces xylene in the original technique.


  1. Prepare films from the centre of a 1 day old colony or from the edge of a 2 day colony.
  2. Air-dry the film and fix with minimal heating.
  3. Flood the slide with aqueous 5% w/v malachite green and heat with a flaming alcohol swab until steam rises. Do not boil.
  4. Leave for 2 minutes without re-heating.
  5. Wash the slide with running water and blot dry.
  6. Flood the slide with 0.3% w/v Sudan black in 70% ethyl alcohol. Leave for 15 minutes.
  7. Wash the slide with running Citroclear from a wash bottle for 5 seconds.
  8. Blot dry using filter paper.
  9. Flood the slide with aqueous 0.5% w/v safranin for 20 seconds.
  10. Wash under running water.
  11. Blot dry and examine under the microscope using the oil immersion lens. A blue filter may be used to accentuate the appearance of the lipid granules but this will give a blue colour cast to the red of the cytoplasm.

‡ Citroclear is available from:
H.D. Supplies
Tel: +44 (0)1296 431920

Characteristic appearance of B. cereus vegetative cells.
(i) Cells are 4-5 micron long and 1.0-1.5 micron wide with square ends and rounded corners.
(ii) The spores stain pale green to mid green, are central or paracentral in position and do not swell the sporangium.
(iii) Lipid globules are black and the vegetative cytoplasm red.
The appearance, together with the typical colony form, confirms the identification of Bacillus cereus.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and Polymixin B Supplement at 2-8°C. Use before the expiry date on the label.
The prepared medium may be stored at 2-8°C.

Dehydrated medium: Dark straw/yellow, free-flowing powder
Prepared medium: Green, opaque gel

Quality control

Positive control:

Expected results

Bacillus cereus ATCC® 10876

Good growth; peaock blue colonies with precipitate and peacock blue medium

Negative controls:


Bacillus subtilis ATCC® 6633 *

Growth; straw coloured colonies

Escherichia coli ATCC® 25922 * Inhibited
* This organism is available as a Culti-Loop®

On this medium Bacillus cereus is indistinguishable from Bacillus thuringiensis.
Identify Bacillus cereus by colony form, colour, egg yolk hydrolysis and confirm with cell and spore morphology.14
Occasional strains of Bacillus cereus show weak or negative egg yolk reactions.

1. Holbrook R. and Anderson J.M. (1980) Can. J. Microbiol., 26 (7) 753-759.
2. Brit. Med. J., 15 January, 1972, 189.
3. Brit. Med. J., 22 September. 1973. 647.
4. Mortimer P.R. and McCann G., 25 May, 1974, Lancet, 1043-1045.
5. Davenport R. and Smith C. (1952) Brit. J. Ophthal. 36. 39.
6. Bouza E., Grant S., Jordan C., Yook R. and Sulit H. (1979) Arch. Ophthalmol. 97. 498-499.
7. Akesson A., HedstrÎm S.A. and Ripa T. (1991) Scand. J. Inf. Dis. 23. 71--77.
8. Wohlgemuth K., Kirkbride C.A., Bicknell E.J. and Ellis R.P. (1972) J. Amer. Vet. Med. Ass., 161. 1691-1695.
9. Donovan K.O. (1958) J. Appl. Bacteriol., 21 (1) 100-103.
10. Mossel D.A.A., Koopman M.J. and Jongerius E. (1967) J. Appl. Microbiol., 15 (3) 650-653.
11. Supplied by A.J. Seward, Pharm. Mfrs. and Distrib., UAC House, 8--16 Blackfriars Road, London SE1.
12. Ashby G.K. (1938) Science, 87, 433-435.
13. Burdon K.L. (1946) J. Bacteriol., 52. 665-678.
14. DeÄk T. and TimÄr E. (1988) Int. J. Food Microbiology. 6. 115-125.

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