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BACILLUS CEREUS SELECTIVE AGAR BASE
Code: CM0617
A selective and diagnostic medium for the isolation and enumeration of Bacillus cereus
Typical Formula* | gm/litre |
Peptone | 1.0 |
Mannitol | 10.0 |
Sodium chloride | 2.0 |
Magnesium sulphate | 0.1 |
Disodium hydrogen phosphate | 2.5 |
Potassium dihydrogen phosphate | 0.25 |
Bromothymol blue | 0.12 |
Sodium pyruvate | 10.0 |
Agar | 15.0 |
pH 7.2 ± 0.2 @ 25°C |
|
POLYMYXIN B SUPPLEMENT
Code: SR0099
Vial contents | SR0099E | per litre |
Polymyxin B | 50,000IU | 100,000IU |
Directions
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.
Description
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.
Technique
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.
Procedure
‡ Citroclear is available from:
H.D. Supplies
Aylesbury
Buckinghamshire
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.
Appearance
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 |
Precautions
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.
References
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.