MFLP-42 Isolation and Enumeration of the Bacillus Cereus Group in Foods
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ID： |
5BBB51C4F50C4EF0ABA7E365C98F8D43 |
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文件大小(MB)： |
0.09 |
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页数： |
12 |
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日期： |
2012-3-2 |
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Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-42,May 2011,Health Products and Food Branch,Ottawa,Isolation and enumeration of the Bacillus cereus group in foods,Donna Douey1, Irene Iugovaz2, and Rick Szabo3,1Calgary Laboratory, Canadian Food Inspection Agency, 3650 36th St. N.W., Calgary, AB, T2L 2L1,2Food Microbiology, Health Products and Food Branch (HPFB), Health Canada, 1001, St-Laurent ouest,Longueuil, Québec, J4K 1C7,3Microbiology Evaluation Division, Bureau of Microbial Hazards, Health Products and Food Branch,Health Canada, Postal Locator 2204E, Ottawa, Ontario, K1A 0L2,E-mail: micro_methods_committee@hc-sc.gc.ca,1. Application,This method is applicable to the isolation, identification and enumeration of the Bacillus cereus group (with,limitations as described in the method) in foods in accordance with the requirements of Sections 4 and 7 of the,Food and Drugs Act. The revised method replaces MFLP-42, dated April, 2003.,2. Description,The method has been shown to produce satisfactory results with naturally-contaminated meats, vegetables,dairy products, cereals and dried foods (8.5).,3. Principle,The Bacillus cereus group is widely distributed in nature, consisting of B. cereus, B. anthracis, B. thuringiensis,B. mycoides, B. pseudomycoides, and B. weihenstephanensis. B. mycoides, B. pseudomycoides, and B.,weihenstephanensis are relatively straightforward to differentiate from the other members of the B. cereus,group and are thus not considered further in this document.,B. cereus is not easily distinguished from other closely related organisms in the B. cereus group. B.,thuringiensis is thought to be nearly identical to B. cereus, with the exception of the production of the protein,toxin crystals, which are encoded by the cry genes. The difficulties that may be encountered in identifying,carriage or expression of cry makes distinguishing B. cereus and B. thuringiensis laborious and complicated.,B. anthracis is non-motile and non-hemolytic. Atypical strains of B. cereus are variable in expression of,motility and hemolysis and further testing may be necessary to identify the isolates. Consider the source of,the sample when identifying the isolates as B. cereus. Only B. cereus and B. thuringiensis are likely to occur,naturally in food products.,B. cereus is commonly found in a variety of foods and when it grows to high numbers in a food (> 106/g),sufficient enterotoxin may be produced resulting in foodborne illness. This method determines the presence of,B. cereus group isolates by plating known quantities of (dilutions of) a food sample onto a selective agar. After,MFLP-42,- 2 - May 2011,Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php,incubation, presumptive B. cereus colonies are selected and subjected to confirmatory testing. From the,results obtained, the number of presumptive B. cereus per g or mL of the food is calculated.,4. Definition of terms,See Appendix A of Volume 3 (8.4),5. Collection of samples,See Appendix B of Volume 3 (8.4),6. Materials and special equipment,The following media and reagents (1-5) are commercially available and are to be prepared and sterilized,according to the manufacturer's instructions. See also Appendix G of Volume 3, and Section 9 for the formula,of individual media.,1) Peptone Water diluent (PW),2) Citrate solution, 2%, warmed to 45EC (for cheese),3) Trypticase Soy Broth (TSB),4) Nutrient Agar plates,5) Polymyxin Pyruvate Egg Yolk Mannitol Bromthymol Blue Agar (PEMBA Medium),6) Blood Agar plates (TSB agar with 5% sheep blood),7) Sporulation broth (9.1) or TSA-MnSO4 agar (optional),8) Staining solutions (optional): Malachite Green, 5% aqueous solution; Safranin, 0.5% aqueous,solution; Sudan Black B, 0.3% in 70% ethanol; Xylol,9) Basic fuchsin, 0.5% aqueous solution OR TB Carbol-fuchsin ZN stain (Difco) [protein toxin crystals],Note: Both Basic Fuchsin and TB Carbol-fuchsin ZN stains are toxic and possibly,carcinogenic. Use appropriate safety precautions. It is recommended that,commercially-available products be purchased.,10) Methanol [protein toxin crystals],11) BC Motility Medium (9.2),12) Rapid identification kits (optional),13) Control cultures, ATCC or equivalent,14) Blender, stomacher or equivalent,15) Microscope,16) Incubators capable of maintaining 30 and 35EC,MFLP-42,- 3 - May 2011,Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/re……
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