MFLP-46 Isolation of Thermophilic Campylobacter from Foods
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CEA41183D35A4A2095067F7F00CE171F |
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0.12 |
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页数： |
13 |
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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/food-aliment.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-46,March 2002,HEALTH PRODUCTS AND FOOD BRANCH,OTTAWA,ISOLATION OF THERMOPHILIC CAMPYLOBACTER FROM FOOD,D. Medeiros and L. Hofmann,Microbiology Research Division,Bureau of Microbial Hazards, Food Directorate,Postal locator: 2204A2,Ottawa, K1A 0L2,E-mail: Lisa_Hofmann@hc-sc.gc.ca,1. APPLICATION,This method is applicable to the determination of the thermotolerant and microaerophilic bacteria of the genus,Campylobacter in foods to determine compliance with the requirements of Section 4 and 7 of the Food and,Drugs Act. This revised method replaces MFLP-46, dated July 1998.,2. PRINCIPLE,The procedure consists of four stages. The initial handling of the food at the enrichment stage varies,according to the type of food under investigation.,2.1 Selective Enrichment,The food is initially inoculated into a selective enrichment medium designed to slow or inhibit the,growth of competing microorganisms while favouring the growth of campylobacters.,2.2 Colony Formation on Selective Agars,Selectively enriched cultures are streaked onto selective agars specifically developed for the isolation,and identification of campylobacters. Presumptive colonies are further identified by examining for,characteristic morphology and motility using phase contrast microscopy.,2.3 Purification,The isolates are purified on an appropriate selective blood or charcoal agar plate.,2.4 Identification,MFLP-46,March 2002,-2-,Presumptive campylobacters are identified by biochemical reactions, tolerance to some chemicals and,antibiotics, and by growth temperatures. Several biotypes are determined by biochemical reactions.,Serotyping schemes for identifying heat-labile antigens (Lior’s scheme) and heat-stable antigens,(Penner’s scheme) have been developed, however the antisera required are not readily available. If,serotyping is required cultures may be sent to Health Canada’s Laboratory Center for Disease Control,in Winnipeg, Manitoba. Various nucleic acid-based and antibody-based rapid methods for the,identification of Campylobacter are commercially available (9.1, 9.2, 9.4),3. DEFINITION,See Appendix A of Volume 3.,4. COLLECTION OF SAMPLES,4.1 Sampling,See Appendix B of Volume 3.,4.1.1 Holding and Shipping,For holding and shipping, place the sample units in sealed containers at 4oC until analysis.,With raw milk samples, supplement with (a) 5% cysteine or (b) 0.01% sodium bisulfite and,either 0.15% sodium thioglycolate or an atmosphere of 100% nitrogen. Sample units of frozen,products shall be kept frozen.,4.1.2 For environmental samples, follow MFLP-41.,5. MATERIALS AND SPECIAL EQUIPMENT,The following media and reagents (1-10) are commercially available and are to be prepared and sterilized,according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 9.1 for the,formula of individual media.,1) Brucella Broth,2) Brucella Semi-solid Agar,3) Preston Agar (Also known as Campylobacter Agar Base),4) Mueller Hinton Agar,5) Mueller Hinton Agar with Blood,6) Wilkins Chalgren Anaerobic Blood Agar,7) Wilkins Chalgren Anaerobic Broth,8) Campylobacter Agar with Charcoal and Deoxycholate plus supplement (CCDA) (Oxoid),9) SIM media,10) Nitrate reagents [N(1-naphthyl)-ethylene diamine@2 HCl and Sulfanilic Acid],11) Each of the following biochemicals prepared in a base of Brucella Semi-solid Media: 1% glucose, 1%,nitrite, 1% glycine and 3.5% sodium chloride,MFLP-46,March 2002,-3-,12) Park and Sanders Enrichment Broth,13) Rapid H2S Test Medium (for biotyping),14) Oxidase Test strips or Tetramethylparaphenylenediamine@2 HCl reagent,15) Gas Mixture (10% CO2, 5% O2, 85% N2) or CampyPak 2 envelope (BBL) or Gas Generating Kit for,campylobacters (Oxoid) or Anaerocult C (Merck),16) Nalidixic Acid (30μg) disks, Cephalothin (30μg) disks or E-test strips (Oxoid),17) Sodium Hippurate,18) Liquid Nitrogen,19) Dimethyl Sulfoxide,20) Hydrogen peroxide,21) Lead acetate strips,22) Vacuum Leak Detector (Electro-Technic Products),23) Cryotubes,24) Positive control (ATCC or equivalent),25) Shaking Incubator (37oC and 42oC),26) CO2 Incubator (37oC and 42oC) (optional),NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or,waterbaths are maintained at the recommended temperatures. Where 35°C is recommended in the,text of the method, the incubator may be 35 +/-1.0°C. Similarly, lower temperatures of 30 or 25/C may,be +/- 1.0°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is,imp……
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