MFHPB-26 Enumeration of Escherichia coli in Foods by the Hydrophobic Grid-Membrane Filter Method (HGMF) Method
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6AD73078A9CB4835A94F9C48C1AC1D39 |
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0.03 |
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页数： |
7 |
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日期： |
2012-3-2 |
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Published on the Food Directorate’s (Health Canada) website at http://www.hc-sc.gc.ca/food-aliment,Government of Canada Gouvernement du Canada,HPB Method MFHPB-26,March 2001,HEALTH PROTECTION BRANCH,OTTAWA,ENUMERATION OF ESCHERICHIA COLI IN FOODS BY THE,HYDROPHOBIC GRID-MEMBRANE FILTER (HGMF) METHOD,P.I. Peterkin, A.N. Sharpe, and H. Wang,Bureau of Microbial Hazards,Health Products and Food Branch,Postal locator: 2204A2,Ottawa, Ont. K1A 0L2,E-mail: Haiyan_Wang@hc-sc.gc.ca,1. APPLICATION,The method may be used for enumeration of E. coli biotype 1 in foods other than bean or alfalfa sprouts, to,determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an,Official Method for a food is specified, that method shall be followed. This revised method replaces MFHPB-,26, dated April 1997.,2. DESCRIPTION,The method has been shown to produce satisfactory results with beef and ground beef, chicken and deboned,chicken, fish, nuts, black pepper, cheese and green beans (9.2, 9.3, 9.4). The HGMF method was granted,Official Final Action Status by the Association of Official Analytical Chemists (9.2). Since this time it has,been used for the enumeration of E. coli biotype 1 in most foods and food ingredients, generally without,reports of problems. The exceptions are bean and alfalfa sprouts, in which large populations of indolepositive,Klebsiella spp. preclude an accurate determination of E. coli.,3. PRINCIPLE,The HGMF analysis takes 24-30h and yields counts that are higher and more precise than the Most Probable,Number method (MFHPB-19), and equivalent to or higher than the Direct Plating Method (MFHPB-27) (9.4).,A single dilution gives an accurate count over a wide range of contamination levels. Counting precision may,be better than on conventional plates or membrane filters because the HGMF reduces the effect of individual,visual acuity on the count (9.5). If a low count is expected, the detection limit can be lowered by filtering,more of the suspension.,The enumeration of presumptive E. coli is based on incubation at a selective temperature and the,demonstration of indole production through formation of pink to red grid cells (9.5). It detects those E. coli,classed as late lactose fermenting or anaerogenic (approx. 10% of strains) which are missed by the MPN,method, but does not detect indole-negative E. coli (3-5% of strains). The specificity of the test is high;,MFHPB-26,- 2 - March 2001,overall confirmation rates of indole-positive colonies on membrane filters is 95% for E. coli biotype 1 (IMViC,++--), while a further 3.4% were confirmed as faecal coliforms on the basis of IMViC +++- or +--- reactions,(9.1). This level of specificity was independently confirmed (9.6). Thus, confirmation of E. coli should not,normally be necessary. Since the indole staining reaction kills bacterial cells, further examination of the,sample would require replication of the HGMF prior to staining.,4. DEFINITION OF TERMS,See Appendix A of Volume 2.,5. COLLECTION OF SAMPLES,See Appendix B of Volume 2.,6. MATERIALS AND SPECIAL EQUIPMENT,The media listed below (items 3-5) are commercially available and are to be prepared and sterilized,according to the manufacture's instructions.,1) HGMF (1600 grid-cell, 0.45 μm pore size). (Available as ISO-GRID filters from Oxoid Ltd., Nepean,Ont.),2) Membrane filter forceps (Millipore Corp., Bedford, MA.),3) Peptone water (0.1%) (PW) or Peptone/Tween 80 diluent (PT),4) Nutrient agar (NA) plates (if a resuscitation step is required),5) Tryptone bile agar (TBA) plates,6) Enzyme solutions (Appendix E of Volume 2; 9.7) as required for some food products,7) Stomacher/or equivalent,8) Whatman No. 1 or equivalent filter papers, 7 cm dia,9) Indole reagent,10) Spreadfilter with funnel plus pipet-tip prefilters (Filtaflex Ltd., P.O. Box 1224, Almonte, Ont.), or ISOGRID,filtration unit (Oxoid, Nepean, Ont.),11) HGMF Linecounter for manual counting (Available from Oxoid Ltd., Nepean, Ont.)(optional),12) An incubator capable of maintaining 44.50C,NOTE: It is the responsibility of each laboratory to ensure that the temperature of the incubators or water,baths are maintained at the recommended temperatures. Where 35EC is recommended in the text,of the method, the incubator may be at 35 +/-1.0E C. Similarly, lower temperatures of 30 or 25 may,be +/- 1.0EC. However, where higher temperatures are recommended, such as 43 or 45.5EC, it is,imperative that the incubators or water baths be maintained within 0.5EC due to potential lethality,of higher temperatures on the microorganism being isolated.,MFHPB-26,- 3 - March 2001,7. PROCEDURE,Analyze each sample u……
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