MFLP-37 Part 1_ Detection of Halophilic Vibrio Species in Seafood_Part 2_ Detection of Vibrio Cholerae
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BC70C63E874B4A06A0044F202064E149 |
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0.08 |
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27 |
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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_e.html,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-37,October 2006,HEALTH PRODUCTS AND FOOD BRANCH,OTTAWA,PART 1: DETECTION OF HALOPHILIC VIBRIO SPECIES IN SEAFOOD,Microbiological Methods Committee,Microbiology Evaluation Division,Bureau of Microbial Hazards, Food Directorate,Health Products and Food Branch, Health Canada,Postal Locator: 2204A1,Ottawa, Ontario K1A 0L2,E-mail: Don_Warburton@hc-sc.gc.ca,1. APPLICATION,The method may be used for the isolation and enumeration of Vibrio parahaemolyticus, Vibrio vulnificus and,other halophilic Vibrio spp. in seafood to determine compliance with the requirements of Sections 4 and 7 of the,Food and Drugs Act. This revised method replaces MFHPB-15 dated April 1997 and MFLP-57 dated,September 1996.,2. DESCRIPTION,The genus Vibrio consists of Gram negative, rod- or curved-rod shaped facultative anaerobes. Most species are,oxidase positive and halophilic; that is, they require additional sodium chloride in growth media. The exceptions,are V. cholera and V. mimicus (non-halophilic). Many Vibrio spp., including Vibrio cholera, Vibrio,parahaemolyticus and Vibrio vulnificus are human pathogens.,3. PRINCIPLE,Detection of Vibrio parahaemolyticus and Vibrio vulnificus, as well as other halophilic vibrios, requires three,successive phases: (i) enrichment in selective medium, (ii) plating out onto isolation agars and presumptive,identification, and (iii) confirmation with biochemical, serological and pathogenicity tests. This method is based,on a procedure published in the Bacteriological Analytical Manual (BAM) (8.9).,4. DEFINITION OF TERMS,See Appendix A of Volume 3,5. COLLECTION OF SAMPLES,See Appendix B of Volume 3,MFLP-37,October 2006,2,6. MATERIALS AND SPECIAL EQUIPMENT,The following media and reagents (1-27) 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 8.1 for the formula,of individual media.,Note: Media for the isolation of halophilic Vibrio spp. require added NaCl (2-3% final concentration). V. cholerae grow,well in media with 0-3% NaCl. Add NaCl to the media noted below to achieve a final 2-3% concentration of NaCl.,Salt content of media follows in square brackets [NaCl %].,(1) Alkaline peptone salt broth (APS) [3%],(2) Phosphate buffered saline (PBS) [0.85%],(3) Alkaline peptone water (APW) [1%],(4) Thiosulphate citrate bile salts agar (TCBS) [1%],(5) Modified cellobiose-polymyxin-B-colistin (mCPC) [2%],(6) Blood agar (optional) [0.5%] - optional to add NaCl for halophiles,(7) Mannitol-maltose agar (optional) [2%],(8) T1N2 agar (1% tryptone and 2% NaCl) (optional),(9) 1% Tryptone broths containing 0, 1, 3, 6, 8 and 10% NaCl (T1N0, T1N1, T1N3, T1N6, T1N8, T1N10),(10) Trypticase soy agar (TSA) [0.5%] - add 1.5% NaCl for halophiles,(11) Arginine glucose slant (AGS) [2%],(12) Gelatin agar (GA) [0%],(13) Gelatin agar with 3% NaCl (GS),(14) Decarboxylase basal medium containing arginine, lysine, ornithine - optional to add NaCl for,halophiles,(15) MR-VP broth and V-P reagents - optional to add NaCl to broth for halophiles,(16) Carbohydrates in bromcresol purple broth or OF broth, semi-solid: sucrose, lactose, D-mannitol,mannose, arabinose, cellobiose [0.5%] - optional to add NaCl for halophiles,(17) Wagatsuma agar [7%],(18) Hugh-Leifson glucose broth [3%] OR OF glucose medium, semi-solid [0.5%],(19) Christensen’s urea agar slants [0.5%] - optional to add NaCl for halophiles,(20) Gram stain solutions,(21) Motility test medium [0.5%] - optional to add NaCl for halophiles,(22) Triple sugar iron agar (TSI) [0.5%] - optional to add 2.5% NaCl for halophiles,MFLP-37,October 2006,3,(23) API 20E identification strips (bioMérieux Inc.) or equivalent,(24) Oxidase reagent or commercially available test strips,(25) O-nitrophenyl-?D galactoside (ONPG) test solution or commercial product,(26) Sterile mineral oil,(27) Positive and negative control cultures (ATCC or equivalent),(28) 2% NaCl in distilled water (for dilution and inoculation of API),(29) Incubators, 35°C, 40°C and 42°C,(30) Blender, stomacher or equivalent,Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators and waterbaths are,maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubator or,waterbath 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 ……
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