MFLP-73 The Isolation and Enumeration of Vibrio vulnificus from Fish and Seafoods
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37F1CBEE1F274B6D96E4D156C08BD2CB |
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0.07 |
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18 |
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日期： |
2012-3-2 |
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Published by: POLYSCIENCE PUBLICATIONS, P.O. Box 1606, Station St-Martin, Laval, Quebec,Canada H7V 3P9. TEL.: 1-800-840-5870. FAX: (450) 688-1930.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-73,April 1995,HEALTH PROTECTION BRANCH,OTTAWA,THE ISOLATION AND ENUMERATION OF,Vibrio vulnificus FROM FISH AND SEAFOODS,S. Stavric and B. Buchanan,Research Division, Bureau of Microbial Hazards, Food Directorate,Health Canada, Ottawa ON, K1A 0L2,1. APPLICATION,This method is applicable to the detection of Vibrio vulnificus in foods to determine compliance with the,requirements of Sections 4 and 7 of the Food and Drugs Act.,2. DESCRIPTION,The method has been shown to produce satisfactory results with fish, shellfish and crustaceans (9.2). Vibrio,vulnificus is an obligate halophile, and a salt concentration of 0.5 to 3.0% is necessary to maintain good growth.,CAUTION: V. vulnificus is one of the most lethal human pathogens for individuals with underlying liver disease,(9.6). Infection from this organism can occur by ingestion of a contaminated food, or by direct entry into an open,wound. As a safety precaution, it is recommended that protective gloves be worn during this procedure.,3. PRINCIPLE,Detection of this microorganism requires three successive phases: (i) enrichment in a nonselective broth, (ii) plating,out on selective media with presumptive identification, and (iii) confirmation by biochemical and serological tests.,This is a quantitative method based on a most probable number (MPN) procedure published recently in the,Bacteriological Analytical Manual of the United States Food and Drug Administration (9.2).,4. DEFINITION OF TERMS.,See Appendix A of Volume 3.,5. COLLECTION OF SAMPLES,5.1 See Appendix B of Volume 3.,5.2 If the analysis is not performed immediately upon the arrival of samples in the laboratory, store samples in,the refrigerator (0-4EC) or frozen, depending on the nature of the product. If this method is used for,environmental surveys (e.g. freshly harvested shellfish or fish), and analysis is performed with minimal delay,after collection of samples, it is preferable not to refrigerate them. At low temperature, V. vulnificus can,enter into a viable but nonculturable state (9.7).,MFLP-73,- 2 - April 1995,6. MATERIALS AND SPECIAL EQUIPMENT,Final concentration of NaCl for V. vulnificus should be between 2% and 3% (w/v). NaCl may need to be added to,commercially made media.,1) Phosphate buffered saline (PBS),2) Alkaline peptone water (APW),3) Thiosulfate-citrate-bile salts-sucrose agar (TCBS),4) Modified cellobiose-polymyxin-colistin agar (mCPC),5) Tryptone salt agar (T1N1),6) Trypticase soy agar supplemented with 1.5% NaCl (TSA-2%),7) Gelatin agar (GA),8) Gelatin salt agar (GSA),9) Tris-EDTA-Triton X-100 (TET) buffer or formalinized PBS (FPBS),10) Antiflagellar V. vulnificus serum (Available from Dr. R. Siebeling, Department of Microbiology, Louisiana,State University, Baton Rouge. LA. 70803 U.S.A., Tel. no. (504) 388-2601, Fax No. (504) 388-2597).,11) Oxidase reagent,12) Arginine glucose slants (AGS),13) Kovacs reagent,14) Triple sugar iron agar (TSI),15) o-Nitrophenyl-$-D-galactosidase (ONPG) reagent,16) Hugh-Leifson glucose broth,17) Bromcresol purple broths, with individually added carbohydrates e.g. glucose, lactose, sucrose, cellobiose,salicin, mannose, and arabinose,18) Methyl red-Voges Proskauer broth (MR-VP) and reagents,19) Falkow's decarboxylase basal medium, with or without individually added amino acids e.g. lysine, ornithine,and arginine.,20) Tryptone broth with or without added NaCl to final concentrations of 0%, 3%, 6%, 8%, and 10%.,21) Trypticase soy broth (TSB) and agar (TSA),22) Motility test medium,23) EIA monoclonal antibody (Available from Aquatic Diagnostic, Inc. 3405 SW 40th Blvd., Box 304, Gainesville,FL. 32608, U.S.A.). For EIA solutions, see Sect. 10.,MFLP-73,- 3 - April 1995,24) Heart infusion broth (HIB) supplemented with 1% NaCl,25) Waring blender, or equivalent.,26) Incubators capable of maintaining 35±0.5EC, 40±0.5EC and 42±0.5EC.,7. PROCEDURE,Each sample unit should be analyzed individually or the analytical units may be composited. For a detailed,description of the washing and opening of shellfish for analysis, see reference 9.4. To prevent hand injury, use,heavy cotton or chain mail gloves for shucking. For a flow chart of the entire analytical procedure, see Fig. 1,Schedule for Sample Analysis. Carry out the test in accordance with the following instructions:,7.1 Handling of Sample Units,7.1.1 In the laboratory, prior to analysis, keep sample units refrigerated (0-4EC) or frozen, depending on the,nature of t……
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