MFHPB-20.pdf

Published on the Food Directorates (Health Canadas) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.php Government of Canada Gouvernement du Canada HPB MethodMFHPB-20 March 2009HEALTH PRODUCTS AND FOOD BRANCHOTTAWAISOLATION AND IDENTIFICATION OF SALMONELLA FROM FOOD AND ENVIRONMENTAL SAMPLESAnne ReidResearch DivisionHealth Products and Food Branch Health Canada, Postal locator: 2204E Ottawa, Ontario, K1A 0K9e-mail: Anne_Reidhc-sc.gc.caMicrobiological Methods CommitteeBureau of Microbial Hazards, Food Directorate,Health Products and Food Branch, Health CanadaPostal Locator: 2204EOttawa, Ontario K1A 0K91.APPLICATIONThis method is applicable to the detection of viable Salmonella in foods and environmental samples todetermine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and specificRegulations (as summarized in the Interpretive Summary (7.15). This revised method replaces MFHPB-20, dated April 1998. Due to health and safety issues surrounding the use of selenite cystine, this revisedmethod should also be used in place of the Official Methods (Volume 1 of this Compendium) such asMFO-6, MFO-10, MFO-11 and MFO-12.This revised method includes the following major changes in the analytical approach for the detection offoodborne Salmonella spp.:1)The incorporation of Appendix J (November 2003) allowing the substitution of RVSenrichment broth for SC enrichment broth for most food commodities.2)The incorporation of MFHPB-20A (April 2002), for the analysis of seed used tomanufacture sprouts.3)The inclusion of other selective media, including chromogenic media.MFHPB-20- 2 -March 20092.PRINCIPLEThe procedure consists of six distinct stages.2.1Non-Selective Enrichment (Pre-enrichment)The initial handling of the food and the non-selective enrichment stage (pre-enrichment) varyaccording to the type of food examined (7.6). The test sample is inoculated into a non-inhibitoryliquid medium to favour the repair and growth of stressed or sublethally-injured salmonellaearising from exposure to heat, freezing, desiccation, preservatives, high osmotic pressure or widetemperature fluctuations (7.1, 7.6).2.2Selective EnrichmentReplicate portions of each pre-enrichment culture are inoculated into two enrichment media tofavour the proliferation of salmonellae through a selective inhibition of the growth of competingmicroorganisms (7.4). The enrichment broth RVS (7.25 - 7.29) may be substituted for SC brothfor most food commodities (see the “Note” in Section 5, point 8 for exceptions).2.3Selective PlatingEnriched cultures are streaked onto selective and differential agars for the isolation of salmonellae(7.5, 7.30).2.4PurificationPresumptive Salmonella isolates are purified on MacConkey agar plates. This removes thepossibility of viable but inhibited organisms from the selective agars contaminating the culture infurther tests.2.5Biochemical ScreeningIsolates are screened using determinant biochemical reactions.2.6Serological IdentificationPolyvalent and/or grouping somatic antisera are used to support the identification of isolates asSalmonella. Cultures should be sent to a reference typing centre for complete serotyping.3.DEFINITION OF TERMSSee Appendix A of Volume 2. 4.COLLECTION OF SAMPLES4.1SamplingSee Appendix B of Volume 2.For routine sampling and analysis, 5 sample units of at least 100 g each should be collected fromeach lot unless stipulated in Table I. Refer to the Interpretive Summary (7.15) for Guidelines andRegulations specific to the foods being analysed for Salmonella.For additional information concerning sampling plans in relation to degrees of risk and conditionsof use refer to ICSMF (7.16). MFHPB-20- 3 -March 20095.MATERIALS AND SPECIAL EQUIPMENTNote: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as “ISO/IEC 17025:2005 (or latest version):General Requirements for the Competence of Testing and Calibration Laboratories.The media and reagents listed below are commercially available and are to be used, prepared and/orsterilized according to the manufacturers instructions. See Appendix G of Volume 2 for the mediaformula.Note: If the analyst uses any variations of the media listed here (either product that is commercially available ormade from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.1)Nutrient Broth (NB)2)Trypticase (Tryptic, Tryptone) Soy Broth3)Brilliant Green Water4)Buffered Peptone Water (BPW)5)Skim Milk Medium6)Tetrathionate Brilliant Green Broth (TBG)7)Selenite Cystine Broth (SC) (optional) 8)Rappaport-Vassiliadis Soya Peptone Broth (RVS)NOTE:a.USE OF SELENITE CYSTINE BROTH: Due to safety and environmental issues, SC brothshould be replaced with RVS broth in all methods used for the detection of Salmonella in foods,food ingredients and environmental samples, under most circumstances. SC broth must b