1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationMicrobiology of food and animal feed Horizontal method for the detection, enumeration and serotyping of SalmonellaPart 2: Enumeration by a miniaturized most probable number techn
2、iquePD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-National forewordThis Published Document is the UK implementation of CEN ISO/TS 6579-2:2012. It super
3、sedes BS EN ISO 6579:2002+A1:2007 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the
4、necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012 ISBN 978 0 580 77125 5 ICS 07.100.30Compliance with a British Standard cannot confer immunity from legal obligations.This Published Doc
5、ument was published under the authority of the Standards Policy and Strategy Committee on 30 November 2012.Amendments issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for Re
6、saleNo reproduction or networking permitted without license from IHS-,-,-TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 6579-2 November 2012 ICS 07.100.30 Supersedes EN ISO 6579:2002English Version Microbiology of food and animal feed - Horizontal method for the d
7、etection, enumeration and serotyping of Salmonella - Part 2: Enumeration by a miniaturized most probable number technique (ISO/TS 6579-2:2012) Microbiologie des aliments - Mthode horizontale pour la recherche, le dnombrement et le srotypage des salmonella - Partie 2: Dnombrement par une technique mi
8、niaturise du nombre le plus probable (ISO/TS 6579-2:2012) Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis, zur Zhlung und zur Serotypisierung von Salmonellen - Teil 2: Zhlung unter Anwendung eines miniaturisierten Verfahrens der wahrscheinlichsten Keimzahl (IS
9、O/TS 6579-2:2012) This Technical Specification (CEN/TS) was approved by CEN on 16 July 2012 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the quest
10、ion whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It is permissible to keep conflicting national standard
11、s in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Mace
12、donia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUR
13、OPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN ISO/TS 6579-2:2012: EPD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization P
14、rovided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-CEN ISO/TS 6579-2:2012 (E) 2 Contents Page Foreword 3PD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleN
15、o reproduction or networking permitted without license from IHS-,-,-CEN ISO/TS 6579-2:2012 (E) 3 Foreword This document (CEN ISO/TS 6579-2:2012) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods
16、” the secretariat of which is held by DIN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 6579:2002.
17、According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France
18、, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO/TS 6579-2:2012 has been approved by CEN as a CE
19、N/TS ISO 6579-2:2012 without any modification. PD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-ISO/TS 6579-2:2012(E) ISO 2012 All rights reserved iiiCont
20、ents PageIntroduction v1 Scope 12 Normative references . 13 Terms and definitions . 24 Principle . 24.1 General . 24.2 Pre-enrichment in non-selective liquid medium . 24.3 Enrichment on a selective semi-solid medium . 24.4 Selective plating and identification 24.5 Confirmation . 24.6 Calculation of
21、most probable number . 35 Culture media and sera 35.1 General . 35.2 Culture media . 36 Apparatus and glassware . 37 Sampling 48 Preparation of test sample 49 Procedure 49.1 Test portion, initial suspension . 49.2 Dilution and pre-enrichment in non-selective liquid medium . 59.3 Selective enrichment
22、 on a semi-solid medium . 59.4 Selective plating 59.5 Biochemical and serological confirmation . 610 Expression of results . 811 Test report . 8Annex A (informative) Composition and preparation of culture media and reagents . 9Annex B (informative) Diagram of procedure .17Bibliography .18PD CEN ISO/
23、TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-ISO/TS 6579-2:2012(E)IntroductionThe procedure described is based on the method reported in Reference 1.The enumerat
24、ion procedure described here concerns a miniaturized most probable number (MPN) technique. For this mini-MSRV (modified semi-solid RappaportVassiliadis) MPN technique, the volume of primary dilution tested is less than the volume in the detection method specified in ISO 6579:2002 + Cor.1:2004 + Amd.
25、1:2007.5For this reason, the sensitivity of the mini-MSRV technique is lower than in these detection methods (Reference 1). The detection limit of the mini-MSRV method is approximately 1 cfu/g, but can vary according to Salmonella serovar and per matrix. For the previously mentioned detection method
26、s, this is typically 1 cfu/25 g (0,04 cfu/g). For samples with (very) low numbers of Salmonella spp. (1 cfu/g), it is possible that the mini-MSRV procedure is not sufficiently sensitive. If a quantitative result is requested for samples likely to contain such low numbers (and tested negative with th
27、is mini-MSRV technique, for example), it is advisable to enumerate with a “conventional” MPN technique (not miniaturized). For other samples, the mini-MSRV method can have an advantage over the conventional MPN technique because the performance of the miniaturized MPN technique can take less time an
28、d need fewer resources (due to small amounts). ISO 2012 All rights reserved vPD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-Microbiology of food and ani
29、mal feed Horizontal method for the detection, enumeration and serotyping of Salmonella Part 2: Enumeration by a miniaturized most probable number techniqueWARNING In order to safeguard the health of laboratory personnel, it is essential that tests for detecting Sal-monella, are only undertaken in pr
30、operly equipped laboratories, under the control of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials.Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safe
31、ty aspects, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory condi-tions.1 ScopeThis part of ISO 6579 gives a method for the enumeration of Salmonella spp. present in: prod
32、ucts intended for human consumption and for the feeding of animals; environmental samples in the area of food production and food handling; animal faeces; environmental samples from the primary production stage;by calculation of the most probable number (MPN).The method is based on miniaturization o
33、f the dilution, pre-enrichment and selective enrichment steps. The selective enrichment medium, modified semi-solid RappaportVassiliadis (MSRV), is intended for the detection of motile salmonellae and is not appropriate for the detection of non-motile salmonellae.It is possible that the method is le
34、ss appropriate to enumerate Salmonella ser. Typhi and Salmonella ser. Paratyphi.The method is not appropriate for the enumeration of Salmonella spp. in (very) low contaminated samples (1 cfu/g).NOTE The number of non-motile salmonellae is generally low in most of the matrices relevant for this metho
35、d. In this note, examples are given for samples from primary production. The non-motile Salmonella biovars of Salmonella Gallinarum (Salmonella Gallinarum biovar gallinarum and Salmonella Gallinarum biovar pullorum) do not seem to survive long in environmental samples and are therefore rarely detect
36、ed in faecal or environmental (such as dust) samples (regardless of the method). The number of other non-motile Salmonella serovars in faecal samples seems to be generally low. For example, in Reference 4 in which approximately 1 000 faecal samples of poultry layer flocks and approximately 900 faeca
37、l samples of broiler flocks were analysed, less than 1 % of the total number of samples were positive in a selective broth and at the same time negative on MSRV medium (and likely to be non-motile). Similar results were found in a Dutch study with ca 3 200 faecal samples of pigs (unpublished data).
38、On the other hand, in the case of the study reported in Reference 4, up to almost 40 % of positive samples would not have been detected (i.e. false negatives) if only a selective broth (in this case RappaportVassiliadis) had been used instead of a semi-solid medium.2 Normative referencesThe followin
39、g referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 6887 (all parts), Microbiology of food and animal feeding
40、stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examinationTECHNICAL SPECIFICATION ISO/TS 6579-2:2012(E) ISO 2012 All rights reserved 1PD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot
41、for ResaleNo reproduction or networking permitted without license from IHS-,-,-ISO/TS 6579-2:2012(E)ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinationsISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on
42、preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratoryISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practical guidelines on p
43、erformance testing of culture media3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1Salmonellamicroorganism which forms typical or less typical colonies on solid selective media and which displays specific biochemical and serological characterist
44、icsNOTE Suitable tests for the specific biochemical and serological characteristics are specified in this part of ISO 6579.3.2count of Salmonellanumber of Salmonella spp. found per millilitre or per gram of a test sample or per surface area or on an object (e.g. bootsocks)4 Principle4.1 GeneralThe e
45、numeration of Salmonella spp. in the MPN format necessitates four successive stages (4.2 to 4.5).4.2 Pre-enrichment in non-selective liquid mediumPreparation of a 10-1dilution of the sample in buffered peptone water (BPW) (initial suspension).Addition of the initial suspension to the first (empty) r
46、ow of three wells of a 12-well microtiter plate.Inoculation of the second row of three wells containing non-selective pre-enrichment broth (BPW) with a specified quantity, from the first row in a 12-well microtiter plate.Inoculation of the third, fourth and if necessary more rows of three wells cont
47、aining BPW.Incubation of the 12-well microtiter plates at 37 C for 18 h.4.3 Enrichment on a selective semi-solid mediumSubculturing of each well obtained in 4.2 in a well containing a semi-solid agar (MSRV).Incubation at 41,5 C (MSRV) for 24 h. If MSRV is negative after 24 h, the plate is incubated
48、for a further 24 h.4.4 Selective plating and identificationFrom the (suspect) cultures (the highest dilutions) obtained in 4.3, a selective solid medium xyloselysinedeoxycholate (XLD) agar is inoculated and incubated at 37 C for 24 h.4.5 ConfirmationColonies of presumptive Salmonella obtained in 4.4 are confirmed by means of appropriate biochemical and serological tests.2 ISO 2012 All rights reservedPD CEN ISO/TS 6579-2:2012Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license
