EN ISO TS 6579-2-2012 en Microbiology of food and animal feed - Horizontal method for the detection enumeration and serotyping of Salmonella - Part 2 Enumeration by a miniaturized .pdf

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1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationMicrobiology of food andanimal feed Horizontalmethod for the detection,enumeration and serotyping of SalmonellaPart 2: Enumeration by a miniaturizedmost probable number technique

2、PD CEN ISO/TS 6579-2:2012PD CEN ISO/TS 6579-2:2012Incorporating corrigendum February 2013National forewordThis Published Document is the UK implementation of CEN ISO/TS 6579-2:2012. It supersedes BS EN ISO 6579:2002+A1:2007,which is withdrawn.The UK participation in its preparation was entrusted to

3、Technical CommitteeAW/9, Microbiology.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions of acontract. Users are responsible for its correct application. The British Standards Inst

4、itution 2012.Published by BSI Standards Limited 2012.ISBN 978 0 580 77125 5ICS 07.100.30Compliance with a British Standard cannot confer immunityfrom legal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 30 November 2012.Amendmen

5、ts issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN ISO/TS 6579-2:2012.PD CEN ISO/TS 6579-2:2012 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation ofCEN ISO/TS 6579-2:2012.Amendments/corrigenda issued since publicationDate Text affected28 February

6、2013 Implementation of CEN correction notice 19 December 2012ritish Standards Ins itution 2013.lished by BSI Standards Limited 2013.ISBN 978 0 580 82081 6ICS 07.100.30National forewordThis Published Document is the UK implementation of CEN ISO/TS 6579-2:2012. It supersedes BS EN ISO 6579:2002+A1:200

7、7,which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be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 necessary provisions of a contra

8、ct. Users are responsible for its correct application. The British Standards Institution 2012.Published by BSI Standards Limited 2012.ISBN 978 0 580 77125 5ICS 07.100.30Compliance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the au

9、thority of the Standards Policy and Strategy Committee on 30 November 2012.Amendments issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN ISO/TS 6579-2:2012TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 6579-2 November 2012 ICS 07.100.30 Supersedes

10、EN ISO 6579:2002English Version Microbiology of food and animal feed - Horizontal method for the detection, 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 rec

11、herche, le dnombrement et le srotypage des salmonella - Partie 2: Dnombrement par une technique miniaturise 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 - Te

12、il 2: Zhlung unter Anwendung eines miniaturisierten Verfahrens der wahrscheinlichsten Keimzahl (ISO/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

13、two years the members of CEN will be requested to submit their comments, particularly on the question 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

14、 at national level in an appropriate form. It is permissible to keep conflicting national standards 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, Bulg

15、aria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Tu

16、rkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES 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. CE

17、N ISO/TS 6579-2:2012: EPD CEN ISO/TS 6579-2:2012CEN 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” the secretariat of

18、 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. According to the CEN

19、/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, Germany, Greece, H

20、ungary, 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 CEN/TS ISO 6579-2:2012

21、 without any modification. PD CEN ISO/TS 6579-2:2012CEN 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” the secretaria

22、t 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. According to the

23、 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, Germany, Greec

24、e, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endors ment notice The text of ISO/TS 6579-2:2012 has been approved by CEN as a CEN/TS ISO 6579-2:

25、2012 without any modification. PD CEN ISO/TS 6579-2:2012ISO/TS 6579-2:2012(E) ISO 2012 All rights reserved iiiContents 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

26、a selective semi-solid medium . 24.4 Selective plating and identification 24.5 Confirmation . 24.6 Calculation of 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

27、, initial suspension . 49.2 Dilution and pre-enrichment in non-selective liquid medium . 59.3 Selective enrichment 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 prep

28、aration of culture media and reagents . 9Annex B (informative) Diagram of procedure .17Bibliography .18PD CEN ISO/TS 6579-2:2012ISO/TS 6579-2:2012(E)IntroductionThe procedure described is based on the method reported in Reference 1.The enumeration procedure described here concerns a miniaturized mos

29、t 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.1:2007.5For this reason, the sensitivity of the mini-MSR

30、V 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 methods, this is typically 1 cfu/25 g (0,04 cfu/g). For sample

31、s 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 this mini-MSRV technique, for example), it is advisable to

32、 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 and need fewer resources (due to small amounts). ISO 2012

33、All rights reserved vPD CEN ISO/TS 6579-2:2012ISO/TS 6579-2:2012(E)IntroductionThe procedure described is based on the method reported in Reference 1.The enumeration procedure described here concerns a miniaturized most probable number (MPN) technique. For this mini-MSRV (modified semi-solid Rappapo

34、rtVassiliadis) 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.1:2007.5For this reason, the sensitivity of the mini-MSRV technique is lower than in these detection methods (Reference 1). The detection

35、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 methods, 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 mi

36、ni-MSRV procedure is not sufficiently sensitive. If a quantitative result is requested for samples likely to contain such low numbers (and tested negative with this mini-MSRV technique, for example), it is advisable to enumerate with a “conventional” MPN technique (not miniaturized). For other sampl

37、es, 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 and need fewer resources (due to small amounts). ISO 2012 All rights reserved vPD CEN ISO/TS 6579-2:2012Microbiology of food and animal feed

38、 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 properly e

39、quipped 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 safety aspec

40、ts, 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: products int

41、ended 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 of the di

42、lution, 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 less appro

43、priate 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 method. In th

44、is 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 detected in fa

45、ecal 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 faecal sample

46、s 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). On the o

47、ther 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 following refere

48、nced 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 stuffs P

49、reparation 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 6579-2:2012ISO/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 preparation and production of culture media Part 1: General guidelines on quality ass