1、Microbiology of the food chain Horizontal method for the detection of Cronobacter spp.BS EN ISO 22964:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National forewordThis British Standard is the UK implementation of EN ISO 22964:2017.The UK participation in its
2、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 contract. Users are responsible for its correct applica
3、tion. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 85632 7ICS 07.100.30; 67.100.01Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strat
4、egy Committee on 31 May 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN ISO 22964:2017EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 22964April 2017ICS 67.100.01; 07.100.30EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHE
5、S KOMITEE FR NORMUNGCEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN Ref. No. EN ISO 22964:2017: EAll rights of exploitation in any form and by any means reserved worldwide for CEN national MembersMicrobiology of the food chain - Horizontal method for the detection of Cronob
6、acter spp. (ISO 22964:2017) Microbiologie de la chane alimentaire - Mthode horizontale pour la recherche de Cronobacter spp. (ISO 22964:2017) Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von Cronobacter spp. (ISO 22964:2017) This European Standard was approved by CEN on
7、13 March 2017.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be
8、 obtained on application to the CEN-CENELEC Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to th
9、e CEN-CENELEC Management Centre has the same status as the official versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Irel
10、and, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.English VersionEN ISO 22964:2017 (E)European forewordThis document (EN ISO 22964:2017) has been prepared by Technical Co
11、mmittee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, a
12、t the latest by October 2017, and conflicting national standards shall be withdrawn at the latest by October 2017.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
13、 or all such patent rights.This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association.According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this Eu
14、ropean Standard: Austria, Belgium, Bulgaria, 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, Serbia, Slo
15、vakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.Endorsement noticeThe text of ISO 22964:2017 has been approved by CEN as EN ISO 22964:2017 without any modification.iiBS EN ISO 22964:2017ISO 22964:2017(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms an
16、d definitions . 14 Abbreviated terms 25 Principle 25.1 Non-selective pre-enrichment in BPW . 25.2 Enrichment in a selective medium (CSB) . 25.3 Plating out and identification on chromogenic agar (CCI agar) . 25.4 Confirmation . 26 Culture media and reagents 27 Equipment and consumables 27.10 Pestle
17、and mortar. . 38 Sampling 39 Preparation of test sample . 310 Procedure (as shown in Annex A) . 310.1 Test portion 310.2 Pre-enrichment . 410.3 Enrichment . 410.4 Isolation of presumptive Cronobacter spp. . 410.5 Confirmation . 410.5.1 General 410.5.2 Purification of colonies . 410.5.3 Biochemical c
18、onfirmation 510.6 Interpretation of biochemical results 611 Expression of results 612 Performance characteristics of the method . 612.1 Interlaboratory study 612.2 Sensitivity 712.3 Specificity 712.4 LOD50713 Test report . 7Annex A (normative) Diagram of the test procedure 8Annex B (normative) Compo
19、sition, preparation and performance testing of culture media and reagents 9Annex C (informative) Distinction of Cronobacter spp. from other genera .17Annex D (informative) Method validation studies and performance characteristics 19Bibliography .22 ISO 2017 All rights reserved iiiContents PageBS EN
20、ISO 22964:2017ISO 22964:2017(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interes
21、ted in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical C
22、ommission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documen
23、ts should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible
24、for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the
25、 convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in t
26、he Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.This document was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/275, Food Analysis Horizontal methods, in collaboration with ISO Technical Committee ISO/TC 34, Food produc
27、ts, Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between ISO and CEN (Vienna Agreement).This first edition cancels and replaces ISO/TS 22964:2006, which has been technically revised with the following changes: the scope has been extended to Cronobacter s
28、pp. detection in food products for humans and feeding animals and environmental samples and the title changed accordingly; the enrichment broth, modified lauryl sulfate tryptose broth (mLST), has been replaced by Cronobacter selective broth (CSB); the isolation agar, Enterobacter sakazakii isolation
29、 agar (ESIA) has been replaced by chromogenic Cronobacter isolation (CCI) agar; several confirmation tests have been replaced by other tests according to Table 1 of this document.iv ISO 2017 All rights reservedBS EN ISO 22964:2017ISO 22964:2017(E)IntroductionThis document describes a horizontal meth
30、od for the detection of Cronobacter spp. in food, in animal feed and in environmental samples. The main changes, listed in the foreword, introduced in this document compared to ISO/TS 22964:2006 are considered as major (see ISO 174682).Because of the large variety of food and feed products, this hor
31、izontal method may not be appropriate in every detail for certain products. In this case, different methods, which are specific to these products, may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this horizontal method as far as
32、 possible.When this document is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products.The harmonization of test methods cannot be
33、 immediate and, for certain groups of products, International Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped that when such standards are reviewed they will be changed to comply with this document so that eventually the only remainin
34、g departures from this horizontal method will be those necessary for well-established technical reasons. ISO 2017 All rights reserved vBS EN ISO 22964:2017This page deliberately left blankMicrobiology of the food chain Horizontal method for the detection of Cronobacter spp.1 ScopeThis document speci
35、fies a horizontal method for the detection of Cronobacter spp.Subject to the limitations discussed in the introduction, this document is applicable to food products and ingredients intended for human consumption and the feeding of animals, and environmental samples in the area of food production and
36、 food handling.2 Normative referencesThe following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced docum
37、ent (including any amendments) applies.ISO 6887 (all parts), Microbiology of the food chain Preparation of test samples, initial suspension and decimal dilutions for microbiological examinationISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiologi
38、cal examinationsISO 11133, Microbiology of food, animal feed and water Preparation, production, storage and performance testing of culture media3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.ISO and IEC maintain terminological databases for use in
39、 standardization at the following addresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp3.1Cronobacter spp.microorganisms which form typical colonies on chromogenic Cronobacter isolation (CCI) agar10and which display the
40、 biochemical characteristics described, when tests are carried out in accordance with this document3.2detection of Cronobacter spp.determination of Cronobacter spp. (3.1) in a particular mass or volume of product or surface area when tests are carried out in accordance with this documentINTERNATIONA
41、L STANDARD ISO 22964:2017(E) ISO 2017 All rights reserved 1BS EN ISO 22964:2017ISO 22964:2017(E)4 Abbreviated termsFor the purposes of this document, the following abbreviations apply.BPW buffered peptone waterCCI chromogenic Cronobacter isolationCSB Cronobacter selective brothTSA tryptone soya agar
42、5 Principle5.1 Non-selective pre-enrichment in BPWA test portion is inoculated into BPW, then incubated between 34 C and 38 C for 18 h 2 h.NOTE Cronobacter spp. can be present in small numbers accompanied by other Enterobacteriaceae such as E. cloacae that could interfere with their detection.5.2 En
43、richment in a selective medium (CSB)The selective enrichment medium is inoculated with the culture obtained in 5.1 and incubated at 41,5 C 1 C for 24 h 2 h.5.3 Plating out and identification on chromogenic agar (CCI agar)The chromogenic agar is streaked for isolation with the enrichment culture obta
44、ined in 5.2 and incubated at 41,5 C 1 C for 24 h 2 h.5.4 ConfirmationTypical colonies are selected from the chromogenic agar, purified on a non-selective agar such as TSA and biochemically characterized.6 Culture media and reagentsFor current laboratory practice, see ISO 7218 and ISO 11133.Compositi
45、on of culture media and reagents and their preparation are described in Annex B.For performance testing of culture media see ISO 11133 and/or Annex B.7 Equipment and consumablesDisposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.Usual microbiolog
46、ical laboratory equipment (see ISO 7218) and, in particular, the following.7.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).As specified in ISO 7218.7.2 Incubators, capable of operating in the range 34 C to 38 C, 37 C 1 C and 41,5 C 1 C.2 ISO 2017 All rights reservedBS EN
47、ISO 22964:2017ISO 22964:2017(E)7.3 Sterile loops, of approximate diameter 3 mm (10 l volume), and of 1 l volume, and inoculation needle or wire.7.4 pH meter, having an accuracy of calibration of 0,1 pH unit at 25 C.7.5 Flasks and bottles, with closures, of suitable capacities for use in the preparat
48、ion of enrichment broths and agars and their storage.7.6 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.7.7 Tubes (plugged or with caps) or culture bottles, of appropriate capacity, with non-toxic metallic caps with liners or plastic disposable caps (s
49、ee ISO 7218).7.8 Petri dishes, of diameter approximately 90 mm.7.9 Spectrophotometer, capable of measuring absorption of light with a wavelength of 405 nm.7.10 Pestle and mortar.7.11 Refrigerators, capable of operating at 5 C 3 C7.12 Water baths, capable of operating between 47 C and 50 C and at 37 C 1 C.7.13 Drying cabinet (or oven ventilated by convection), capable of being maintained between 25 C and 50 C.8 SamplingSampling is not part of the method specified in this document. See the specif
