1、BS EN ISO 16649-3:2015Microbiology of the foodchain Horizontal methodfor the enumeration ofbeta-glucuronidase-positiveEscherichia coliPart 3: Detection and most probablenumber technique using 5-bromo-4-chloro-3-indolyl-D-glucuronideIncorporating corrigendum February 2017(ISO 16649-3:2015)BSI Standar
2、ds PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 16649-3:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 16649-3:2015. It supersedes DD ISO/TS 16649-3:2005 which is withdrawn.The UK participation in its preparation was ent
3、rusted 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 application. The British S
4、tandards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 97525 7 ICS 07.100.30 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 201
5、5.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dImplementation of ISO corrected text 15 December28 February 20172016: See ISO foreword for detailsEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16649-3 May 2015 ICS 07.100.30 English Version Microbiology of the fo
6、od chain - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronide (ISO 16649-3:2015, Corrected version 2016-12-15) Microbiologie de la chane alimentaire - Mthode horiz
7、ontale pour le dnombrement des Escherichia coli bta-glucuronidase positive - Partie 3: Recherche et technique du nombre le plus probable utilisant le bromo-5-chloro-4-indolyl-3 -D-glucuronate (ISO 16649-3:2015, Version corrige 2016-12-15) Mikrobiologie der Lebensmittelkette - Horizontales Verfahren
8、zur Zhlung von -Glucuronidase-positiven Escherichia coli - Teil 3: Nachweis und Bestimmung der wahrscheinlichsten Keimzahl unter Verwendung von 5-Brom-4-Chlor-3-Indol-D-Glucuronid (ISO 16649-3:2015, korrigierte Fassung 2016-12-15) This European Standard was approved by CEN on 16 April 2015. CEN memb
9、ers 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 obtained on applicati
10、on 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 the CEN-CENELEC Managem
11、ent 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, Ireland, Italy, Latvia,
12、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 EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 1
13、7, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16649-3:2015 EEN ISO 16649-3:2015 (E) European foreword This document (EN ISO 16649-3:2015) has been prepared by Technical Committee ISO/TC 34 “Food produc
14、ts” 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, at the latest by November 2015
15、, and conflicting national standards shall be withdrawn at the latest by November 2015. 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
16、. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, G
17、ermany, 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 16649-3:2015, Corrected version 2016-12-15 has bee
18、n approved by CEN as EN ISO 16649-3:2015 without any modification. BS EN ISO 16649-3:2015iiISO 16649-3:2015(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 24 Principle 24.1 Detection method . 24.2 Enumeration method 34.2.1 Inoculation of three or five tubes o
19、f double strength liquid selective enrichment medium 5.3.1.1 a) with an equal volume of the test sample if the initial product is liquid, or with an equal volume of the initial suspension in the case of other products. 35 Dilution fluids and culture media . 35.1 General . 35.2 Dilution fluids 35.3 C
20、ulture media . 35.3.1 Minerals modified glutamate medium (selective enrichment medium) . 45.3.2 Tryptone bile glucoronide agar (second selective enrichment medium) . 55.3.3 Performance testing for the quality assurance of the culture media . 56 Apparatus and glassware 67 Sampling 68 Preparation of t
21、est sample . 79 Procedure. 79.1 Detection method . 79.1.1 Test portion, initial suspension, and dilutions . 79.1.2 Incubation of selective enrichment medium . 79.1.3 Subculturing 79.1.4 Secondary incubation 79.1.5 Examination of the plates . 79.1.6 Interpretation . 79.2 Enumeration method 79.2.1 Tes
22、t portion, initial suspension, and dilutions . 79.2.2 Inoculation of the selective enrichment medium 89.2.3 Incubation 89.2.4 Subculturing 89.2.5 Second incubation 89.2.6 Examination of the plates . 89.2.7 Interpretation . 810 Expression of results 910.1 Detection method . 910.2 Enumeration method 9
23、11 Precision . 912 Test report . 9Bibliography .10 ISO 2015 All rights reserved iiiContents PageBS EN ISO 16649-3:2015ISO 16649-3:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing
24、International Standards is normally carried out through ISO technical committees. Each member body interested 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 liaiso
25、n with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (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 Direct
26、ives, Part 1. In particular the different approval criteria needed for the different types of ISO documents 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
27、 of the elements of this document may be the subject of patent rights. ISO shall not be held responsible 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 declarat
28、ions received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about
29、 ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9, Microbiology.This first edition cancels and replaces ISO/TS 16649-3:2
30、005, which has been technically revised.ISO 16649 consists of the following parts, under the general title Microbiology of the food chain Horizontal method for the enumeration of -glucuronidase positive Escherichia coli: Part 1: Colony-count technique at 44 C using membranes and 5-bromo-4-chloro-3-i
31、ndolyl-D-glucuronide Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl-D-glucuronide Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronideThis corrected version of ISO 16649-3:2015 incorporates the following corrections: in 4.1.4, f
32、irst line, “(22 2) h” has been replaced with “(21 3) h”; in 4.2.5, first line, “(22 2) h” has been replaced with “(21 3) h”; Table 1 has been replaced; Table 2 has been replaced; in 9.1.4, first line, “(22 2) h” has been replaced with “(21 3) h”; in 9.2.5, first line, “(22 2) h” has been replaced wi
33、th “(21 3) h”; in 9.2.5, second line, “three” has been replaced with “six”.iv ISO 2015 All rights reservedBS EN ISO 16649-3:2015ISO 16649-3:2015(E)IntroductionBecause of the large variety of food and feed products, this horizontal method might not be appropriate in every detail for certain products.
34、 In this case, different methods which are specific to these products might be used if absolutely necessary, for justified technical reasons. Nevertheless, every attempt will be made to apply this horizontal method as far as possible.When this part of ISO 16649 is next reviewed, account will be take
35、n of all information 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 immediate and for certain groups of products, International Standards a
36、nd/or national standards might 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 part of ISO 16649 so that eventually, the only remaining departures will be those necessary for well-established te
37、chnical reasons. ISO 2015 All rights reserved vBS EN ISO 16649-3:2015Microbiology of the food chain Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronideWARNING Strains
38、 of Escherichia coli that do not grow at 44 C and, in particular, those that are -glucuronidase negative, such as Escherichia coli O157 and some other strains of pathogenic E. coli, will not be detected by the method described in this part of ISO 16649.1 ScopeThis part of ISO 16649 specifies a horiz
39、ontal method for the detection and enumeration of -glucuronidase positive Escherichia coli, by means of the liquid-medium culture technique and calculation of the most probable number (MPN) after incubation at (37 1) C, then at (44 1) C. This part of ISO 16649 is applicable to the following: product
40、s intended for human consumption and the feeding of animals; environmental samples in the area of food production and food handling.The method is suitable for the enumeration of cells of E. coli that might have been subjected to stress arising from dehydration, freezing, and exposure to a saline (su
41、ch as marine) environment or damage by disinfectants such as chlorine-containing products.A limitation of the applicability of this part of ISO 16649 is imposed by the susceptibility of the method to a large degree of variability. The method is intended to be applied and the results interpreted in t
42、he light of the information given in Clause 11.This method has not been fully evaluated for all matrices (e.g. for milk and milk products). ISO 7251 is intended to be used for milk and milk products.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in thi
43、s document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples,
44、 initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutionsISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for micr
45、obiological examination Part 2: Specific rules for the preparation of meat and meat productsISO 6887-3, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 3: Specific rules for the preparation of f
46、ish and fishery productsINTERNATIONAL STANDARD ISO 16649-3:2015(E) ISO 2015 All rights reserved 1BS EN ISO 16649-3:2015ISO 16649-3:2015(E)ISO 6887-4, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination P
47、art 4: Specific rules for the preparation of miscellaneous productsISO 6887-5, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 5: Specific rules for the preparation of milk and milk productsISO
48、6887-6, Microbiology of food and animal feed Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 6: Specific rules for the preparation of samples taken at the primary production stageISO 7218, Microbiology of food and animal feeding stuffs Gener
49、al requirements and guidance for microbiological 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.3.1-glucuronidase positive Escherichia colistrains of E. coli which, at 44 C, form typical blue or blue green colonies on tryptone bile glucuronide medium (TBX) under the conditions
