1、BSI Standards PublicationBS 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-glucuronideBS EN ISO 16649-3:2015 BRITISH STANDARDNati
2、onal 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 It supersedes DD ISO/TS 16649-3:2005 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be obt
3、ained 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 Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 79725 5 ICS 07.100.30 Compliance
4、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 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUR
5、OPISCHE NORM EN ISO 16649-3 May 2015 ICS 07.100.30 English Version Microbiology 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-glucuronide (ISO 16649-3
6、:2015) Microbiologie de la chane alimentaire - Mthode horizontale 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) Mikrobiologie der Lebensmittel
7、kette - Horizontales Verfahren 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)This European Standard was approved by CEN on 16 April 2015. CEN member
8、s 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 application
9、 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 Managemen
10、t 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, Li
11、thuania, 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 17,
12、 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 EBS EN ISO 16649-3:2015EN ISO 16649-3:2015 (E) 3 Foreword This document (EN ISO 16649-3:2015) has been prepared by Technical Committee ISO/TC 34
13、 “Foodproducts” in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” thesecretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identicaltext or by endorsement, at the latest by Nov
14、ember 2015, and conflicting national standards shall be withdrawnat the latest by November 2015. Attention is drawn to the possibility that some of the elements of this document may be the subject of patentrights. CEN and/or CENELEC shall not be held responsible for identifying any or all such paten
15、t rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, CzechRepublic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, Fra
16、nce, 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 16649-3:2015 has been approved by CEN as EN IS
17、O 16649-3:2015 without any modification. BS EN ISO 16649-3:2015ISO 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 of double strength liquid sel
18、ective 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 Culture media . 35.3.1 Minera
19、ls 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 test sample . 79 Procedure. 7
20、9.1 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 Test portion, initial suspensio
21、n, 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 911 Precision . 912 Test repo
22、rt . 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 International Standards is n
23、ormally 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 liaison with ISO, also take part i
24、n 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 Directives, Part 1. In particular
25、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 of the elements of this doc
26、ument 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 declarations received (see www.iso.o
27、rg/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 ISOs adherence to the WTO p
28、rinciples 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 16649-3:2005, which has been technica
29、lly 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-indolyl-D-glucuronide Part 2:
30、 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-glucuronideiv ISO 2015 All rights reservedBS EN ISO 16649-3:2015ISO 16649-3:2015(E)IntroductionBecause of the large variety of food a
31、nd feed products, this horizontal method might not be appropriate in every detail for certain products. 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
32、horizontal method as far as possible.When this part of ISO 16649 is next reviewed, account will be taken 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 harmoniz
33、ation of test methods cannot be immediate and for certain groups of products, International Standards and/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
34、16649 so that eventually, the only remaining departures will be those necessary for well-established technical reasons. ISO 2015 All rights reserved vBS EN ISO 16649-3:2015ISO 16649-3:2015(E)BS EN ISO 16649-3:2015Microbiology of the food chain Horizontal method for the enumeration of beta-glucuronid
35、ase-positive Escherichia coli Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronideWARNING Strains 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 strai
36、ns 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 horizontal method for the detection and enumeration of -glucuronidase positive Escherichia coli, by means of the liquid-medium culture technique and calculation
37、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: products intended for human consumption and the feeding of animals; environmental samples in the area of food production and food handling.The method is suitable f
38、or the enumeration of cells of E. coli that might have been subjected to stress arising from dehydration, freezing, and exposure to a saline (such as marine) environment or damage by disinfectants such as chlorine-containing products.A limitation of the applicability of this part of ISO 16649 is imp
39、osed by the susceptibility of the method to a large degree of variability. The method is intended to be applied and the results interpreted in the 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 inte
40、nded to be used for milk and milk products.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of th
41、e referenced document (including any amendments) applies.ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal di
42、lutionsISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 2: Specific rules for the preparation of meat and meat productsISO 6887-3, Microbiology of food and animal feeding stuffs Prepa
43、ration of test samples, initial suspension and decimal dilutions for microbiological examination Part 3: Specific rules for the preparation of fish 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, Microb
44、iology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 4: Specific rules for the preparation of miscellaneous productsISO 6887-5, Microbiology of food and animal feeding stuffs Preparation of test samples, i
45、nitial suspension and decimal dilutions for microbiological examination Part 5: Specific rules for the preparation of milk and milk productsISO 6887-6, Microbiology of food and animal feed Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 6: S
46、pecific rules for the preparation of samples taken at the primary production stageISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinationsISO 11133, Microbiology of food, animal feed and water Preparation, production, storage and per
47、formance 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
48、the conditions specified in the procedure3.2enumeration of -glucuronidase positive Escherichia colidetermination of the most probable number of -glucuronidase positive E. coli per millilitre or gram of sample when the test is carried out in accordance with the specified procedure4 Principle4.1 Detec
49、tion method4.1.1 A liquid selective enrichment medium is inoculated with a specified quantity of test sample if the initial product is liquid or with a specified quantity of the initial suspension in the case of other products.4.1.2 The tube is incubated at (37 1) C for (24 2) h. The tube is examined for acid production, indicating lactose fermentation.4.1.3 If the tube has given rise to acid production, it is subcultured onto tryptone bile glucuronide agar (5.3.2).4.1.4 Incubation of
