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1、BRITISH STANDARD BS ISO 7251:2005 Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of presumptive Escherichia coli Most probable number technique ICS 07.100.30 ? Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontro
2、lled Copy, (c) BSI BS ISO 7251:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 5 May 2005 BSI 5 May 2005 ISBN 0 580 43482 6 National foreword This British Standard reproduces verbatim ISO 7251:2005 and implements it as the UK national st
3、andard. It supersedes BS 5763-8:1994 and BS ISO 11866-1:1997 which are withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its
4、secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or
5、of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text;
6、 present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover
7、, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 13 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun,
8、 na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference number ISO 7251:2005(E) OSI 5002 INTERNATIONAL STANDARD ISO 7251 Third edition 2005-02-01 Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of presumptive Escherichia coli M
9、ost probable number technique Microbiologie des aliments Mthode horizontale pour la recherche et le dnombrement dEscherichia coli prsums Technique du nombre le plus probable BS ISO 7251:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS
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15、0:00 2006, Uncontrolled Copy, (c) BSI IS:1527 O5002(E) I SO 5002 All irthgs ersedevr iii Contents Page Forewordiv Introduction v 1 Scope1 2 Normative references .1 3 Terms and definitions.2 4 Principle.2 4.1 Detection method2 4.2 Enumeration method2 5 Diluent, culture media and reagent.3 6 Apparatus
16、 and glassware.5 7 Sampling6 8 Preparation of test sample.6 9 Procedure.6 9.1 Detection method6 9.2 Enumeration method7 10 Expression of results8 10.1 Detection method8 10.2 Enumeration method8 11 Test report8 Annex A (normative) MPN table10 Bibliography .13 BS ISO 7251:2005 Licensed Copy: sheffield
17、un sheffieldun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:1527 O5002(E) iv I SO 5002 All irthgs ersedevr Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Int
18、ernational 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 liaison w
19、ith ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of techni
20、cal committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn
21、to the possibility that some 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. ISO 7251 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. This third editi
22、on cancels and replaces the second edition (ISO 7251:1993), and also ISO 11866-1:1997 and IDF 170-1:1999. Clauses 4, 9 and 10 of ISO 7251:1993 have been technically revised. The main changes are as follows: the temperature of the second incubation is now 44 C 1 C (see 4.2.5); detection (9.1) and enu
23、meration (9.2) of presumptive E. coli are covered; the use of five tubes per dilution is allowed for some specific products (see 9.2.2.1); some products (dairy products) may hinder the collection of gas (see 9.1.2 and 9.2.2.5); the MPN calculation refers to ISO 7218 (see Clause 10). BS ISO 7251:2005
24、 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:1527 O5002(E) I SO 5002 All irthgs ersedevr v Introduction Because of the large variety of products within this field of application, these guidelines may not be appropriate in every detail
25、 for certain products, and for some other products it may be necessary to use different methods. Nevertheless, it is hoped that in all cases every attempt will be made to apply the provided guidelines as far as possible and that deviations from them will only be made if absolutely necessary for tech
26、nical reasons. When this International Standard is next reviewed, account will be taken of all information then available regarding the extent to which the guidelines have been followed and the reasons for deviations from them in the case of particular products. The harmonization of test methods can
27、not be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with these guidelines. In cases where International Standards already exist for the product to be tested, they should be followed, but it is hoped that when suc
28、h standards are reviewed they will be changed to comply with this International Standard so that eventually the only remaining departures from these guidelines will be those necessary for well-established technical reasons. BS ISO 7251:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:5
29、4:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI INTENRATIONAL TSANDADR IS:1527 O5002(E) I SO 5002 All irthgs ersedevr 1 Microbiology of food and animal feeding stuffs Horizontal method for the
30、detection and enumeration of presumptive Escherichia coli Most probable number technique 1 Scope This International Standard gives general guidelines for the detection and enumeration of presumptive Escherichia coli by means of the liquid-medium culture technique and calculation of the most probable
31、 number (MPN) after incubation at 37 C, then at 44 C. This International Standard is applicable to products intended for human consumption and the feeding of animals, and environmental samples in the area of food production and food handling. WARNING Some pathogenic strains of Escherichia coli do no
32、t grow at 44 C. A limitation of the applicability of this International Standard is imposed by the susceptibility of the method to a large degree of variability (see Clause 10). 2 Normative references The following referenced documents are indispensable for the application of this document. For date
33、d 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, initial suspension and decimal dilutions for microbiological
34、 examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 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 prepa
35、ration of meat and meat products ISO 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 fish and fishery products ISO 6887-4, Microbiology of food a
36、nd animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products, and fish and fishery products ISO 7218, Microbiology of foo
37、d and animal feeding stuffs General rules for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination BS ISO 7251:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun
38、 Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:1527 O5002(E) 2 I SO 5002 All irthgs ersedevr ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of
39、 culture media in the laboratory ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media 3 Terms and definitions For the purposes of this document, the following term
40、s and definitions apply. 3.1 presumptive Escherichia coli bacteria which at 44 C ferment lactose with the production of gas, and which at 44 C produce indole from tryptophan, when the test is carried out in accordance with the method specified in this International Standard 3.2 enumeration of presum
41、ptive Escherichia coli most probable number of E. coli per millilitre or per gram of the test sample when the test is carried out according to the method specified in this International Standard 4 Principle 4.1 Detection method 4.1.1 A liquid selective enrichment medium is inoculated with a specifie
42、d quantity of the initial suspension of the test sample. 4.1.2 The tube is incubated at 37 C for up to 48 h. The tube is examined for gas production after 24 h and 48 h. 4.1.3 If the tube has given rise to opacity, cloudiness or gaseous emission, it is subcultured to a tube containing a liquid selec
43、tive medium (EC broth). 4.1.4 The tube obtained in 4.1.3 is incubated at 44 C for up to 48 h. The tube is examined for gas production after 24 h and 48 h. 4.1.5 If the tube of medium obtained in 4.1.4 has given rise to gaseous emission, it is subcultured to a tube containing indole-free peptone wate
44、r. 4.1.6 The tube obtained in 4.1.5 is incubated at 44 C for 48 h. The tube is examined for indole production resulting from the degradation of tryptophan in the peptone constituent. 4.1.7 Tubes showing opacity, cloudiness or gas production in the liquid selective enrichment medium (4.1.1) and whose
45、 subcultures have produced gas in the EC broth and indole in the peptone water at 44 C are considered to contain presumptive Escherichia coli. The results are given as the “presence“ or “absence“ of presumptive Escherichia coli in x g or x ml of product. 4.2 Enumeration method 4.2.1 Three tubes of d
46、ouble-strength liquid selective enrichment medium are inoculated with a specified quantity of the initial suspension. 4.2.2 Three tubes of single-strength liquid enrichment medium are inoculated with a specified quantity of the initial suspension. BS ISO 7251:2005 Licensed Copy: sheffieldun sheffiel
47、dun, na, Sun Nov 26 02:54:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:1527 O5002(E) I SO 5002 All irthgs ersedevr 3 Then, under the same conditions, another three tubes of the single-strength medium are inoculated with a specified quantity of decimal dilutions of the initial suspension. 4.2.3 T
48、he tubes of double- and single-strength medium are incubated at 37 C for up to 48 h. The tubes are examined for gas production after 24 h and 48 h. 4.2.4 Each tube of double-strength medium that has given rise to opacity, cloudiness or gaseous emission, and each tube of single-strength medium that h
49、as given rise to gaseous emission, is subcultured to a tube containing a liquid selective medium (EC broth). 4.2.5 The tubes obtained in 4.2.4 are incubated at 44 C for up to 48 h. The tubes are examined for gas production after 24 h and 48 h. 4.2.6 Each tube of medium obtained in 4.2.5 that has given rise to gaseous emission is subcultured to a tube containing indole-free peptone water. 4.2.7 The tubes obtained