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 BS ISO 7251:2005 This British Standard was published under the authority of the Standards
2、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 standard. It supersedes BS 5763-8:1994 and BS ISO 11866-1:1997 which are withdrawn. The UK participation in its
3、 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 secretary. Cross-references The British Standards which implement international publications referred to in t
4、his 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 of British Standards Online. This publication does not purport to include all the necessary provisions of a c
5、ontract. 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; present to the responsible international/European committee any enquiries on the interpretation, or proposal
6、s 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, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 13 and a back cover. The
7、 BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments Reference number ISO 7251:2005(E) OSI 5002INTERNATIONAL STANDARD ISO 7251 Third edition 2005-02-01 Microbiology of food and animal feeding stuffs H
8、orizontal method for the detection and enumeration of presumptive Escherichia coli Most probable number technique Microbiologie des aliments Mthode horizontale pour la recherche et le dnombrement dEscherichia coli prsums Technique du nombre le plus probable BSISO7251:2005IS:1527 O5002(E) DPlcsid Fre
9、mia ihTs PDF file mya ctnoian emdebt dedyfepcaes. In ccacnadrow eitA hebods licnesilop gnic,y tihs file mirp eb yatnde iv roweb detu slahl ton ide ebtlnu deess teh typfecaes wihce era hml era deddebicsnede ti dna onstlalde t noeh comuptfrep reromign tide ehtin.g In wodlnidaot gnihs fil,e trapise atp
10、ecc tiereht nser ehnopsiiblity fo nto ifnriigngn Aebods licnesilop gnic.y ehT ISO tneClar Secrteirata caceptl on siibality in this .aera Ai ebods a tedarmfo kra Aebod SystemI snctaropro.de teDials fo teh sfotwcudorp erats sut deo crtaee tihs PDF file cna f ebi dnuon tlareneG eh Ifnler oatit evt oeh
11、file; tP ehDc-Frtaeino marapterew stpo ereimizde fro irptni.gn Evyre cera neeb sah takne tsne oeru taht teh file is siutlbae fosu re yb ISO memdob rebeis. In tlnu ehikletneve y ttah lborp aem lertait gno it is f,dnuo plsaee ifnrom ttneC ehlar Secterirata ta teh serddaig sleb nevwo. ISO 5002 All irth
12、gs erse.devr lnUeto sswrehise specified, on trap fo this lbupictaion maeb y cudorperro de tuilizi den yna form ro na ybm ynae,s lecetrinoc ro mceinahcla, incliduntohp gcoiypodna gn micrfoilm, wittuoh repmissii now nritign from ietI rehSa Ot tsserdda eh ebolw or ISOs memreb i ydobn the cnuotfo yr tts
13、euqer ehe.r ISO cirypothg fofice saCe tsopale 65 eneG 1121-HC 02 av leT. 4 + 10 947 22 1 11 xaF0 947 22 14 + 9 74 E-mial coirypthgis.o gro We bwww.is.o groii ISO 5002 Allr ithgsr esedevrBSISO7251:2005IS:1527 O5002(E) I SO 5002 All irthgs ersedevr iiiContents Page Foreword iv Introduction v 1 Scope 1
14、 2 Normative references . 1 3 Terms and definitions. 2 4 Principle . 2 4.1 Detection method 2 4.2 Enumeration method 2 5 Diluent, culture media and reagent . 3 6 Apparatus and glassware. 5 7 Sampling 6 8 Preparation of test sample. 6 9 Procedure. 6 9.1 Detection method 6 9.2 Enumeration method 7 10
15、Expression of results 8 10.1 Detection method 8 10.2 Enumeration method 8 11 Test report 8 Annex A (normative) MPN table 10 Bibliography . 13 BSISO7251:2005IS:1527 O5002(E) iv I SO 5002 All irthgs ersedevrForeword ISO (the International Organization for Standardization) is a worldwide federation of
16、national standards bodies (ISO member bodies). The work of preparing 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. Inte
17、rnational organizations, governmental and non-governmental, in liaison with 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 th
18、e rules given in the ISO/IEC Directives, Part 2. The main task of technical 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 b
19、y at least 75 % of the member bodies casting a vote. 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 for identifying any or all such patent rights. ISO 7251 was prepared by Technical Committee ISO
20、/TC 34, Food products, Subcommittee SC 9, Microbiology. This third edition 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 th
21、e second incubation is now 44 C 1 C (see 4.2.5); detection (9.1) and enumeration (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);
22、the MPN calculation refers to ISO 7218 (see Clause 10). BSISO7251:2005IS:1527 O5002(E) I SO 5002 All irthgs ersedevr vIntroduction Because of the large variety of products within this field of application, these guidelines may not be appropriate in every detail for certain products, and for some oth
23、er 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 technical reasons. When this International
24、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 cannot be immediate, and for certain group
25、s 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 such standards are reviewed they will be c
26、hanged 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. BSISO7251:2005INTENRATIONAL TSANDADR IS:1527 O5002(E)I SO 5002 All irthgs ersedevr 1Microbiology of food and animal
27、 feeding stuffs Horizontal method for the 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 tech
28、nique and calculation of the most probable 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 pa
29、thogenic strains of Escherichia coli do not 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
30、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 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension
31、 and decimal dilutions for microbiological 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 examin
32、ation Part 2: Specific rules for the preparation 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 p
33、roducts ISO 6887-4, Microbiology 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 products other than milk and milk products, meat and meat products, and fish and fish
34、ery products ISO 7218, Microbiology of food 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 BSISO7251:2005IS:1527
35、O5002(E) 2 I SO 5002 All irthgs ersedevrISO/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 culture media in the laboratory ISO/TS 11133-2, Microbiology of food
36、 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 terms and definitions apply. 3.1 presumptive Escherichia coli bacteria wh
37、ich 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 presumptive Escherichia coli most probable number of E. coli per millilitre
38、 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 specified quantity of the initial suspension of the test sample. 4.1.2 The tu
39、be 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 selective medium (EC broth). 4.1.4 The tube obtained in 4.1.3 is incubated
40、 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 water. 4.1.6 The tube obtained in 4.1.5 is incubated at 44 C for 48 h. Th
41、e 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 subcultures have produced gas in the EC broth and indole in the pept
42、one 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 double-strength liquid selective enrichment medium are inoculated with
43、 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. BSISO7251:2005IS:1527 O5002(E) I SO 5002 All irthgs ersedevr 3Then, under the same conditions, another three tubes of the
44、 single-strength medium are inoculated with a specified quantity of decimal dilutions of the initial suspension. 4.2.3 The 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-streng
45、th medium that has given rise to opacity, cloudiness or gaseous emission, and each tube of single-strength medium that has 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.
46、 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 in 4.2.6 are incubated at 44 C for 48 h. The tubes are examined
47、for indole production resulting from the degradation of tryptophan in the peptone constituent. 4.2.8 The most probable number of presumptive Escherichia coli is determined by means of the MPN table (see Annex A), according to the number of tubes of single- and double-strength medium whose subculture
48、s have produced gas in the EC broth and indole in the peptone water at 44 C. 5 Diluent, culture media and reagent For current laboratory practice, see ISO 7218. 5.1 Diluent In general, see ISO 6887-1, but for milk products see ISO 8261. 5.2 Selective enrichment medium (lauryl sulfate broth) 5.2.1 Co
49、mposition a) Double-strength medium b) Single-strength medium Enzymatic digest of plant and animal tissues 40,0 g 20,0 g Lactose 10,0 g 5,0 g Dipotassium monohydrogen phosphate (K 2 HPO 4 ) 5,5 g 2,75 g Potassium dihydrogen phosphate (KH 2 PO 4 ) 5,5 g 2,75 g Sodium chloride 10,0 g 5,0 g Sodium lauryl sulfate CH 3 (CH 2 ) 11 OSO 3 Na 0,2 g 0,1 g Water 1 000 ml 1 000 ml 5.2.2 Preparation Diss
