1、BRITISH STANDARD BS EN ISO 16654:2001 Incorporating Corrigendum No. 1 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli 0157 The European Standard EN ISO 16654:2001 has the status of a British Standard ICS 07.100.30 NO COPYING WITHOUT BSI PERMISSI
2、ON EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN ISO 16654:2001 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 August 2001 BSI 07-2001 ISBN 0
3、580 37719 9 National foreword This British Standard is the official English language version of EN ISO 16654:2001. It is identical with ISO 16654:2001. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: A list of organiza
4、tions represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by
5、using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immun
6、ity from legal obligations. aid enquirers to understand the text; 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
7、in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to v, a blank page, pages 1 to 13, the annex ZA page, an inside back cover and a back cover. The BSI copyright notice displayed in t
8、his document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments 13344 Corrigendum No. 1 July 2001 Incorporation of annex ZAEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16654 May 2001 ICS 07.100.30 English version Microbiology of food a
9、nd animal feeding stuffs - Horizontal method for the detection of Escherichia coli O 157 (ISO 16654:2001) Microbiologie des aliments - Mthode horizontale pour la recherche des Escherichia coli O 157 (ISO 16654:2001) Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren fr den Na
10、chweis von Escherichia coli O 157 (ISO 16654:2001) This European Standard was approved by CEN on 1 May 2001. 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 altera
11、tion. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the 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 translati
12、on under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
13、 Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CEN All rights of exploitation in any form and b
14、y any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16654:2001 EForeword The text of the International Standard ISO 16654:2001 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Ho
15、rizontal 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 2001, and conflicting national standards shall be withdrawn at the latest by No
16、vember 2001. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands
17、, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 16654:2001 was approved by CEN as a European Standard without any modification. NOTE: Normative references to International Standards are listed in annex ZA (normative
18、). EN ISO 16654:2001Reference number ISO 16654:2001(E) INTERNATIONAL STANDARD ISO 16654 First edition 2001-05-01 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 Microbiologie des aliments Mthode horizontale pour la recherche des Escherichia
19、 coli O157 EN ISO 16654:2001ii iii Contents Page Foreword.iv Introduction.v 1 Scope 1 2 Normative references 1 3 Term and definition .1 4 Principle2 5 Culture media, reagents and antisera .2 6 Apparatus and glassware .7 7 Sampling.8 8 Preparation of test sample8 9 Procedure (see annex A)8 9.1 Test p
20、ortion and initial suspension .8 9.2 Enrichment .8 9.3 Immunomagnetic separation (IMS)8 9.4 Plating out onto selective agars and identification of E. coli O157 colonies9 9.5 Confirmation.10 9.6 Further characterization11 10 Quality assurance11 10.1 Test strains for quality assurance purposes11 10.2
21、Culture method 11 11 Expression of results 11 12 Test report 11 Annex A (normative) Diagram of procedure 12 Bibliography13 EN ISO 16654:2001 Annex ZA (normative) Normative references to international publications with their relevant European publications.14iv Foreword ISO (the International Organiza
22、tion 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 interested in a subject for which a technical committee has been established
23、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 Commission (IEC) on all matters of electrotechnical standardization. In
24、ternational Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. 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
25、 member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 16654 was prepared by Technic
26、al Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. Annex A forms a normative part of this International Standard. EN ISO 16654:2001v Introduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain pro
27、ducts. In this case, different methods 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 possible. When this International Standard is next reviewed, account will be tak
28、en 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 immediate, and for certain groups of products International Stan
29、dards 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 International Standard so that eventually the only remaining departures from this horizontal method will be tho
30、se necessary for well-established technical reasons. EN ISO 16654:2001blank 1 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 WARNING Escherichia coli O157 can cause severe life-threatening illness and has a low infective dose. Laboratory-a
31、cquired infections have been reported. In order to safeguard the health of laboratory personnel, it is essential that the whole of this method be carried out only by skilled personnel using good laboratory practices and preferably working in a containment facility. Relevant national Health and Safet
32、y Regulations relating to this organism must be adhered to. Care must be taken in the disposal of all infectious materials. 1 Scope This International Standard specifies a horizontal method for the detection of Escherichia coli serogroup O157. Subject to the limitations discussed in the introduction
33、, this International Standard is applicable to products intended for human consumption or for animal feeding stuffs. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated refe
34、rences, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated re
35、ferences, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for micro
36、biological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions. ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Term and definition For the purposes of this International Standard, the follow
37、ing term and definition applies. 3.1 Escherichia coli O157 E. coli O157 microorganisms which form typical colonies on the surface of the plating-out medium used in this International Standard, and which produce indole and agglutinate specifically with antiserum against the O157 antigen NOTE 1 Sorbit
38、ol-positive E. coli O157 strains are not detected on CT-SMAC (5.2) media. NOTE 2 Some indole-negative mutations have been found. EN ISO 16654:20012 4P r i n c i p l e The detection of Escherichia coli O157 necessitates four successive stages (see annex A). a) Enrichment of the test portion homogeniz
39、ed in modified tryptone soya broth containing novobiocin (mTSB + N) with incubation at 41,5 C 1 C for 6 h and subsequently for a further 12 h to 18h. b) Separation and concentration of microorganisms by means of immunomagnetic particles coated with antibodies to E. coli O157. c) Isolation by subcult
40、ure of the immunomagnetic particles with adhering bacteria onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC) and the users choice of a second selective isolation agar. d) Confirmation of sorbitol-negative colonies from CT-SMAC and colonies typical of E. coli O157 on the second isolation agar
41、, by indole production and agglutination with E. coli O157 antiserum. NOTE Further characterization, by for example pathogenic markers, of the positive isolates can be obtained by forwarding them to an appropriate reference laboratory. 5 Culture media, reagents and antisera For current laboratory pr
42、actices, see ISO 7218. 5.1 Enrichment medium: Modified tryptone soya broth with novobiocin (mTSB + N) See reference 1. 5.1.1 Modified tryptone soya broth (mTSB) 5.1.1.1 Composition Enzymatic digest of casein 17,0 g Enzymatic digest of soya 3,0 g D(+)-glucose 2,5 g Bile salts No. 3 1,5 g Sodium chlor
43、ide 5,0 g Dipotassium hydrogen phosphate (K 2 HPO 4 ) 4,0 g Water 1000 ml 5.1.1.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the pH, using the pH-meter (6.6), if necessary, so that after sterilization it is 7,4 0,2 at 25 C. Dis
44、pense the medium in appropriate amounts in flasks or bottles (6.7). Sterilize for 15 min in the autoclave (6.1) set at 121 C. EN ISO 16654:2001 3 5.1.2 Novobiocin solution 5.1.2.1 Composition Novobiocin 0,45 g Water 100 ml 5.1.2.2 Preparation Dissolve the novobiocin in the water and sterilize by mem
45、brane filtration. Prepare on the day of use. 5.1.2.3 Preparation of the complete medium Immediately before use, add 1 ml or 4 ml of novobiocin solution (5.1.2) to either 225 ml or 900 ml of cooled mTSB (5.1.1). The final concentration of novobiocin is 20 mg per litre of mTSB. 5.2 First selective iso
46、lation medium: Cefixime tellurite sorbitol MacConkey agar (CT-SMAC) See reference 2. 5.2.1 Base medium 5.2.1.1 Composition Enzymatic digest of casein 17,0 g Enzymatic digest of animal tissues 3,0 g Sorbitol 10,0 g Bile salts No. 3 1,5 g Sodium chloride 5,0 g Neutral Red 0,03 g Crystal Violet 0,001 g
47、 Agar 9gto1 8g a Water 1000 ml a Depending on the gel strength of the agar. 5.2.1.2 Preparation Dissolve the basic components or the complete dehydrated base in the water by boiling. Adjust the pH (6.6), if necessary, so that after sterilization it is 7,1 0,2 at 25 C. Sterilize for 15 min in the aut
48、oclave (6.1) set at 121 C. 5.2.2 Potassium tellurite solution 5.2.2.1 Composition Potassium tellurite for bacteriological use 0,25 g Water 100 ml EN ISO 16654:20014 5.2.2.2 Preparation Dissolve the potassium tellurite in the water and sterilize by membrane filtration. This solution may be stored at
49、room temperature for up to 1 month, but discard it if a white precipitate forms. 5.2.3 Cefixime solution 5.2.3.1 Composition Cefixime 5,0 mg Water 100,0 ml 5.2.3.2 Preparation Dissolve the cefixime in the water and sterilize by membrane filtration. NOTE Cefixime may need to be dissolved in ethanol. This solution may be stored at 3 C 2 C for 1 week. 5.2.4 Complete medium 5.2.4.1 Composition Base medium (5.2.1) 1 000 ml Potassium tellurite solution (5.2.2) 1,0 ml Cefixime solution (5.2.3) 1,0 ml 5.2.4.2 Preparation Either cool the freshly s
