1、BRITISH STANDARD Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli 0157 The European Standard EN IS0 16654:2001 has the status of a British Standard ICs 07.100.30 BS EN IS0 16654:ZOOl Incorporating Corrigendum No. 1 o NO COPYING WITHOUT BSI PERM
2、ISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN IS0 16654:2001 Products and Services Sector Committee, was published Amd. No. Date under the authority of the Standards Committee and 13344 July 2001 comes into effect on 15 August 2001 Corrigendum No. 1 National foreword Comments Incorporation of an
3、nex ZA This British Standard is the official English language version of EN IS0 16654:2001. It is identical with IS0 16654:2001. The UK participation in its preparation was entrusted to Technical Committee AWI9, Microbiology, which has the responsibility to: - - aid enquirers to understand the text;
4、 present to the responsible internationalEuropean 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. - A list of organizations represented on this committee
5、 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 using the “Find facility of the B
6、SI 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 immunity from legal obligations. Summar
7、y of pages This document comprises a front cover, an inside front cover, the EN IS0 title page, the EN IS0 foreword page, the IS0 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 this document indi
8、cates when the document was last issued. O O BSI 07-2001 ISBN O 580 37719 9 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM O EN IS0 16654 May 2001 ICs 07.100.30 English version Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O 157 (IS0 1665
9、4:20“1) Microbiologie des aliments - Mthode horizontale pour la recherche des Escherichia coli O 157 (IS0 16654:2001) Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren fr den Nachweis von Escherichia coli O 157 (IS0 16654:2001) This European Standard was approved by CEN on 1
10、 May 2001. CEN members are bound to comply with the CENKENELEC 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 obt
11、ained 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 translation under the responsibility of a CEN member into its own language and notified to the Management Ce
12、ntre 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, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN
13、 COMMlTiEE FOR STANDARDIZATION COMITE EUROPEEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 8-1050 Brussels O 2001 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN IS0 16654:2001 E EN IS0 16
14、654:2001 Foreword The text of the International Standard IS0 16654:2001 has been prepared by Technical Committee ISOT-C 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods“, the secretariat of which is held by DIN. This European St
15、andard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the late-f by November 2001, and conflicting national standards shall be withdrawn at the latestby November 2001. According to the CENKENELEC Internal Regulations, the national s
16、tandards 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, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorse
17、ment notice The text of the International Standard IS0 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). INTERNATIONAL STANDARD EN IS0 16654:2001 IS0 16654 First edition 2001 -05-0
18、1 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O1 57 Microbiologie des aliments - Mthode horizontale pour la recherche des Escherichia coli 0157 Reference number IS0 16654:2001 (E) EN IS0 16654:2001 I Contents Page Foreword . iv Introductio
19、n . v 1 2 3 4 5 6 7 8 9 9.1 9.2 9.3 9.4 9.5 9.6 10 10.1 10.2 11 12 Scope 1 Normative references 1 Term and definition . 1 Culture media, reagents and antisera . 2 Sampling . 8 Procedure (see annex A) 8 Principle 2 Apparatus and glassware . 7 Preparation of test sample 8 Test portion and initial susp
20、ension . 8 Enrichment . 8 Immunomagnetic separation (IMS) 8 Plating out onto selective agars and identification of E. coli 0157 colonies 9 Confirmation . 10 Further characterization 11 Quality assurance 11 Test strains for quality assurance purposes 11 Culture method 11 Expression of results 11 Test
21、 report 11 Annex A (normative) Diagram of procedure 12 Bibliography 13 Annex ZA (normative) Normative references to international publications with their relevant European publications . 14 . 111 EN IS0 16664:2001 Foreword IS0 (the International Organization for Standardization) is a worldwide feder
22、ation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 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 commit
23、tee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordanc
24、e with the rules given in the ISOAEC Dirp?tives, 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 member bodies casting a vote. O Attention is d
25、rawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. IS0 shall not be held responsible for identifying any or all such patent rights. International Standard IS0 16654 was prepared by Technical Committee ISO/TC 34, Food products, Subcom
26、mittee SC 9, Microbiology. Annex A forms a normative part of this International Standard. iv EN IS0 16654:ZOOl Introduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain products. In this case, different methods spec
27、ific 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 taken of all information then available regard
28、ing the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of partic4-v products. The harmonization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already
29、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 those necessary for well-established technical
30、reasons. r V - - EN IS0 16654:2001 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli 0157 WARNING - Escherichia coli 0157 can cause severe life-threatening illness and has a low infective dose. Laboratory-acquired infections have been reported.
31、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 practicer pd preferably working in a containment facility. Relevant national Health and Safety Regulations relating to this organism
32、must be adhered to. Care must be taken in the disposal of all infectious materials. O 1 Scope This International Standard specifies a horizontal method for the detection of Escherichia coli serogroup O1 57. Subject to the limitations discussed in the introduction, this International Standard is appl
33、icable 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 references, subsequent amendments to, or
34、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 references, the latest edition of the n
35、ormative document referred to applies. Members of IS0 and IEC maintain registers of currently valid International Standards. IS0 6887-1, Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: Ge
36、neral rules for the preparation of the initial suspension and decimal dilutions. IS0 721 8, Microbiology of food and animal feeding stuffs - General rules for microbiological examinations. 3 Term and definition For the purposes of this International Standard, the following term and definition applie
37、s. 3.1 Escherichia coli O1 57 E. coli 0157 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 O1 57 antigen NOTE 1 Sorbitol-positive E. coli0157 stra
38、ins are not detected on CT-SMAC (5.2) media. 0 NOTE 2 Some indole-negative mutations have been found. 1 EN IS0 16654:2001 4 Principle The detection of Escherichia coli0157 necessitates four successive stages (see annex A). a) Enrichment of the test portion homogenized in modified tryptone soya broth
39、 containing novobiocin (mTSB + N) with incubation at 41,5 OC f 1 OC 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. coli0157. c) Isolation by subculture of the immunomagnetic parti
40、cles 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 O1 57 on the second isolation agar, by indole production and agg
41、lutination with E. coli01 57 antiserum. NOTE them to an appropriate reference laboratory. Further characterization, by for example pathogenic markers, of the positive isolates can be obtained by forwarding 5 Culture media, reagents and antisera For current laboratory practices, see IS0 721 8. 5.1 En
42、richment medium: Modified tryptone soya broth with novobiocin (mTSB + N) See reference l. 5.1.1 5.1 .1.1 Composition Modified tryptone soya broth (mTSB) Enzymatic digest of casein 17,o Enzymatic digest of soya 3,O g D(+)-glucose 2,s g Bile salts No. 3 1,s g Sodium chloride 5,O g 4,o 9 Water 1 O00 ml
43、 Dipotassium hydrogen phosphate (K,HPO,) 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 f 0,2 at 25 OC. Dispense the medium in appropriat
44、e amounts in flasks or bottles (6.7). Sterilize for 15 min in the autoclave (6.1) set at 121 OC. 2 EN IS0 16654:2001 0 5.1.2 Novobiocin solution 5.1.2.1 Composition Novobiocin Water 100 ml I 5.1.2.2 Preparation Dissolve the novobiocin in the water and sterilize by membrane filtration. Prepare on the
45、 day of use. 5.1.2.3 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 .I). The final concentration of novobiocin is 20 mg per litre of mTSB. 5.2 Preparation of the complete medium First selective isolation medium: Cefixime telluri
46、te sorbitol MacConkey agar (CT-SMAC) O See reference 2. 5.2.1 Base medium 5.2.1.1 Composition O Enzymatic digest of casein 17,o Enzymatic digest of animal tissues 310 g Sorbitol 1010 9 Bile salts No. 3 1,5 9 Sodium chloride 5,O g Neutral Red 0103 g Crystal Violet 0,001 g Agar Water 1 O00 ml a 9g to
47、18 g 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,l f 0,2 at 25 OC. Sterilize for 15 min in the autoclave (6.1) set at 121
48、 OC. 5.2.2 Potassium tellurite solution 5.2.2.1 Composition Potassium tellurite for bacteriological use 025 g 100 ml O Water 3 EN IS0 16654:2001 5.2.2.2 Preparation Dissolve the potassium tellurite in the water and sterilize by membrane filtration. This solution may be stored at room temperature for
49、 up to 1 month, but discard it if a white precipitate forms. 5.2.3 Cefixime solution 5.2.3.1 Composition Cef ixime 530 mg Water 100,O ml 5.2.3.2 Preparation Dissolve the cefixime in the water and sterilize by membrane filtration. NOTE This solution may be stored at 3 OC f 2 OC for 1 week. 5.2.4 Complete medium 5.2.4.1 Composition Cefixime may need to be dissolved in ethanol. Base medium (5.2.1) Potassium tellurite solution (5.2.2) Cefixime solution (5.2.3) 1000ml 1,0 ml 1,0 ml 5.2.4.2 Preparation Either cool the freshly sterilized base medium (5.2.1) to between 44 OC a
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