Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157 (ISO 16654:2001)

This International Standard specifies a horizontal method for the detection of Escherichia coli serogroup O157.
Subject to the limitations discussed in the introduction, this International Standard is applicable to products
intended for human consumption or for animal feeding stuffs.

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den Nachweis von Escherichia coli O157 (ISO 16654:2001)

Diese Internationale Norm legt ein horizontales Verfahren zum Nachweis von Escherichia coli (E. coli) Serogruppe 0 157 fest. Vorbehaltlich der in der Einleitung angefu~hrten Einschra~nkunten, ist diese Internationale Norm auf Lebensmittel und Futtermittel anwendbar.

Microbiologie des aliments - Méthode horizontale pour la recherche des Escherichia coli O157 (ISO 16654:2001)

La présente Norme internationale spécifie une méthode horizontale pour la recherche des Escherichia coli sérogroupe O157.  Soumise aux restrictions énoncées dans l'introduction, la présente Norme internationale est applicable aux produits destinés à la consommation humaine ou aux produits alimentaires.

Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje Escherichia coli O157 (ISO 16654:2001)

Ta mednarodni standard določa horizontalno metodo za ugotavljanje prisotnosti mikroorganizma Escherichia coli seroskupine O157.
V skladu z omejitvami, obravnavanimi v uvodu, ta mednarodni standard velja za proizvode, namenjene za prehrano ljudi ali za živalsko krmo.

General Information

Status
Published
Publication Date
31-Aug-2002
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Sep-2002
Due Date
01-Sep-2002
Completion Date
01-Sep-2002

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SLOVENSKI STANDARD
SIST EN ISO 16654:2002
01-september-2002
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje Escherichia coli
O157 (ISO 16654:2001)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection of
Escherichia coli O157 (ISO 16654:2001)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den
Nachweis von Escherichia coli O157 (ISO 16654:2001)
Microbiologie des aliments - Méthode horizontale pour la recherche des Escherichia coli
O157 (ISO 16654:2001)
Ta slovenski standard je istoveten z: EN ISO 16654:2001
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16654:2002 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16654:2002

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SIST EN ISO 16654:2002
EUROPEAN STANDARD
EN ISO 16654
NORME EUROPÉENNE
EUROPÄISCHE NORM
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 (ISO
16654:2001)
Microbiologie des aliments - Méthode horizontale pour la Mikrobiologie von Lebensmitteln und Futtermitteln -
recherche des Escherichia coli O 157 (ISO 16654:2001) Horizontales Verfahren für den Nachweis 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 alteration. 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 translation
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, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16654:2001 E
worldwide for CEN national Members.

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SIST EN ISO 16654:2002
Page 2
EN ISO 16654:2001
Foreword
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 - Horizontal 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 November 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, 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).

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SIST EN ISO 16654:2002
Page 3
EN ISO 16654:2001
Annex ZA (normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions
of any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications,
indicated by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 6887-1 1999 Microbiology of food and animal EN ISO 6887-1 1999
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 dilutions

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SIST EN ISO 16654:2002

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SIST EN ISO 16654:2002
INTERNATIONAL ISO
STANDARD 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 — Méthode horizontale pour la recherche des
Escherichia coli O157
Reference number
ISO 16654:2001(E)
©
ISO 2001

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
PDF disclaimer
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© ISO 2001
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body
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Printed in Switzerland
ii © ISO 2001 – All rights reserved

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
Contents Page
Foreword.iv
Introduction.v
1 Scope .1
2 Normative references .1
3 Term and definition .1
4 Principle.2
5 Culture media, reagents and antisera .2
6 Apparatus and glassware .7
7 Sampling.8
8 Preparation of test sample.8
9 Procedure (see annex A).8
9.1 Test portion 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 colonies.9
9.5 Confirmation.10
9.6 Further characterization.11
10 Quality assurance.11
10.1 Test strains for quality assurance purposes.11
10.2 Culture method .11
11 Expression of results .11
12 Test report .11
Annex A (normative) Diagram of procedure .12
Bibliography.13
© ISO 2001 – All rights reserved iii

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
Foreword
ISO (the International Organization 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 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.
International 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 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 Technical Committee ISO/TC 34, Food products,
Subcommittee SC 9, Microbiology.
Annex A forms a normative part of this International Standard.
iv © ISO 2001 – All rights reserved

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
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 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 taken 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
Standards 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 those necessary for well-established
technical reasons.
© ISO 2001 – All rights reserved v

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SIST EN ISO 16654:2002

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SIST EN ISO 16654:2002
INTERNATIONAL STANDARD ISO 16654:2001(E)
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-acquired 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 Safety 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, 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 references, 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 references, 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 microbiological 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 following 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 Sorbitol-positive E. coli O157 strains are not detected on CT-SMAC (5.2) media.
NOTE 2 Some indole-negative mutations have been found.
© ISO 2001 – All rights reserved 1

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
4Principle
The detection of Escherichia coli O157 necessitates four successive stages (see annex A).
a) Enrichment of the test portion homogenized 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 subculture of the immunomagnetic particles with adhering bacteria onto cefixime tellurite sorbitol
MacConkey agar (CT-SMAC) and the user's 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, 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 practices, 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 chloride 5,0 g
Dipotassium hydrogen phosphate (K HPO ) 4,0 g
2 4
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.
Dispense the medium in appropriate amounts in flasks or bottles (6.7).
Sterilize for 15 min in the autoclave (6.1) set at 121 °C.
2 © ISO 2001 – All rights reserved

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
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 membrane 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 isolation 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
a
Agar
9gto18 g
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 autoclave (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
© ISO 2001 – All rights reserved 3

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SIST EN ISO 16654:2002
ISO 16654:2001(E)
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 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 sterilized base medium (5.2.1) to between 44 �C and 47 �C (6.5), or melt it by steaming th
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