Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method (ISO 10272-1:2017)

This part of the standard describes the detection of Campylobacter spp. (Reference document EN/ISO 10272 -1)

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren (ISO 10272-1:2017)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Campylobacter spp. - Partie 1 : Méthode de recherche (ISO 10272-1:2017)

L'ISO 10272-1:2017 spécifie une méthode horizontale de recherche par enrichissement ou ensemencement direct de Campylobacter spp. Il s'applique:
-      aux produits destinés à la consommation humaine;
-      aux produits destinés à l'alimentation des animaux;
-      aux échantillons environnementaux prélevés dans les secteurs de la production et de la manutention des aliments; et
-      aux échantillons au stade de la production primaire tels que les matières fécales des animaux, la poussière et les prélèvements de surface.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Campylobacter spp. - 1. del: Metoda za ugotavljanje prisotnosti (ISO 10272-1:2017)

Ta del standarda opisuje metodo za ugotavljanje prisotnosti Campylobacter spp. (referenčni dokument je standard EN/ISO 10272 -1).

General Information

Status
Published
Public Enquiry End Date
29-Apr-2015
Publication Date
15-Aug-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Aug-2017
Due Date
06-Oct-2017
Completion Date
16-Aug-2017

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SLOVENSKI STANDARD
SIST EN ISO 10272-1:2017
01-september-2017
1DGRPHãþD
SIST EN ISO 10272-1:2006
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 1. del: Metoda za ugotavljanje
prisotnosti (ISO 10272-1:2017)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 1: Detection method (ISO 10272-1:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren (ISO 10272-1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 1 : Méthode de recherche (ISO 10272-
1:2017)
Ta slovenski standard je istoveten z: EN ISO 10272-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 10272-1:2017

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SIST EN ISO 10272-1:2017


EN ISO 10272-1
EUROPEAN STANDARD

NORME EUROPÉENNE

July 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 10272-1:2006
English Version

Microbiology of the food chain - Horizontal method for
detection and enumeration of Campylobacter spp. - Part 1:
Detection method (ISO 10272-1:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche et le dénombrement de Verfahren zum Nachweis und zur Zählung von
Campylobacter spp. - Partie 1 : Méthode de recherche Campylobacter spp. - Teil 1: Nachweisverfahren (ISO
(ISO 10272-1:2017) 10272-1:2017)
This European Standard was approved by CEN on 1 May 2017.

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 CEN-CENELEC 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 CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10272-1:2017 E
worldwide for CEN national Members.

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SIST EN ISO 10272-1:2017
EN ISO 10272-1:2017 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 10272-1:2017
EN ISO 10272-1:2017 (E)
European foreword
This document (EN ISO 10272-1:2017) has been prepared by Technical Committee ISO/TC 34 “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 January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 10272-1:2006.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 10272-1:2017 has been approved by CEN as EN ISO 10272-1:2017 without any
modification.
3

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SIST EN ISO 10272-1:2017

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SIST EN ISO 10272-1:2017
INTERNATIONAL ISO
STANDARD 10272-1
Second edition
2017-06
Microbiology of the food chain —
Horizontal method for detection and
enumeration of Campylobacter spp. —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Campylobacter spp. —
Partie 1: Méthode de recherche
Reference number
ISO 10272-1:2017(E)
©
ISO 2017

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Enrichment in selective liquid medium . 2
4.2.1 Detection procedure A . 2
4.2.2 Detection procedure B . 2
4.2.3 Detection procedure C . 2
4.3 Isolation on selective solid medium . 3
4.3.1 Detection procedure A . 3
4.3.2 Detection procedure B . 3
4.3.3 Detection procedure C . 3
4.3.4 Detection procedure A, B and C . 3
4.4 Confirmation . 3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure. 4
9.1 General . 4
9.2 Test portion and initial suspension . 5
9.2.1 General. 5
9.2.2 Detection procedure A . 5
9.2.3 Detection procedure B . 5
9.2.4 Detection procedure C . 5
9.3 Enrichment . 6
9.3.1 Detection procedure A . 6
9.3.2 Detection procedure B . 6
9.4 Isolation . 6
9.4.1 Detection procedure A . 6
9.4.2 Detection procedure B . 6
9.4.3 Detection procedures A, B and C . 6
9.5 Confirmation of Campylobacter . 6
9.5.1 General. 6
9.5.2 Selection of colonies for confirmation . 7
9.5.3 Examination of morphology and motility . 7
9.5.4 Study of aerobic growth at 25 °C . 7
9.5.5 Detection of oxidase activity . 7
9.5.6 Interpretation . 7
9.6 Identification of Campylobacter species (optional) . 8
9.6.1 General. 8
9.6.2 Detection of catalase activity. 8
9.6.3 Detection of hippurate hydrolysis . 8
9.6.4 Detection of indoxyl acetate hydrolysis . 8
9.6.5 Interpretation . 9
10 Expression of results . 9
11 Performance characteristics of the method . 9
© ISO 2017 – All rights reserved iii

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

11.1 Interlaboratory study . 9
11.2 Sensitivity . 9
11.3 Specificity . 9
11.4 LOD .
50 9
12 Test report .10
Annex A (normative) Diagram of procedures .11
Annex B (normative) Culture media and reagents .12
Annex C (informative) Method validation studies and performance characteristics .21
Bibliography .24
iv © ISO 2017 – All rights reserved

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www . i so .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN), Technical
Committee CEN/TC 275, Food Analysis — Horizontal methods, in collaboration with ISO Technical
committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 10272-1:2006), which has been technically
revised with the following main changes:
— samples from the primary production stage have been added to the scope;
— the detection method was extended to include the option of a second enrichment broth (Preston
broth), primarily to overcome problems with background flora resistant to third generation
ß-lactams (like cefoperazone in Bolton broth);
— the detection method was extended to include the option of direct plating on mCCDA;
— the note on the use of closed containers with reduced headspace as an alternative to incubation in a
microaerobic atmosphere has been deleted;
— the confirmation tests on study of microaerobic growth at 25 °C and aerobic growth at 41,5 °C were
replaced by the study of aerobic growth at 25 °C;
— performance testing for the quality assurance of the culture media has been added to Annex B;
— performance characteristics have been added to Annex C.
A list of all parts in the ISO 10272 series can be found on the ISO website.
© ISO 2017 – All rights reserved v

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO 10272-1:2006
are considered as minor (see ISO 17468).
Because of the large variety of food and feed products, this horizontal method may not be appropriate
in every detail 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 this horizontal
method as far as possible and that deviations from this will only be made if absolutely necessary for
technical reasons.
When this document 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
in the case of particular products. The harmonization of test methods cannot be immediate and, for
certain group 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 document so that eventually the only remaining departures from this
horizontal method will be those necessary for well-established technical reasons.
vi © ISO 2017 – All rights reserved

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SIST EN ISO 10272-1:2017
INTERNATIONAL STANDARD ISO 10272-1:2017(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 1:
Detection method
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for detecting Campylobacter are only undertaken in properly equipped laboratories, under the
control of a skilled microbiologist, and that great care is taken in the disposal of all incubated
materials. Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety aspects, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
1 Scope
This document specifies a horizontal method for the detection by enrichment or direct plating of
Campylobacter spp. It is applicable to
— products intended for human consumption,
— products intended for animal feeding,
— environmental samples in the area of food and feed production, handling, and
— samples from the primary production stage such as animal faeces, dust, and swabs.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www.e lectropedia. org/
— ISO Online browsing platform: available at http:// www. iso. org/o bp
© ISO 2017 – All rights reserved 1

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

3.1
Campylobacter
microorganism forming characteristic colonies on solid selective media when incubated in a
microaerobic atmosphere at 41,5 °C, and which possesses the characteristic morphology and motility
and biochemical and growth properties described when the tests are conducted in accordance with
this document
Note 1 to entry: This document targets the thermotolerant Campylobacter species relevant to human health. The
most frequently encountered and relevant to human health thermotolerant species are Campylobacter jejuni and
Campylobacter coli. However, other species have been described (Campylobacter lari, Campylobacter upsaliensis
and others).
3.2
detection of Campylobacter
determination of the presence or absence of Campylobacter (3.1) in a defined quantity of product, when
the test is conducted in accordance with this document
4 Principle
4.1 General
The detection of Campylobacter requires three successive stages as specified in Annex A.
Depending on the type of sample and the purpose of the test, three different detection procedures can
be used:
— detection procedure A: Detection of Campylobacter by enrichment, in samples with low numbers
of campylobacters and low level of background microflora and/or with stressed campylobacters;
— detection procedure B: Detection of Campylobacter by enrichment, in samples with low numbers
of campylobacters and high level of background microflora;
— detection procedure C: Detection of Campylobacter by direct plating, in samples with high numbers
of campylobacters.
4.2 Enrichment in selective liquid medium
4.2.1 Detection procedure A
The test portion is added to the liquid enrichment medium (Bolton broth).
It is incubated in a microaerobic atmosphere at 37 °C for 4 h to 6 h and then at 41,5 °C for 44 h.
4.2.2 Detection procedure B
The test portion is added to the liquid enrichment medium (Preston broth).
It is incubated in a microaerobic atmosphere at 41,5 °C for 24 h.
4.2.3 Detection procedure C
Enrichment technique is not used.
2 © ISO 2017 – All rights reserved

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

4.3 Isolation on selective solid medium
4.3.1 Detection procedure A
From the enrichment culture obtained in 4.2, two selective solid media are inoculated:
— modified Charcoal Cefoperozone Deoxycholate agar (mCCD agar);
— any other solid selective Campylobacter medium using different selective principles from those in
mCCD agar.
4.3.2 Detection procedure B
From the enrichment culture obtained in 4.2, the selective mCCD agar is inoculated.
4.3.3 Detection procedure C
The test portion is plated directly or after suspending in an appropriate amount of liquid onto the
selective mCCD agar.
4.3.4 Detection procedure A, B and C
The selective solid media are incubated at 41,5 °C in a microaerobic atmosphere and examined after
44 h to detect the presence of suspect Campylobacter colonies.
4.4 Confirmation
The suspect Campylobacter colonies are examined for morphology and motility using a microscope
and sub-cultured on a non-selective blood agar, and then confirmed by detection of oxidase activity
and an aerobic growth test at 25 °C. Optionally, the Campylobacter species are identified by specific
biochemical tests and/or molecular methods.
5 Culture media and reagents
For current laboratory practice, see ISO 7218 and ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
For performance testing of culture media, see Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Incubators, capable of operating at 25 °C ± 1 °C, 37 °C ± 1 °C and 41,5 °C ± 1 °C.
6.2 Water bath, capable of operating at 37 °C ± 1 °C.
6.3 Sterile loops, of 10 µl volume and of 1 µl volume, and inoculation needle or wire.
A nickel/chromium loop is not suitable for use in the oxidase test (see 9.5.5).
6.4 Microscope, preferably with phase contrast (for observing the characteristic morphology and
motility of Campylobacter).
© ISO 2017 – All rights reserved 3

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SIST EN ISO 10272-1:2017
ISO 10272-1:2017(E)

6.5 Apparatus suitable for achieving a microaerobic atmosphere, with oxygen content of
5 % ± 2 %, carbon dioxide 10 % ± 3 %, optional hydrogen ≤10 %, with the balance nitrogen.
The appropriate microaerobic atmosph
...

SLOVENSKI STANDARD
oSIST prEN ISO 10272-1:2015
01-april-2015
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti in
števila Campylobacter spp. - 1. del: Metoda za ugotavljanje prisotnosti (ISO/DIS
10272-1:2015)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 1: Detection method (ISO/DIS 10272-1:2015)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren (ISO/DIS 10272-1:2015)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 1 : Méthode de recherche (ISO/DIS
10272-1:2015)
Ta slovenski standard je istoveten z: prEN ISO 10272-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 10272-1:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 10272-1:2015

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oSIST prEN ISO 10272-1:2015
DRAFT INTERNATIONAL STANDARD
ISO/DIS 10272-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2015-02-12 2015-05-12
Microbiology of food and animal feed — Horizontal method
for detection and enumeration of Campylobacter —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de
Campylobacter spp. —
Partie 1: Méthode de recherche
ICS: 07.100.30
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the European Committee for Standardization
(CEN), and processed under the CEN lead mode of collaboration as defined in the
Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member
bodies for a parallel five month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments
received, will be submitted to a parallel two-month approval vote in ISO and
THIS DOCUMENT IS A DRAFT CIRCULATED
formal vote in CEN.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 10272-1:2014(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2014

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oSIST prEN ISO 10272-1:2015
ISO/DIS 10272-1:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

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oSIST prEN ISO 10272-1:2015
ISO/DIS 10272-1
Contents Page
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle. 2
5 Culture media and reagents . 3
6 Apparatus and glassware . 4
7 Sampling. 4
8 Preparation of test sample . 5
9 Procedure (see diagram in Annex B) . 5
10 Expression of results . 9
11 Accuracy (precision) of the method . 9
12 Test report . 10
Annex A (normative) Diagram of detection . 11
Annex B (normative) Composition and preparation of culture media and reagents . 13
Annex C (informative) Results of interlaboratory study . 21
Bibliography . 24

© ISO 2012 – All rights reserved iii

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oSIST prEN ISO 10272-1:2015
ISO/DIS 10272-1
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 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 by 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 10272-1 was prepared by the European Committee for Standardization (CEN), in collaboration with
Technical Committee ISO/TC 34, Food Products, Subcommittee SC 9, Microbiology.
This second edition cancels and replaces the first edition (ISO 10272-1:2006), which has been technically
revised and performance characteristics have been added.
ISO 10272 consists of the following parts, under the general title Microbiology of the food chain — Horizontal
method for detection and enumeration of Campylobacter:
 Part 1: Detection method
 Part 2: Colony-count technique
iv © ISO 2012 – All rights reserved

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oSIST prEN ISO 10272-1:2015
ISO/DIS 10272-1
Introduction
This horizontal method may not be appropriate in every detail for certain of the wide variety of food and feed
products that exist, while for some products it may be necessary to use different methods. Nevertheless, it is
hoped that in all cases attempts will be made to apply this horizontal method as far as possible and that
deviations will only be made if absolutely necessary for technical reasons.
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 in the case of particular products. The harmonization of test methods cannot be immediate and, for certain
group 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.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 10272-1

Microbiology of food and animal feed — Horizontal method for
detection and enumeration of Campylobacter — Part 1:
Detection method
1 Scope
This part of ISO 10272 describes a horizontal method for the detection by enrichment or direct plating of
Campylobacter.
It is applicable to products intended for human consumption or for the feeding of animals, and to
environmental samples in the area of food production and food handling, subject to the limitations stated in the
Introduction. It can also be applied to intestinal contents or faecal samples from animals, especially poultry.
2 Normative references
The following referenced documents are indispensable for 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 (all parts), Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination
ISO 7218 Microbiology of food and animal feeding stuffs — General rules for microbiological examinations
ISO 11133 Microbiology of food, animal feed and water - Preparation, production, storage and performance
testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Campylobacter
microorganisms forming characteristic colonies on solid selective media when incubated in a microaerobic
atmosphere at 41,5 °C and which possess the characteristic morphology and motility and biochemical and
growth properties described when the tests are conducted in accordance with this part of ISO 10272
NOTE to entry The most frequently encountered species are Campylobacter jejuni and Campylobacter coli. Other species
have, however, been described (Campylobacter lari, Campylobacter upsaliensis and others).
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3.2
detection of Campylobacter
determination of the presence or absence of Campylobacter (3.1) in a defined quantity of product, when the
test is conducted in accordance with this part of ISO 10272.
4 Principle
4.1 General
Depending on the type of product and the purpose of the test three different detection procedures can be
used:
A Detection of Campylobacter by enrichment, in products with low numbers of campylobacters and low level
of background microflora and/or with stressed campylobacters, e.g. cooked or frozen products.
B Detection of Campylobacter by enrichment, in products with low numbers of campylobacters and high level
of background microflora, e.g. raw (poultry) meats or raw milk.
C Detection of Campylobacter by direct plating, in products with high numbers of campylobacters, e.g. faeces,
poultry caecal contents or raw poultry meat.
4.2 Enrichment in selective liquid medium
Detection procedure A :
The test portion is added to the liquid enrichment medium (Bolton broth) and homogenized.
It is incubated in a microaerobic atmosphere at 37 °C for 4 h to 6 h and then at 41,5 °C for 44 h  4 h.
Detection procedure B:
The test portion is added to the liquid enrichment medium (Preston broth) and homogenized.
It is incubated in a microaerobic atmosphere at 41,5 °C for 24 h  2 h.
Detection procedure C:
Enrichment technique is not used.
4.3 Direct plating
Detection procedures A and B:
Direct plating is not used.
Detection procedure C:
The test portion is plated directly or after suspending in an appropriate amount of liquid onto modified charcoal
cefoperazone deoxycholate agar (mCCD agar).
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4.4 Isolation and selection for confirmation
Detection procedure A:
From the enrichment culture obtained in 4.2, two selective solid media are inoculated:
 modified charcoal cefoperazone deoxycholate agar (mCCD agar);
 any other solid selective Campylobacter medium using different selective principles from those in mCCD
agar.
Detection procedure B :
From the enrichment culture obtained in 4.2, the selective medium, modified charcoal cefoperazone
deoxycholate agar (mCCD agar) is inoculated.
Detection procedure A, B and C:
The selective solid media are incubated at 41,5 °C in a microaerobic atmosphere and examined after
44 h  4 h to detect the presence of colonies presumed because of their characteristics to be Campylobacter.
4.5 Confirmation
The colonies presumed to be Campylobacter are examined for morphology and motility using a microscope
and sub-cultured on a non-selective blood agar, and then confirmed by detection of oxidase and an aerobic
growth test at 25°C. Optionally, the Campylobacter species are identified by specific biochemical tests and/or
molecular methods.
5 Culture media and reagents
5.1 General
For current laboratory practice, see ISO 11133.
NOTE Because of the large number of culture media and reagents and for the clarity of the text, their compositions
and preparations are given in Annex B.
5.2 Liquid enrichment medium (detection procedure A): Bolton broth
See B.1.
5.3 Liquid enrichment medium (detection procedure B): Preston broth
See B.2.
5.4 Selective plating medium: Modified charcoal cefoperazone deoxycholate agar (mCCD
agar)
See B.3.
5.5 Selective plating medium using different selective principles from those in mCCD agar
(e.g. Preston agar, Butzler agar, etc.)

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rd
NOTE Selective principles in mCCD agar in this case mainly refer to the use of 3 generation ß-lactams like
cefoperazone.
5.6 Confirmation and identification media and reagents
5.6.1 Blood agar
See B.4.
5.6.2 Reagent for the detection of oxidase
See B.5.
5.6.3 Hydrogen peroxide solution, 3 % (volume fraction)
5.6.4 Reagents for the detection of hydrolysis of hippurate
See B.6.
5.6.5 Indoxyl acetate discs
See B.7.
6 Apparatus and glassware
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Incubators, capable of operating at 25 °C  1 °C, 37 °C  1 °C and 41,5 °C  1 °C.
6.2 Water bath, capable of operating at 37 °C  1 °C.
6.3 Sterile loops, of platinum/iridium, nickel/chromium or plastic, approximately 3 mm in diameter, and
wires of the same material, or a glass or plastic rod. A nickel/chromium loop is not suitable for use in the
oxidase test (see 9.4.5).
6.4 Microscope, preferably with phase contrast (for observing the characteristic morphology and motility of
Campylobacter).
6.5 Apparatus suitable for achieving a microaerobic atmosphere with oxygen content of 5 %  2 %,
carbon dioxide 10 %  3 %, optional hydrogen ≤10 %, with the balance nitrogen. The appropriate
microaerobic atmosphere can be obtained using gastight jars and gas-generating kits, following precisely the
manufacturer's instructions. Alternatively, the jar or incubator may be filled with an appropriate gas mixture
prior to incubation.
7 Sampling
It is important that the laboratory receives a sample which is truly representative and has not been damaged
or changed during transport or storage.
Sampling is not part of the method specified in this part of ISO 10272. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with sampling of the
product concerned, it is recommended that the parties concerned come to an agreement on this subject.
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Since Campylobacter is very sensitive to freezing but survives best at low temperatures, samples to be tested
should not be frozen, but stored at +3 °C  2 °C and subjected to analysis as rapidly as possible. Also take
care to prevent the samples from drying.
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product
concerned. If there is no specific International Standard, it is recommended that the parties concerned come
to an agreement on this subject.
9 Procedure (see diagram in Annex B)
9.1 General
Depending on the type of product and the purpose of the test one or more of three different detection
procedures is/are used:
 Detection procedure A: Detection of Campylobacter by enrichment, in products with low numbers of
campylobacters and low level of background microflora and/or with stressed campylobacters, e.g. cooked
or frozen products.
 Detection procedure B: Detection of Campylobacter by enrichment, in products with low numbers of
campylobacters and high level of background microflora, e.g. raw (poultry) meats or raw milk.
 Detection procedure C: Detection of Campylobacter by direct plating, in products with high numbers of
campylobacters, e.g. faeces, poultry caecal contents or raw poultry meat. This can be used in
2
combination with Part 2 of this standard in order to count numbers of cfu per g, per ml, or per cm of
campylobacters in the test material.
If little information is available concerning the best method for the particular type of sample to be tested, then
use procedure C, in parallel with procedure(s) A and/or B.
rd
In general procedure B is useful for products that contain significant numbers of microflora resistant to 3
generation ß-lactams like cefoperazone, which is used in Bolton broth (procedure A) as well as in mCCD agar,
but not in Preston broth (procedure B).
9.2 Test portion, initial suspension and dilutions
9.2.1 See the suitable part of ISO 6887.
9.2.2 Detection procedure A
In general, for preparing the initial suspension, introduce a quantity x of the test portion (mass or volume) into
nine times its volume of the enrichment medium Bolton broth (5.2), so as to obtain a test portion/enrichment
medium ratio of 1:10 (mass/volume, mass/mass or volume/volume), and homogenize (see ISO 7218).
9.2.3 Detection procedure B
In general, for preparing the initial suspension, introduce a quantity x of the test portion (mass or volume) into
nine times its volume of the enrichment medium Preston broth (5.3), so as to obtain a test portion/enrichment
medium ratio of 1:10 (mass/volume, mass/mass or volume/volume), and homogenize (see ISO 7218).
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9.2.4 Detection procedure C
9.2.4.1 For caecal or faecal samples use a loop (6.3) or a sterile swab to bring some of the well-mixed
sample material onto the first half of a mCCD agar plate (5.4). Use another loop to streak out on the second
half of the plates.
9.2.4.2 For all other samples, add an appropriate amount of liquid (e.g. peptone salt solution or Preston
broth), for example 1 to 1 w/w, mix well, and either streak the plate using a loop (6.3), or dispense a suitable
volume and spread it over the mCCD agar plate (5.4).
NOTE Using a second plating medium with selective agents different from those in mCCD agar (5.5) could improve
rd
Campylobacter detection, especially in the presence of background flora resistant to 3 generation ß-lactams like
cefoperazone.

9.3 Enrichment
9.3.1 Detection procedure A
Incubate the initial suspension (9.2.2) in a microaerobic atmosphere (6.5) at 37°C (6.1) for 4 h to 6 h, then at
41,5 °C (6.1) for 44 h  4 h.
9.3.2 Detection procedure B
Incubate the initial suspension (9.2.3) in a microaerobic atmosphere (6.5) at 41,5 °C (6.1) for 24 h  2 h.
9.4 Isolation
9.4.1 Detection procedure A
Using the culture obtained in the enrichment medium (9.3.1), inoculate with a sterile loop (6.3) the surface of
the first selective isolation medium, mCCD agar (5.4).
Proceed in the same manner with the second Campylobacter selective isolation medium chosen (5.5).
9.4.2 Detection procedure B
Using the culture obtained in the enrichment medium (9.3.2), inoculate with a sterile loop (6.3) the surface of
the isolation medium, mCCD agar (5.4).
9.4.3 Detection procedures A, B and C
Incubate the plates (9.2.4, 9.4.1, 9.4.2) at 41,5 °C (6.1) in a microaerobic atmosphere (6.5).
9.4.4 After 44 h  4 h of incubation, examine the plates for typical and/or suspect colonies of
Campylobacter.
Typical colonies are greyish on mCCD agar, often with a metallic sheen, and are flat and moist, with a
tendency to spread. Colonies spread less on drier agar surfaces. Other forms of colonies may occur.
9.5 Confirmation of Campylobacter
9.5.1 General
As Campylobacter rapidly loses culturability in air, follow the procedure described in 9.5.2 to 9.5.5 without
delay.
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9.5.2 Selection of colonies for confirmation
9.5.2.1 For confirmation, take from each plate of each selective medium (9.4.4) at least one colony
considered to be typical or suspected as being Campylobacter and up to a maximum of five colonies if the first
is negative.
9.5.2.2 Streak each of the selected colonies onto a blood agar plate (5.6.1) in order to allow the
development of well-isolated colonies. Incubate the plates in a microaerobic atmosphere at 41,5 °C (6.1) for
24 h to 48 h. Use well-isolated freshly grown colonies for examination of morphology and motility (9.5.3),
absence of aerobic growth at 25 °C (9.5.4) and the presence of oxidase (9.5.5).
9.5.3 Examination of morphology and motility
9.5.3.1 Examine a freshly grown colony from the blood agar plate (9.5.2.2) for morphology and motility
using a microscope (6.4).
9.5.3.2 Retain for further examination all cultures (9.5.2.2) in which curved bacilli with a spiralling
“corkscrew” motility are found (9.5.3.1).
9.5.4 Study of aerobic growth at 25 °C
Using the colonies isolated in 9.5.2.2, inoculate with the aid of a loop (6.3) the surface of a blood agar plate
(5.5.1).
Incubate the plate at 25 °C (6.1) aerobically for 44 h  4 h.
Examine the plate for absence of growth of colonies.
9.5.5 Detection of oxidase
Using a platinum/iridium or plastic loop or glass rod (6.3), take a portion of a well-isolated colony from each
individual plate (9.5.2.2) and streak it onto a filter paper moistened with the oxidase reagent (5.6.2); the
appearance of a mauve, violet or deep blue colour within 10 s indicates a positive reaction. If a commercially
available oxidase test kit is used, follow the manufacturer's instructions.
Confirm the results using positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control), Escherichia coli WDCM 00013 (negative control).
9.5.6 Interpretation
Campylobacter gives results in accordance with Table 1.
Table 1 — Characteristics of Campylobacter
1
Morphology (9.5.3) small curved bacilli
1
Motility (9.5.3) Characteristic corkscrew darting
Aerobic growth at 25 °C (9.5.4)

Oxidase (9.5.5) +
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Older cultures may rapidly lose their characteristic shape and motility and turn into less motile cocoid-like
forms.

Campylobacter is present if at least one colony presents the above characteristics.
NOTE As an alternative or additionally to the confirmation and identification tests described in this standard, other
tests (polymerase chain reaction (PCR) tests, serological methods, alternate biochemical methods, etc.) may be used
under the conditions described in ISO 7218.
9.6 Identification of Campylobacter species (optional)
9.6.1 General
Among the Campylobacter spp. growing at 41,5 °C, the most frequently encountered species are
Campylobacter jejuni and Campylobacter coli. Other species have, however, been described (Campylobacter
lari, Campylobacter upsaliensis and others); the characteristics given in Table 2 permit their differentiation.
9.6.2 Detection of catalase
For each colony selected in 9.5.2.2, deposit a loop of culture into a drop of hydrogen peroxide solution (5.5.3)
on a clean microscope slide.
The test is positive if bubbles appear within 30 s.
Confirm the results using positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control), Enterococcus faecalis WDCM 00087 (negative control).
9.6.3 Detection of hippurate hydrolysis
For each colony selected in 9.5.2.2, use a loop (6.3) with a heavy inoculum to prepare a suspension in a tube
of appropriate size containing 0,4 ml of a sodium hippurate solution (B.6.1), taking care not to incorporate any
agar.
Shake in order to mix thoroughly and incubate for 2 h in a water bath set at 37 °C (6.2) or 4 h in an incubator
set at 37 °C (6.1).
Carefully add 0,2 ml of a ninhydrin solution (B.6.2) on top of the sodium hippurate solution. Do not shake.
Interpret after incubation of an additional 10 min in the water bath set at 37 °C (6.2) or in an incubator set at
37 °C (6.1).
A dark violet colour indicates a positive reaction.
A pale violet colour or no colour change indicates a negative reaction.
Confirm the results using positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control), Campylobacter coli WDCM 00004 (negative control).
9.6.4 Detection of indoxyl acetate hydrolysis
Place a colony selected in 9.5.2.2 on an indoxyl acetate disc (B.7) and add a drop of sterile distilled water. A
loopful of colony material is required for a clear reaction.
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If the indoxyl acetate is hydrolysed, a colour change to dark blue occurs within 5 min to 10 min. No colour
change indicates hydrolysis has not taken place.
Confirm the results using positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control), Campylobacter lari WDCM 00204 (negative control).
If commercially available indoxyl acetate discs are used, follow the manufacturer’s instructions.
9.6.5 Interpretation
Campylobacter species growing at 41,5 °C may be identified at a species level according to Table 2.
Table 2 — Characteristics of Campylobacter species
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase (9.6.2) + + + – or weak
1
Hydrolysis of hippurate (9.6.3) + – – –
Indoxyl acetate (9.6.4) + + – +
Key: + = positive; – = negative
1
Some hippurate-negative C. jejuni strains have been reported
10 Expression of results
According to the interpretation of the results, indicate whether Campylobacter is detected or not detected in
the test portion examined (See ISO 7218).
11 Accuracy (precision) of the method
11.1 Interlaboratory study
The accuracy (precision) of the method was determined in an interlaboratory study to determine the
specificity, sensitivity and the LOD of the method. The data are summarised in Annex C. The values derived
50
from the interlaboratory study may not be applicable to food types other than those given in Annex C.
11.2 Sensitivity
The sensitivity is defined as the number of samples found positive divided by the number of samples tested at
a given level of contamination. The results are thus dependent on the level of contamination of the sample.
11.3 Specificity
The specificity is defin
...

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