EN 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)

ISO 10272-1:2017 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.

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
• Publication Date: 04-Jul-2017
• Withdrawal Date: 30-Jan-2018
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463/WG 3 - Campylobacter
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 05-Jul-2017
• Due Date: 28-Feb-2014
• Completion Date: 05-Jul-2017
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/381 - Mandate for standardisation addressed to CEN in the field of Methods of analysis of foodstuffs concerning Food Hygiene
• Ref Project: SIST EN 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)

Relations

• Replaces: EN ISO 10272-1:2006 - Microbiology of food and animal feeding stuffs - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method (ISO 10272-1:2006)
• Effective Date: 22-Dec-2008
• Amended By: EN ISO 10272-1:2017/A1:2023 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method - Amendment 1: Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp., the use of growth supplement in Preston broth and changes in the performance testing of culture media (ISO 10272-1:2017/Amd 1:2023)
• Effective Date: 30-Jan-2019

Overview

EN ISO 10272-1:2017 - "Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method" - is a CEN-endorsed ISO standard that specifies a horizontal laboratory method for the detection of Campylobacter species. It applies to food and feed products, environmental samples from food/feed production and handling, and primary-production samples (animal faeces, dust, swabs). The standard defines detection by enrichment or direct plating, and describes confirmation and optional species identification procedures.

Key topics and requirements

• Scope and applicability: Detection method for products intended for human consumption, animal feeding, environmental and primary-production samples in the food chain.
• Principle: Detection via selective enrichment in liquid media or isolation on selective solid media, followed by confirmation and optional species identification.
• Detection procedures: Multiple detection routes (designated A, B, C) covering enrichment and plating approaches to suit different sample types and laboratory workflows.
• Sampling and sample preparation: Requirements for representative sampling and preparation of test portions and initial suspensions tailored to the matrix.
• Isolation and confirmation: Guidance on colony selection, morphology and motility examination, oxidase testing and other confirmation steps; includes a specific check for aerobic growth at 25 °C as part of confirmation.
• Species identification (optional): Tests such as catalase, hippurate hydrolysis and indoxyl acetate hydrolysis are included for further differentiation of Campylobacter spp.
• Performance characteristics: Information on method validation including sensitivity, specificity and limit of detection (LOD), plus interlaboratory study considerations.
• Annexes: Normative diagrams of procedures, lists of culture media and reagents, and informative method validation/performance data.

Practical applications & who uses it

• Food testing laboratories performing routine surveillance for Campylobacter in poultry, meat, dairy and other food matrices.
• Feed producers and testing labs monitoring animal feed safety.
• Primary producers and farm laboratories testing animal faeces, environmental swabs and dust for on-farm surveillance.
• Regulatory agencies and public health laboratories using a harmonized, validated method for compliance testing and outbreak investigations.
• Contract testing and QC labs that need standardized protocols for reporting and traceability.

Using EN ISO 10272-1:2017 helps ensure consistent, comparable results across laboratories and supports food safety risk management related to Campylobacter contamination.

Related standards

• EN/ISO 10272 series (the complementary part covering enumeration of Campylobacter spp. is typically referenced for quantitative analysis).
• Other food microbiology standards and national regulations for food safety testing and laboratory accreditation.

Keywords: EN ISO 10272-1:2017, Campylobacter detection, food microbiology standard, enrichment, selective media, detection method, food chain testing.

Standards Content (Sample)

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
ISO 10272-1:2017(E)
© 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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

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 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

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 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

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 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

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 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 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.
6.6 Sterile Petri dishes, with a diameter of approximately 90 mm, preferably with vents to facilitate
microaerobic incubation.
6.7 Refrigerators, capable of operating at 3 °C ± 2 °C and at 5 °C ± 3 °C.
7 Sampling
Sampling is not part of the method specified in this document. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with the
sampling of the product concerned, it is recommended that the parties concerned come to an agreement
on this subject.
A recommended sampling method is given in ISO/TS 17728 for food and animal feed, in ISO 13307 for
sampling at the primary production stage, in ISO 17604 for sampling of carcasses, and in ISO 18593 for
sampling of surfaces.
It is important that the laboratory receives a sample that is representative and the sample should not
have been damaged or changed during transport or storage.
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 (6.7) 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 from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned: see ISO 6887 (all parts). If there is no specific
International Standard, it is recommended that the parties concerned come to an agreement on this
subject.
9 Procedure
9.1 General
Depending on the type of sample 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 samples 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 samples with low numbers
of campylobacters and high level of background microflora, e.g. raw meats (including poultry) or
raw milk;
— detection procedure C: Detection of Campylobacter by direct plating, in samples with high numbers
of campylobacters, e.g. faeces, poultry caecal contents or raw poultry meat. This can be used in
combination with ISO 10272-2 in order to count numbers of Campylobacter per g, per ml, or per cm
in the test material.
4 © ISO 2017 – All rights reserved

ISO 10272-1:2017(E)
If little information is available concerning the best method for the particular type of sample to be
tested, then use detection procedure C, in parallel with detection procedure(s) A and/or B.
In general, detection procedure B is useful for products (including cooked or frozen) that contain
significant numbers of microflora resistant to third generation ß-lactams like cefoperazone.
Cefoperazone is used in Bolton broth (detection procedure A) as well as in mCCD agar. Preston broth
(detection procedure B) uses different selective principles and is therefore more suitable to suppress
this type of resistant microflora.
9.2 Test portion and initial suspension
9.2.1 General
For preparation of the initial suspension, in the general case, use as diluent the enrichment medium
specified in 9.2.2 or 9.2.3. Pre-warm the enrichment medium to room temperature before use.
In general, an amount of test portion (mass or volume) is mixed with a quantity of enrichment medium
(mass or volume) to yield a tenfold dilution. However, for some types of samples (e.g. boot socks, swabs),
it may be necessary to use another ratio.
This document has been validated for test portions of 10 g (or ml), except for the caecal samples. A
smaller size of test portion may be used, without the need for additional validation/verification,
providing that the same ratio between enrichment broth and test portion is maintained. A larger test
portion than that initially validated may be used, if a validation/verification study has shown that there
are no adverse effects on the detection of Campylobacter.
NOTE Validation can be conducted in accordance with the appropriate documents of ISO 16140 (all parts).
Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017,
Annex D (verification protocol for pooling samples for qualitative tests).
9.2.2 Detection procedure A
In general, for preparing the initial suspension, combine a quantity of 10 g or 10 ml of the test portion
with 90 ml of the enrichment medium Bolton broth (B.2), so as to obtain a 1 in 10 dilution, and
homogenize (see ISO 7218).
9.2.3 Detection procedure B
In general, for preparing the initial suspension, combine a quantity of 10 g or 10 ml of the test portion
with 90 ml of the enrichment medium Preston broth (B.3), so as to obtain a 1 in 10 dilution, and
homogenize (see ISO 7218).
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 (B.4). Use another loop to streak out on the
second half of the plate.
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 in 2 (volume fraction), 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 (B.4).
NOTE Using a second plating medium (B.5) with selective agents different from those in mCCD agar could
improve Campylobacter detection, especially in the presence of background flora resistant to 3rd generation
ß-lactams like cefoperazone.
ISO 10272-1:2017(E)
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 10 µl loop (6.3)
the surface of the first selective isolation medium, mCCD agar (B.4).
Proceed in the same manner with the second Campylobacter selective isolation medium chosen (B.5).
9.4.2 Detection procedure B
Using the culture obtained in the enrichment medium (9.3.2), inoculate with a sterile 10 µl loop (6.3)
the surface of the isolation medium, mCCD agar (B.4).
9.4.3 Detection procedures A, B and C
Incubate the plates (9.2.4, 9.4.1 and 9.4.2) at 41,5 °C (6.1) in a microaerobic atmosphere (6.5).
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 tend to spread less on drier agar surfaces. Other forms of colonies may occur.
NOTE The recognition of colonies of Campylobacter is to a large extent a matter of experience and their
appearance can vary somewhat, not only from strain to strain, but also from batch to batch of the selective
culture medium used.
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.
For a clear distinction between positive and negative confirmation reactions, it is helpful to verify this
with well-characterized positive and negative control strains. Examples of suitable control strains are
[17]
Campylobacter jejuni WDCM 00005 (positive control) and Escherichia coli WDCM 00013 (negative
control).
As an alternative, or in addition, to the confirmation and identification tests described in this document,
other tests (PCR tests, serological methods, matrix-assisted laser desorption/ionization time-of-
flight mass spectrometer (MALDI-TOF-MS) analysis, etc.) can be used, providing the suitability of the
alternative procedure is verified (see ISO 7218).
6 © ISO 2017 – All rights reserved

ISO 10272-1:2017(E)
9.5.2 Selection of colonies for confirmation
9.5.2.1 Select at least one typical or suspect Campylobacter colony (9.4.3) for purification and
confirmation. One confirmed isolate per sample is sufficient. If the first colony is negative, select up to
four more suspect colonies.
If needed, store the original isolation plates (9.4.3) preferably under microaerobic conditions at
5 °C (6.7) for use in further confirmation and/or identification.
9.5.2.2 Streak each of the selected colonies onto a non-selective blood agar plate, e.g. Columbia
blood agar (B.6) in order to allow the development of well-isolated colonies. Incubate the plates in a
microaerobic atmosphere (6.5) 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 activity (9.5.5).
NOTE The suspect colony could be previewed for characteristic morphology and motility before streaking
on blood agar.
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 non-selective
blood agar plate, e.g. Columbia blood agar (B.6).
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 activity
Using a loop (6.3), take a portion of a well-isolated colony from the blood agar plate (9.5.2.2) and streak
it onto a filter paper moistened with the oxidase reagent (B.7); 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.
9.5.6 Interpretation
Campylobacter gives results in accordance with Table 1.
Campylobacter is present if at least one colony presents the characteristics.
ISO 10272-1:2017(E)
Table 1 — Characteristics of Campylobacter
a
Morphology (9.5.3) Small curved bacilli
a
Motility (9.5.3) Characteristic corkscrew darting
Aerobic growth at 25 °C (9.5.4) −
Oxidase activity (9.5.5) +
+ Positive.
−   Negative.
a
Older cultures may rapidly lose their characteristic shape and motility and turn into less motile
coccoid forms.
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. However, other species have been described
(Campylobacter lari, Campylobacter upsaliensis and others); the characteristics given in Table 2 permit
their differentiation.
9.6.2 Detection of catalase activity
For each colony selected in 9.5.2.2, deposit a loop of culture into a drop of hydrogen peroxide solution
(B.8) 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) and Enterococcus faecalis WDCM 00087 (negative
control).
9.6.3 Detection of hippurate hydrolysis
For each colony selected in 9.5.2.2, use a 10 µl 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.9.1), taking care not to
incorporate any agar.
Shake in order to mix thoroughly and incubate for 2 h ± 5 min in a water bath at 37 °C (6.2) or 4 h ± 5 min
in an incubator at 37 °C (6.1).
Carefully add 0,2 ml of a ninhydrin solution (B.9.2) on top of the sodium hippurate solution. Do not shake.
Interpret after incubation of 5 min to 10 min at 37 °C (6.2 or 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) and Campylobacter coli WDCM 00004 (negative
control).
9.6.4 Detection of indoxyl acetate hydrolysis
Place a 1 µl loopful of colony material (9.5.2.2) on an indoxyl acetate disc (B.10) and add a drop of sterile
distilled water.
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ISO 10272-1:2017(E)
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) and 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 species level according to Table 2.
Table 2 — Characteristics of Campylobacter species
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase activity (9.6.2) + + + − or weak
a
Hippurate hydrolysis (9.6.3) + − − −
Indoxyl acetate hydrolysis (9.6.4) + + − +
+ Positive.
−   Negative.
a
Some hippurate-negative C. jejuni strains have been reported.
10 Expression of results
In accordance with the interpretation of the results, indicate Campylobacter detected or not detected in
the test portion examined.
11 Performance characteristics of the method
11.1 Interlaboratory study
The performance characteristics of the method were determined in an interlaboratory study to
determine the specificity, sensitivity and the LOD of the method. The data are summarized in Annex C.
The values derived from the interlaboratory study may not be applicable to food types or strains other
[13]
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 true
positive samp
...