SIST EN ISO 10272-2:2017
(Main)Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique (ISO 10272-2:2017)
Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique (ISO 10272-2:2017)
This part of the standard describes the enumeration of Campylobacter spp. by means of colony count technique. (Reference document ISO/TS 10272 -2)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Campylobacter - Teil 2: Koloniezählverfahren (ISO 10272-2:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Campylobacter spp. - Partie 2 : Technique par comptage des colonies (ISO 10272-2:2017)
L'ISO 10272-2:2017 spécifie une méthode horizontale de dénombrement 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. - 2. del: Tehnika štetja kolonij (ISO 10272-2:2017)
Ta del standarda opisuje metodo za ugotavljanje števila Campylobacter spp. s tehniko štetja kolonij (referenčni dokument je standard ISO/TS 10272 -2).
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10272-2:2017
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 2. del: Tehnika štetja kolonij (ISO 10272
-2:2017)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 2: Colony-count technique (ISO 10272-2:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter - Teil 2: Koloniezählverfahren (ISO 10272-2:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 2 : Technique par comptage des colonies
(ISO 10272-2:2017)
Ta slovenski standard je istoveten z: EN ISO 10272-2:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-2:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10272-2:2017
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SIST EN ISO 10272-2:2017
EN ISO 10272-2
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for
detection and enumeration of Campylobacter spp. - Part 2:
Colony-count technique (ISO 10272-2:2017)
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 2 : Technique par Campylobacter - Teil 2: Koloniezählverfahren (ISO
comptage des colonies (ISO 10272-2:2017) 10272-2:2017)
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 10272-2:2017 E
worldwide for CEN national Members.
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SIST EN ISO 10272-2:2017
EN ISO 10272-2:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 10272-2:2017
EN ISO 10272-2:2017 (E)
European foreword
This document (EN ISO 10272-2:2017) 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 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 10272-2:2017 has been approved by CEN as EN ISO 10272-2:2017 without any
modification.
3
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SIST EN ISO 10272-2:2017
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SIST EN ISO 10272-2:2017
INTERNATIONAL ISO
STANDARD 10272-2
First edition
2017-06
Microbiology of the food chain —
Horizontal method for detection and
enumeration of Campylobacter spp. —
Part 2:
Colony-count technique
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Campylobacter spp. —
Partie 2: Technique par comptage des colonies
Reference number
ISO 10272-2:2017(E)
©
ISO 2017
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(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 10272-2:2017
ISO 10272-2:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Preparation of dilutions . 2
4.3 Enumeration . 2
4.4 Confirmation . 2
5 Culture media and reagents . 2
6 Equipment and consumables . 3
7 Sampling . 3
8 Preparation of test sample . 3
9 Procedure. 4
9.1 Test portion, initial suspension and dilutions . 4
9.2 Inoculation and incubation . 4
9.3 Enumeration of characteristic colonies . 4
9.4 Confirmation of Campylobacter . 4
9.4.1 General. 4
9.4.2 Selection of colonies for confirmation . 5
9.4.3 Examination of morphology and motility . 5
9.4.4 Study of aerobic growth at 25 °C . 5
9.4.5 Detection of oxidase activity . 5
9.4.6 Interpretation . 5
9.5 Identification of Campylobacter species (optional) . 6
9.5.1 General. 6
9.5.2 Detection of catalase activity. 6
9.5.3 Detection of hippurate hydrolysis . 6
9.5.4 Detection of indoxyl acetate hydrolysis . 6
9.5.5 Interpretation . 7
10 Expression of results . 7
11 Performance characteristics of the method . 7
11.1 Interlaboratory study . 7
11.2 Repeatability limit . 7
11.3 Reproducibility limit . 8
12 Test report . 9
Annex A (normative) Diagram of procedure .10
Annex B (normative) Culture media and reagents .11
Annex C (informative) Method validation studies and performance characteristics .16
Bibliography .19
© ISO 2017 – All rights reserved iii
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(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 first edition cancels and replaces ISO/TS 10272-2:2006, which has been technically revised with
the following main changes:
— samples from the primary production stage have been added to the scope;
— serial dilutions are plated in single instead of in duplicate, to be in line with ISO 7218;
— 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.
iv © ISO 2017 – All rights reserved
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(E)
Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO/TS 10272-
2:2006 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.
© ISO 2017 – All rights reserved v
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SIST EN ISO 10272-2:2017
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SIST EN ISO 10272-2:2017
INTERNATIONAL STANDARD ISO 10272-2:2017(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 2:
Colony-count technique
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
enumeration of 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 enumeration 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 10272-2:2017
ISO 10272-2:2017(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 are Campylobacter jejuni and Campylobacter coli.
However, other species have been described (Campylobacter lari, Campylobacter upsaliensis and others).
3.2
enumeration of Campylobacter
determination of the number of colony-forming units (cfu) of Campylobacter (3.1) found per gram, per
millilitre, per square centimetre or per sampling device when the test is conducted in accordance with
this document
4 Principle
4.1 General
The enumeration of Campylobacter requires three successive stages as specified in Annex A.
4.2 Preparation of dilutions
For the preparation of decimal dilutions from the test portion, see ISO 6887.
4.3 Enumeration
The solid selective medium modified Charcoal Cefoperozone Deoxycholate agar (mCCD agar) is
inoculated with a specified quantity of the test portion if the product is liquid or of the initial suspension
in the case of other products.
Other plates are prepared under the same conditions, using decimal dilutions of the test portion or of
the initial suspension.
The plates are incubated at 41,5 °C in a microaerobic atmosphere and examined after 44 h to record the
number 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.
The number of colony-forming units (cfu) of Campylobacter per unit of the test portion is calculated
from the number of confirmed typical colonies per plate.
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.
2 © ISO 2017 – All rights reserved
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(E)
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 9.4.5).
6.4 Microscope, preferably with phase contrast (for observing the characteristic morphology and
motility of Campylobacter).
6.5 Appropriate apparatus 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 and (optional) large size (diameter
approximately 140 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.
© ISO 2017 – All rights reserved 3
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(E)
9 Procedure
9.1 Test portion, initial suspension and dilutions
See ISO 6887 and the specific International Standard dealing with the product concerned.
Prepare a single decimal dilution series from the test portion if the product is liquid or from the initial
suspension in the case of other products.
9.2 Inoculation and incubation
9.2.1 Using a sterile pipette, transfer 0,1 ml of the initial suspension (or sample if liquid) (9.1) to the
mCCD agar plate (B.3). Repeat the procedure using further decimal dilutions if necessary. If only the
initial suspension is used, prepare duplicate plates using an additional agar plate.
When, for certain products, it is necessary to estimate low numbers of Campylobacter, the limit of
enumeration may be lowered by a factor of 10 by examining 1,0 ml of the initial suspension. Distribute
the 1,0 ml of inoculum either on the surface of the agar medium in a large Petri dish (140 mm) or three
regular plates (90 mm). In both cases, prepare duplicates by using two large plates or six regular plates.
9.2.2 Evenly spread the inoculum, as quickly as possible, over the surface of the agar plate, using a
sterile spreader. Avoid touching the sides of the Petri dish with the spreader.
NOTE Drying of the plates is critical to produce countable plates. Each laboratory has to use its own
standardised way to dry the plates in a proper way.
9.2.3 Incubate the plates (9.2.2) at 41,5 °C (6.1) in a microaerobic atmosphere (6.5).
9.3 Enumeration of characteristic colonies
9.3.1 After 44 h ± 4 h of incubation, examine the plates (9.2.3) 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.
9.3.2 Select the plates (9.3.1) containing less than 150 typical or suspect colonies; count these colonies
and record their number as presumptive colonies per dish. Then choose at random five such colonies for
subculturing for the confirmation tests (9.4).
9.4 Confirmation of Campylobacter
9.4.1 General
As Campylobacter rapidly loses culturability in air, follow the procedure described in 9.4.2 to 9.4.5
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
[10]
Campylobacter jejuni WDCM 00005 (positive control) and Escherichia coli WDCM 00013 (negative
control).
4 © ISO 2017 – All rights reserved
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SIST EN ISO 10272-2:2017
ISO 10272-2:2017(E)
As an alternative, or in addition, to the confirmation and
...
SLOVENSKI STANDARD
oSIST prEN ISO 10272-2:2015
01-april-2015
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti in
števila Campylobacter spp. - 2. del: Tehnika štetja kolonij (ISO/DIS 10272-2:2015)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 2: Colony-count technique (ISO/DIS 10272-2:2015)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter - Teil 2: Koloniezählverfahren (ISO/DIS 10272-2:2015)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 2 : Technique par comptage des colonies
(ISO/DIS 10272-2:2015)
Ta slovenski standard je istoveten z: prEN ISO 10272-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 10272-2:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 10272-2:2015
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oSIST prEN ISO 10272-2:2015
DRAFT INTERNATIONAL STANDARD
ISO/DIS 10272-2
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 2:
Colony-count technique
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de
Campylobacter spp. —
Partie 2: Technique par comptage des colonies
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 10272-2:2014(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 10272-2:2015
ISO/DIS 10272-2:2014(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 10272-2:2015
ISO/DIS 10272-2
Contents Page
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle. 1
5 Culture media and reagents . 2
6 Apparatus and glassware . 3
7 Sampling. 3
8 Preparation of test sample . 3
9 Procedure (see diagram in Annex A) . 3
10 Expression of results . 6
11 Precision of the method . 9
12 Test report . 10
Annex A (normative) Diagram of procedure . 11
Annex B (normative) Composition and preparation of culture media and reagents . 12
Bibliography . 19
© ISO 2012 – All rights reserved iii
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oSIST prEN ISO 10272-2:2015
EN ISO/NP 10272-2
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 10272-2 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/TS 10272-2:2006), 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
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Introduction
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 Technical Specification 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 10272-2
Microbiology of food and animal feed — Horizontal method for
detection and enumeration of Campylobacter — Part 2: Colony-
count technique
1 Scope
This part of ISO 10272 describes a horizontal method for the enumeration 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.
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/Amd1 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 The most frequently encountered species are Campylobacter jejuni and Campylobacter coli. Other species
have, however, been described (Campylobacter lari, Campylobacter upsaliensis and others).
3.2
enumeration of Campylobacter
determination of the number of colony-forming units (cfu) of Campylobacter (3.1) found per millilitre, per gram
2
or per cm of test sample when the test is conducted in accordance with this part of ISO 10272.
4 Principle
4.1 Preparation of dilutions
For the preparation of decimal dilutions from the test sample, see ISO 6887.
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4.2 Enumeration
The solid selective medium, modified charcoal cefoperazone deoxycholate agar (mCCD agar), is inoculated
with a specified quantity of the test sample if the product is liquid, or of the initial suspension in the case of
other products.
Other plates are prepared under the same conditions, using decimal dilutions of the test sample or of the initial
suspension.
The plates are incubated at 41,5 °C in a microaerobic atmosphere and examined after 44 h 4 h to record the
number of colonies presumed because of their characteristics to be Campylobacter.
4.3 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.
2
The number of colony-forming units (cfu) Campylobacter per ml,per g or per cm of the test sample is
calculated from the number of confirmed typical colonies per plate.
5 Culture media and reagents
5.1 General
For current laboratory practice, see ISO11133.
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 Diluent
See ISO 6887.
5.3 Selective plating medium: Modified charcoal cefoperazone deoxycholate agar (mCCD
agar)
See B.1.
5.4 Confirmation and identification media and reagents
5.4.1 Blood agar
See B.2.
5.4.2 Reagent for the detection of oxidase
See B.3.
5.4.3 Hydrogen peroxide solution, 3 % (volume fraction)
5.4.4 Reagents for the detection of hydrolysis of hippurate
See B.4.
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5.4.5 Indoxyl acetate discs
See B.5.
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 9.4.3).
6.4 Microscope, preferably with phase contrast (for observing the characteristic morphology and motility of
Campylobacter).
6.5 Appropriate apparatus 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.
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 A)
9.1 Test portion, initial suspension and dilutions
See ISO 6887 and the specific International Standard dealing with the product concerned.
Prepare a single decimal dilution series from the test sample if the product is liquid, or from the initial
suspension in the case of other products.
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9.2 Inoculation and incubation
9.2.1 Using a sterile pipette, transfer 0,1 ml of the initial suspension (or sample if liquid) (9.1) to the mCCD
agar plate (5.3). Repeat the procedure using further decimal dilutions if necessary. If only the initial
suspension is used, also prepare duplicate plates using an additional agar plate.
When, for certain products, it is necessary to estimate low numbers of Campylobacter, the limit of
enumeration may be lowered by a factor of 10 by examining 1,0 ml of the initial suspension. Distribute the
1 ml of inoculum either on the surface of the agar medium in a large Petri dish (140 mm) or over the surface of
the agar medium in three regular plates (90 mm) using a sterile spreader. In both cases, prepare duplicates by
using two large plates or six regular plates.
9.2.2 Carefully spread the inoculum uniformly and as quickly as possible evenly over the surface of the agar
plate, without touching the sides of the dish, using a sterile spreader. Allow the inoculum to absorb with the
lids in place for about 15 min at room temperature.
9.2.3 Incubate the plates (9.2.1) at 41,5 °C (6.1) in a microaerobic atmosphere (6.5).
9.3 Enumeration of characteristic colonies
9.3.1 After 44 h 4 h of incubation, examine the plates (9.2.3) for typical and/or suspect colonies of
Campylobacter.
The 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.
9.3.2 Select the plates (9.2.3) containing less than 150 typical or suspect colonies; count these colonies
and record their number as presumptive colonies per dish. Then choose at random five such colonies for
subculturing for the confirmation tests (9.4).
9.4 Confirmation of Campylobacter
9.4.1 General
As Campylobacter rapidly loses culturability in air, follow the procedure described in 9.4.2 to 9.4.5 without
delay.
9.4.2 Selection of colonies for confirmation
9.4.2.1 For confirmation, take five presumptive colonies from each dish retained for enumeration (9.3.2).
9.4.2.2 Streak each of the selected colonies onto a blood agar plate (5.4.1) 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 (9.4.3),
absence of aerobic growth at 25 °C (9.4.4) and the presence of oxidase (9.4.5).
9.4.3 Examination of morphology and motility
9.4.3.1 Examine a freshly grown colony from the blood agar plate (9.4.2.2) for morphology and motility
using a microscope (6.4).
9.4.3.2 Retain for further examination all cultures (9.4.2.2) in which curved bacilli with a spiralling
“corkscrew” motility are found (9.4.3.1).
9.4.4 Study of aerobic growth at 25 °C
Using the colonies isolated in 9.4.2.2, inoculate with the aid of a loop (6.3) the surface of a blood agar plate
(5.4.1).
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Incubate the plate at 25 °C (6.1) aerobically for 44 h 4 h.
Examine the plate for absence of growth of colonies.
9.4.5 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 (9.4.2.2) and streak it onto a filter paper moistened with the oxidase reagent (5.4.2); 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).
9.4.6 Interpretation
Campylobacter gives results in accordance with Table 1.
Table 1 — Characteristics of Campylobacter
1
Morphology (9.4.3) small curved bacilli
1
Motility (9.4.3) Characteristic corckscrew darting
Aerobic growth at 25 °C (9.4.4)
Oxidase (9.4.5) +
1
Older cultures may rapidly lose their characteristic shape and motility and turn into less motile cocoid-like forms.
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.
9.5 Identification of Campylobacter species (optional)
9.5.1 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.
9.5.2 Detection of catalase
For each colony selected in 9.4.2.2, deposit a loop of culture into a drop of hydrogen peroxide solution (5.4.3)
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).
9.5.3 Detection of hippurate hydrolysis
For each colony selected in 9.4.2.2, 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.4.1), taking care not to incorporate any
agar.
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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.4.2) on top of the sodium hippurate solution. Do not shake.
Interpret after an additional incubation of 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).
9.5.4 Detection of indoxyl acetate hydrolysis
Place a colony selected in 9.4.2.2 on an indoxyl acetate disc (B.5) and add a drop of sterile distilled water. A
loopful of colony material is required for a clear reaction.
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.
9.5.5 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
+ + + – or weak
Catalase (9.5.2)
1
+
Hydrolysis of hippurate (9.5.3) – – –
Indoxyl acetate (9.5.4) + + – +
Key: + = positive; – = negative
1
Some hippurate-negative C. jejuni strains have been reported.
10 Expression of results
10.1 Count of Campylobacter colonies
Calculate for each of the plates retained for enumeration (9.3.2) the number a of confirmed colonies of
Campylobacter present, using Equation (1):
b
a C
(1)
A
where
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A is the number of presumptive colonies selected for confirmation (9.4.2.1);
b is the number of colonies selected for confirmation A (9.4.2.1) that comply with the confirmation
criteria (9.4.6);
C is the total number of presumptive colonies counted on the dish (9.3.2).
Round off the calculated result to the nearest whole number.
10.2 Method of calculation
10.2.1 General case – Plates containing 10 to 150 colonies of presumptive Campylobacter
For a result to be valid, it is generally considered necessary to count the colonies on at least one dish
containing at least 10 confirmed colonies a (10.1).
Calculate the number N of Campylobacter present in the test sample as a weighted mean from two
successive dilutions using Equation (2):
a
N
(2)
V 1,1d
Where
a is the sum of Campylobacter-confirmed colonies, counted on the plates retained from two
successive dilutions, at least one of which contains a minimum of 10 Campylobacter colonies;
V is the volume of inoculum applied to each dish, in ml;
d is the dilution factor corresponding to the first dilution retained (d = 1 when the undiluted liquid
product (test sample) is used).
Round off the calculated results to two significant figures. In order to do this, if the third figure is less than 5,
do not modify the preceding figure; if the third figure is greater than or equal to 5, increase the preceding
figure by one unit.
Express the result preferably as a number between 1,0 and 9,9 multiplied by the appropriate power of 10, or a
whole number with two significant figures.
Report the result as follows:
number N of Campylobacter per millilitre (liquid products) or per gram (other products).
EXAMPLE Counting has produced the following results:
3
at the first dilution (10 ) retained: 66 colonies;
4
at the second dilution (10 ) retained: 4 colonies.
Testing of selected colonies was carried out:
of the 66 colonies, 5 colonies were tested, 4 of which complied with the criteria; hence a = 53 (see 10.1);
of the 4 colonies: all 4 complied with the criteria; hence a = 4 (see 10.1);
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a
53 4
N 518182 (3)
3
V 1,1d
0,11,110
5
Rounding off the result, the number of Campylobacter is 520 000 or 5,2 10 per ml or per g of product.
10.2.2 Case when any dish (test sample or initial suspension or first dilution) contains less than 10
colonies
Counts from 10 up to the (practical) upper limit of the method are in the optimum precision range. Precision
decreases rapidly as the number of colonies decreases below 10, however. Depending on the purpose of the
test, a lower limit of determination can be defined for counts lower than 10.
Summarizing according to ISO 7218 this means:
If the plate contains less than 10 colonies, but at least 4, calculate the result as given in the general case
(10.2.1), and report it as the estimated number N of Campylobacter per millilitre (liquid products) or per gram
(other products).
If the total is from 3 to 1, the precision of the results is too low and the result shall be reported as:
“Campylobacter is present but less than (4/d V) per ml or g”.
where
0
d is the dilution factor of the initial suspension or of the first dilution inoculated or retained [d = 10 = 1
where the directly inoculated test sample (liquid products) is retained];
V is the volume of the inoculum applied to the plates, in ml.
10.2.3 Case when the dish (test sample or initial suspension or first dilution) contains no colonies
If the dish containing the test sample (liquid products) or the initial suspension (other products) or the first
dilution inoculated or retained does not contain any colonies, report the result as follows:
“less than 1/d V of Campylobacter per ml (liquid products) or per gram (other products)”
where
0
d is the dilution factor of the initial suspension or of the first dilution inoculated or retained [d = 10 = 1
where the directly inoculated test sample (liquid products) is retained];
V is the volume of the inoculum applied to the plates, in ml.
10.2.4 Special cases
Special cases are described in ISO 7218.
10.3 Expression of results as detected or not detected
As an alternative to the number of Campylobacter per amount of sample tested, the result may also be
expressed as Campylobacter detected or Campylobacter not detected in a test portion of x g or x mI of
product, according to the interpretation of the results (9.3.1 and 9.4.6).
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11 Precision of the method
11.1 Interlaboratory study
Results of the interlaboratory study to determine the precision of the method are summarised in Annex C.
Repeatability and reproducibility limits were determined using 5 sample types (broiler caecal material, frozen
spinach, minced meat, raw milk, chicken skin
...
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