Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique (ISO 10272-2:2017)

ISO 10272-2:2017 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.

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).

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04-Jul-2017
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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

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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.
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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
...

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