Microbiology of the food chain - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 2: Colony-count technique (ISO 21528-2:2017, Corrected version 2018-06-01)

ISO 21528-2:2017 specifies a method for the enumeration of Enterobacteriaceae. It is applicable to
-      products intended for human consumption and the feeding of animals, and
-      environmental samples in the area of primary production, food production and food handling.
This technique is intended to be used when the number of colonies sought is expected to be more than 100 per millilitre or per gram of the test sample.
The most probable number (MPN) technique, as included in ISO 21528‑1, is generally used when the number sought is expected to be below 100 per millilitre or per gram of test sample.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die Zählung von Enterobacteriaceae - Teil 2: Koloniezählverfahren (ISO 21528-2:2017, korrigierte Fassung 2018-06-01)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement des Enterobacteriaceae - Partie 2: Technique par comptage des colonies (ISO 21528-2:2017, Version corrigée 2018-06-01)

L'ISO 21528-2:2017 spécifie une méthode pour le dénombrement des Enterobacteriaceae. Elle est applicable:
-      aux produits destinés à l'alimentation humaine et animale, et
-      aux échantillons d'environnement pour la production au stade primaire, la production des aliments et la distribution des aliments.
Cette technique est destinée à être utilisée lorsque le nombre de colonies recherchées est supposé être supérieur à 100 par millilitre ou par gramme d'échantillon pour essai.
La technique du nombre le plus probable (NPP), incluse dans l'ISO 21528‑1, est généralement utilisée lorsque le nombre recherché est censé être inférieur à 100 par millilitre ou par gramme d'échantillon pour essai.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila enterobakterij - 2. del: Metoda štetja kolonij (ISO 21528-2:2017)

Ta del standarda EN ISO 21528 določa metodo za ugotavljanje števila enterobakterij brez predhodne obogatitve. Uporablja se za: – izdelke, namenjene za prehrano ljudi in krmo živali; in – okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano. Ugotavljanje števila se izvaja s štetjem kolonij na trdem mediju po inkubaciji pri 37 °C (ali 30 °C).  Ta tehnika se priporoča, kadar se pričakuje več kot 100 kolonij na mililiter ali gram preskusnega vzorca.

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Publication Date
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6060 - Definitive text made available (DAV) - Publishing
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SLOVENSKI STANDARD
SIST EN ISO 21528-2:2017
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje

prisotnosti in števila enterobakterij - 2. del: Metoda štetja kolonij (ISO 21528-

2:2017)

Microbiology of the food chain - Horizontal method for the detection and enumeration of

Enterobacteriaceae - Part 2: Colony-count technique (ISO 21528-2:2017)

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die

Zählung von Enterobacteriaceae - Teil 2: Koloniezählverfahren (ISO 21528-2:2017)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le

dénombrement des Enterobacteriaceae - Partie 2: Technique par comptage des colonies

(ISO 21528-2:2017)
Ta slovenski standard je istoveten z: EN ISO 21528-2:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 21528-2:2017 en

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

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SIST EN ISO 21528-2:2017
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SIST EN ISO 21528-2:2017
EN ISO 21528-2
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Enterobacteriaceae - Part 2:
Colony-count technique (ISO 21528-2:2017)

Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales

horizontale pour la recherche et le dénombrement des Verfahren für den Nachweis und die Zählung von

Enterobacteriaceae - Partie 2: Technique par comptage Enterobacteriaceae - Teil 2: Koloniezählverfahren (ISO

des colonies (ISO 21528-2:2017) 21528-2:2017)
This European Standard was approved by CEN on 27 April 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 21528-2:2017 E

worldwide for CEN national Members.
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SIST EN ISO 21528-2:2017
EN ISO 21528-2:2017 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 21528-2:2017
EN ISO 21528-2:2017 (E)
European foreword

This document (EN ISO 21528-2:2017) 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 21528-2:2017 has been approved by CEN as EN ISO 21528-2:2017 without any

modification.
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SIST EN ISO 21528-2:2017
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SIST EN ISO 21528-2:2017
INTERNATIONAL ISO
STANDARD 21528-2
Second edition
2017-06
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of
Enterobacteriaceae —
Part 2:
Colony-count technique
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement des Enterobacteriaceae —
Partie 2: Technique par comptage des colonies
Reference number
ISO 21528-2:2017(E)
ISO 2017
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(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 21528-2:2017
ISO 21528-2:2017(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 Preparation of initial suspension and decimal dilutions .................................................................................... 2

4.2 Isolation and selection for confirmation .......................................................................................................................... 2

4.3 Confirmation ............................................................................................................................................................................................. 2

4.4 Calculation .................................................................................................................................................................................................. 2

5 Diluent, culture media and reagent .................................................................................................................................................. 2

6 Equipment and consumables .................................................................................................................................................................. 2

7 Sampling ........................................................................................................................................................................................................................ 3

8 Preparation of test sample ......................................................................................................................................................................... 3

9 Procedure..................................................................................................................................................................................................................... 3

9.1 General ........................................................................................................................................................................................................... 3

9.2 Test portion, initial suspension and dilutions .............................................................................................................. 4

9.3 Inoculation and incubation .......................................................................................................................................................... 4

9.4 Counting and selection of colonies for confirmation ............................................................................................. 4

9.5 Subculturing selected colonies.................................................................................................................................................. 4

9.6 Biochemical confirmation tests ................................................................................................................................................ 5

9.6.1 Oxidase reaction ............................................................................................................................................................... 5

9.6.2 Fermentation test ............................................................................................................................................................ 5

9.6.3 Interpretation of biochemical tests ................................................................................................................. 5

10 Expression of results ........................................................................................................................................................................................ 5

11 Performance characteristics of the method ............................................................................................................................. 5

11.1 Interlaboratory study ........................................................................................................................................................................ 5

11.2 Repeatability limit ................................................................................................................................................................................ 5

11.3 Reproducibility limit .......................................................................................................................................................................... 6

12 Test report ................................................................................................................................................................................................................... 6

13 Quality assurance ................................................................................................................................................................................................ 7

Annex A (normative) Culture media and reagents ................................................................................................................................ 8

Annex B (informative) Method validation studies and performance characteristics ....................................12

Bibliography .............................................................................................................................................................................................................................15

© ISO 2017 – All rights reserved iii
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(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 WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: w w w . i s o .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 21528-2:2004), which has been

technically revised with the following main changes:

— the confirmation step has been changed by replacing glucose agar by glucose OF medium;

— precision data based on the results of an interlaboratory study using the method according to this

revised edition has been included in an informative annex.
A list of all the parts in the ISO 21528 series can be found on the ISO website.
iv © ISO 2017 – All rights reserved
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(E)
Introduction

This document is intended to provide general guidance for the examination of products not dealt

with by existing International Standards and to be taken into account by organizations preparing

microbiological test methods for application to foods or animal feeding stuffs. Because of the large

variety of products within this field of application, these guidelines 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 the guidelines provided

as far as possible and that deviations from them will only be made if absolutely necessary for technical

reasons.

The main changes, listed in the foreword, introduced in this document compared to ISO 21528-2:2004,

are considered as minor changes (see ISO 17468).

The harmonization of test methods cannot be immediate, and for certain groups of products,

International Standards and/or national standards may already exist that do not comply with this

horizontal method. It is hoped that when such standards are reviewed, they will be changed to comply

with this 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 21528-2:2017
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SIST EN ISO 21528-2:2017
INTERNATIONAL STANDARD ISO 21528-2:2017(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Enterobacteriaceae —
Part 2:
Colony-count technique

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for

detecting Enterobacteriaceae 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 method for the enumeration of Enterobacteriaceae. It is applicable to

— products intended for human consumption and the feeding of animals, and

— environmental samples in the area of primary production, food production and food handling.

This technique is intended to be used when the number of colonies sought is expected to be more than

100 per millilitre or per gram of the test sample.

The most probable number (MPN) technique, as included in ISO 21528-1, is generally used when the

number sought is expected to be below 100 per millilitre or per gram of test sample.

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

ISO 18593, Microbiology of food and animal feeding stuffs — Horizontal methods for sampling techniques

from surfaces using contact plates and swabs
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 21528-2:2017
ISO 21528-2:2017(E)
3.1
Enterobacteriaceae

microorganism that forms characteristic colonies on violet red bile glucose agar and that ferment

glucose and show a negative oxidase reaction when the tests are carried out in accordance with the

methods specified in this document
3.2
count of Enterobacteriaceae

number of Enterobacteriaceae found per millilitre or per gram of the test sample or per unit area

swabbed, when the test is carried out according to the method specified in this document

4 Principle
4.1 Preparation of initial suspension and decimal dilutions
An initial suspension and decimal dilutions are prepared from the test sample.
4.2 Isolation and selection for confirmation

Violet red bile glucose (VRBG) 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. An overlay of the same

medium is added. Other plates are prepared under the same conditions, using decimal dilutions of the

test sample or of the initial suspension.
The dishes are incubated at 37 °C (or 30 °C) for 24 h.

NOTE The incubation temperature of 37 °C for enrichment and isolation/enumeration on the plating

medium is generally used when the detection and enumeration of Enterobacteriaceae is for a hygiene indicator.

Alternatively, a temperature of 30 °C can be chosen when the detection or enumeration of Enterobacteriaceae is

conducted for technological purposes and includes psychrotrophic Enterobacteriaceae. In this document, 37 °C

will be used throughout the text.
4.3 Confirmation

Colonies of presumptive Enterobacteriaceae are subcultured onto non-selective medium, and confirmed

by means of tests for the fermentation of glucose and the presence of oxidase.
4.4 Calculation

The number of Enterobacteriaceae per millilitre or gram of the test sample is calculated from the

number of confirmed typical colonies per dish.
5 Diluent, culture media and reagent
For current laboratory practice, see ISO 7218.

Composition of culture media and reagents and their preparation are described in Annex A.

For performance testing of culture media, see ISO 11133 and Annex A.
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 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in ISO 7218.

2 © ISO 2017 – All rights reserved
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(E)
6.2 Incubator, capable of operating at 37 °C ± 1 °C (or 30 °C ± 1 °C).

6.3 Drying cabinet (ventilated by convection) or incubator, capable of operating between 25 °C

and 50 °C.

6.4 Water bath, or similar apparatus, capable of being maintained between 47 °C to 50 °C.

6.5 Test tubes or flasks, of appropriate capacity.

6.6 Petri dishes, made of glass or plastic, with a diameter of approximately 90 mm and (optional)

large size (diameter approximately 140 mm).

6.7 Loops (of diameter approximately 3 mm) and wires, made of platinum/iridium or

nickel/chromium, and/or glass rods, or equivalent sterile disposable loops or inoculating needles.

6.8 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.

6.9 pH-meter, accurate to within ±0,1 pH unit at 25 °C.
6.10 Homogenizer, as specified in ISO 7218.
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.
Recommended sampling techniques are given in:
— ISO/TS 17728 for food and animal feed;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for environmental samples.

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.
8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard appropriate to the

product concerned. If there is no specific International Standard available, it is recommended that the

parties concerned come to an agreement on this subject.
9 Procedure
9.1 General
See ISO 7218.
© ISO 2017 – All rights reserved 3
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(E)
9.2 Test portion, initial suspension and dilutions
See ISO 6887 (all parts).

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.
9.3 Inoculation and incubation

9.3.1 Take one sterile Petri dish (6.6). Using a sterile pipette (6.8), transfer to the dish 1 ml of the test

sample if the product is liquid, or 1 ml of the initial suspension in case of other products.

Repeat the procedure described with the further dilutions, if necessary, using a fresh sterile pipette for

each dilution.

If only the initial suspension is used, then inoculate two plates of this dilution (ISO 7218).

9.3.2 Add into each Petri dish approximately 15 ml of the violet red bile glucose (VRBG) agar (A.2)

which has been prepared then cooled to 47 °C to 50 °C in the water bath (6.4). The time elapsing between

the inoculation of the Petri dishes and the moment when the medium is poured into the dishes shall not

exceed 15 min.

Carefully mix the inoculum with the medium by horizontal movements and allow the medium to

solidify, with the Petri dishes standing on a cool surface.

9.3.3 After complete solidification of the mixture, add a covering layer of approximately 5 ml to 10 ml

of the violet red bile glucose (VRBG) agar (A.2), prepared then cooled as described in 9.3.2, to prevent

spreading growth and to achieve semi-anaerobic conditions. Allow to solidify as described above.

9.3.4 Invert the prepared dishes and incubate them at 37 °C (6.2) for 24 h ± 2 h.

9.4 Counting and selection of colonies for confirmation

Characteristic colonies are pink to red or purple (with or without precipitation haloes).

Select the dishes (see 9.3.4) containing less than 150 characteristic colonies; count these colonies.

Then choose at random five such colonies from each dish for subculturing (see 9.5) for the biochemical

confirmation tests (see 9.6). If there are less than five colonies on the plate, take all presumptive

colonies present.

Spreading colonies shall be considered as single colonies. If less than one-quarter of the dish is overgrown

by spreading, count the colonies on the unaffected part of the dish and calculate by extrapolation the

theoretical number of colonies for the entire plate. If more than one-quarter is overgrown by spreading

colonies, discard the count.

Certain Enterobacteriaceae may cause decolouration of their colonies or of the medium. Therefore,

when no characteristic colonies are present, choose five whitish colonies for confirmation.

9.5 Subculturing selected colonies

Streak the selected colonies (9.4) onto the surface of pre-dried non-selective agar medium (A.3), in a

manner which will allow well-isolated colonies to develop.
Incubate these plates at 37 °C for 24 h ± 2 h.

Select a well-isolated colony from each of the incubated plates for the biochemical confirmation tests

(see 9.6).
4 © ISO 2017 – All rights reserved
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SIST EN ISO 21528-2:2017
ISO 21528-2:2017(E)
9.6 Biochemical confirmation tests
9.6.1 Oxidase reaction

Using a platinum/iridium loop, wire or glass rod (6.7), take a portion of each well-isolated colony (see

9.5) and streak onto a filter paper moistened with the oxidase reagent (A.5) or onto a commercially

available disc or stick. A nickel/chromium loop or wire shall not be used.

Consider the test to be negative when the colour of the filter paper does not turn dark blue purple

within 10 s.
Consult the manufacturer’s instructions for ready-to-use discs or sticks.
9.6.2 Fermentation test

Using a wire (6.7), stab the same colonies selected in 9.5 that gave a negative oxidase test into tubes

containing Glucose OF medium (A.4). Overlay the surface of the medium with minimal 1 cm of sterile

mineral oil (A.6).
Incubate these tubes at 37 °C for 24 h ± 2 h.

If a yellow colour develops throughout the content of the tube, regard the reaction as being positive.

9.6.3 Interpretation of biochemical tests

Colonies that are oxidase-negative and glucose-positive are confirmed as Enterobacteriaceae.

10 Expression of results

See ISO 7218. Calculate and report the results as the number of Enterobacteriaceae in cfu per gram,

millilitre, per square centimetre or per sampling device.
11 Performance characteristics of the method
11.1 Interlaboratory study

Results of the interlaboratory study to determine the precision of the method are summarized

in Annex B. Repeatability and reproducibility limits were determined using five types of food

contaminated at various levels. The values derived from the interlaboratory study may not be applicable

to concentration ranges and food
...

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