EN ISO 21528-1:2017

Microbiology of the food chain - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 1: Detection of Enterobacteriaceae (ISO 21528-1:2017)

ISO 21528-1:2017 specifies a method, with enrichment, for the detection 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 method is applicable
-      when the microorganisms sought are expected to need resuscitation by enrichment, and
-      when the number sought is expected to be below 100 per millilitre or per gram of test sample.
A limitation on the applicability of ISO 21528-1:2017 is imposed by the susceptibility of the method to a large degree of variability (see Clause 11).
NOTE       Enumeration can be carried out by calculation of the most probable number (MPN) after incubation in liquid medium. See Annex A.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die Zählung von Enterobacteriaceae Teil 1: Nachweis von Enterobacteriaceae (ISO 21528-1:2017)

Microbiologie de la chaîne alimentaire - Méthode horizontale par la recherche et le dénombrement des Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae (ISO 21528-1:2017)

L'ISO 21528-1:2017 spécifie une méthode pour la recherche des Enterobacteriaceae avec enrichissement. 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 méthode est applicable:
-      lorsque les micro-organismes recherchés nécessitent une revivification par enrichissement, et
-      lorsque le nombre recherché est supposé être inférieur à 100 par millilitre ou par gramme d'échantillon pour essai.
Des limites sont imposées aux possibilités d'application de l'ISO 21528-1:2017 du fait que ces méthodes sont sujettes à de grandes variations (voir l'Article 11).
NOTE       Le dénombrement peut être effectué en calculant le nombre le plus probable (NPP) après incubation en milieu liquide. Voir l'Annexe A.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila enterobakterij - 1. del: Ugotavljanje prisotnosti enterobakterij (ISO 21528-1:2017)

Ta standard določa metodo najverjetnejšega števila (MPN) s predhodno obogatitvijo za ugotavljanje prisotnosti enterobakterij. Uporablja se za:
– izdelke, namenjene za prehrano ljudi in krmo živali; in
– okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano; in
– vzorce iz primarne proizvodnje.
(referenčni dokument: ISO 21528-1)

General Information

• Status: Published
• Publication Date: 04-Jul-2017
• Withdrawal Date: 30-Jan-2018
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463 - Microbiology of the food chain
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 05-Jul-2017
• Due Date: 01-Mar-2014
• Completion Date: 05-Jul-2017
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/381 - Mandate for standardisation addressed to CEN in the field of Methods of analysis of foodstuffs concerning Food Hygiene
• Ref Project: SIST EN ISO 21528-1:2017 - Microbiology of the food chain - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 1: Detection of Enterobacteriaceae (ISO 21528-1:2017)

Overview

EN ISO 21528-1:2017 - Microbiology of the food chain: Horizontal method for the detection and enumeration of Enterobacteriaceae (Part 1: Detection) - specifies a standardized, enrichment-based laboratory method to detect Enterobacteriaceae in foods, animal feed and related environmental samples. The standard is intended for use when target organisms may require resuscitation by enrichment and when expected counts are below 100 CFU per g or mL. The document also warns that the method can show substantial variability (see Clause 11) and that testing should be done in properly equipped labs by skilled microbiologists.

Key topics and requirements

• Scope of application: Products for human consumption, animal feeding stuffs, and environmental samples from primary production, food processing and handling areas.
• Detection principle: Enrichment in a non-selective medium to resuscitate stressed Enterobacteriaceae, followed by isolation, selection and biochemical confirmation.
• Enrichment and confirmation: The 2017 revision standardizes enrichment in buffered peptone water (BPW) and confirmation in Glucose OF medium (change noted from earlier editions).
• Enumeration option: Enumeration by Most Probable Number (MPN) after incubation in liquid medium is described in Annex A (informative).
• Performance and variability: Clause 11 covers precision, sensitivity and specificity; users should note the method’s susceptibility to variability and apply quality assurance accordingly.
• Safety and laboratory competence: Testing must be performed in appropriate lab facilities under trained personnel with safe disposal of incubated materials.
• Documentation: Procedures for sampling, test portion preparation, incubation, colony selection, subculturing and biochemical confirmation tests are defined; Annexes list media, reagents and validation guidance.

Applications

• Routine screening for Enterobacteriaceae as indicators of hygiene and potential contamination in:
  • Raw and processed foods
  • Animal feed
  • Environmental swabs from production, handling and storage areas
• Food safety monitoring, supplier verification and HACCP-related testing
• Method validation and quality control of culture media and laboratory procedures

Who should use this standard

• Food and feed microbiology laboratories (public and private)
• Quality assurance and food safety teams in manufacturing and processing
• Regulatory and inspection authorities performing compliance testing
• Test method developers and standardization bodies referencing horizontal methods

Related standards

• Other parts of the ISO 21528 series (enumeration and complementary methods) - see the ISO website for the complete list and cross-references.

Keywords: EN ISO 21528-1:2017, Enterobacteriaceae detection, food microbiology standard, enrichment method, MPN enumeration, food safety testing.

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila enterobakterij - 1. del: Ugotavljanje prisotnosti enterobakterij
(ISO 21528-1:2017)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Enterobacteriaceae - Part 1: Detection of Enterobacteriaceae (ISO 21528-1:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die
Zählung von Enterobacteriaceae Teil 1: Nachweis von Enterobacteriaceae (ISO 21528-
1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale par la recherche et le
dénombrement des Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae
(ISO 21528-1:2017)
Ta slovenski standard je istoveten z: EN ISO 21528-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 21528-1
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 1:
Detection of Enterobacteriaceae (ISO 21528-1:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale par la recherche et le dénombrement des Verfahren für den Nachweis und die Zählung von
Enterobacteriaceae - Partie 1: Recherche des Enterobacteriaceae Teil 1: Nachweis von
Enterobacteriaceae (ISO 21528-1:2017) Enterobacteriaceae (ISO 21528-1:2017)
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 21528-1:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 21528-1:2017) has been prepared by Technical Committee ISO/TC 34 “Food
products” in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2018, and conflicting national standards shall
be withdrawn at the latest by January 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document 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 21528-1:2017 has been approved by CEN as EN ISO 21528-1:2017 without any
modification.
INTERNATIONAL ISO
STANDARD 21528-1
Second edition
2017-06
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of
Enterobacteriaceae —
Part 1:
Detection of Enterobacteriaceae
Microbiologie de la chaîne alimentaire — Méthode horizontale par
la recherche et le dénombrement des Enterobacteriaceae —
Partie 1: Recherche des Enterobacteriaceae
Reference number
ISO 21528-1:2017(E)
©
ISO 2017
ISO 21528-1:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

ISO 21528-1:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 Enrichment in non-selective medium . 2
4.2 Isolation and selection for confirmation . 2
4.3 Confirmation . 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 and initial suspension . 4
9.3 Enrichment . 4
9.4 Isolation and selection for confirmation . 4
9.4.1 Isolation . 4
9.4.2 Selection of colonies for confirmation . 4
9.5 Subculturing selected colonies. 4
9.6 Biochemical confirmation tests . 4
9.6.1 Oxidase reaction . 4
9.6.2 Fermentation test . 5
10 Expression of results . 5
11 Precision . 5
11.1 Interlaboratory study . 5
11.2 Sensitivity . 5
11.3 Specificity . 5
12 Test report . 5
13 Quality assurance . 6
Annex A (informative) Enumeration by MPN technique . 7
Annex B (normative) Culture media and reagents .10
Annex C (informative) Method validation studies and performance characteristics .15
Bibliography .17
ISO 21528-1:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www . i so .org/ iso/ foreword .html
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 21528-1:2004), which has been technically
revised with the following main changes:
— the MPN method has become an informative Annex A;
— the pre-enrichment step in BPW followed by enrichment in EE broth has been changed to enrichment
[7]
in BPW and confirmation now takes place in Glucose OF medium instead of using glucose agar;
— performance testing for the quality assurance of the culture media has been added;
— performance characteristics for this method have been added to Annex C.
A list of all the parts in the ISO 21528 series can be found on the ISO website.
iv © ISO 2017 – All rights reserved

ISO 21528-1:2017(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 21528-1:2004
are considered as major (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.
INTERNATIONAL STANDARD ISO 21528-1:2017(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Enterobacteriaceae —
Part 1:
Detection of Enterobacteriaceae
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, with enrichment, for the detection 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 method is applicable
— when the microorganisms sought are expected to need resuscitation by enrichment, and
— when the number sought is expected to be below 100 per millilitre or per gram of test sample.
A limitation on the applicability of this document is imposed by the susceptibility of the method to a
large degree of variability (see Clause 11).
NOTE Enumeration can be carried out by calculation of the most probable number (MPN) after incubation in
liquid medium. See Annex A.
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 21528-1:2017(E)
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
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
detection of Enterobacteriaceae
determination of Enterobacteriaceae (3.1), in a particular mass or volume of product or surface area,
when tests are carried out in accordance with this document
4 Principle
4.1 Enrichment in non-selective medium
Buffered peptone water (BPW) is inoculated with the test portion, and then incubated at 37 °C (or
30 °C) for 18 h.
NOTE The incubation temperature of 37 °C for enrichment and isolation/enumeration on 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.
4.2 Isolation and selection for confirmation
Violet red bile glucose (VRBG) agar is inoculated with the culture obtained after enrichment in
BPW, then incubated at 37 °C. It is examined after 24 h to detect the presence of typical colonies of
presumptive Enterobacteriaceae.
4.3 Confirmation
Typical 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.
5 Diluent, culture media and reagent
For current laboratory practice, see ISO 7218.
Composition of culture media and reagents and their preparation are specified in Annex B.
For performance testing of culture media, see ISO 11133 and Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in ISO 7218.
6.2 Incubator, capable of operating at 37 °C ± 1 °C (or 30 °C ± 1 °C).
2 © ISO 2017 – All rights reserved

ISO 21528-1:2017(E)
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 Containers (e.g. bottles, tubes, flasks), suitable for the sterilization and storage of culture media.
6.6 Test tubes or flasks of appropriate capacity.
6.7 Petri dishes, made of glass or plastics, of diameter 90 mm to 100 mm.
6.8 Loops (of diameter approximately 3 mm) and wires, made of platinum/iridium or
nickel/chromium, or glass rods, or equivalent sterile disposable loops or inoculating needles.
6.9 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.
6.10 pH-meter, accurate to within ±0,1 pH unit at 25 °C.
6.11 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 21528-1:2017(E)
9.2 Test portion and initial suspension
In general, an amount of test portion (mass or volume) is added to a quantity of BPW (mass or volume)
to yield a 10-fold dilution. For example, a 10 g test portion is mixed with 90 ml of BPW.
This document has been validated for test portions
...