SIST EN ISO 16649-3:2015

Microbiology of the food chain - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (ISO 16649-3:2015)

This International Standard specifies a horizontal method for the detection and enumeration of β-glucuronidase positive Escherichia coli, by means of the liquid medium culture technique and calculation of the most probable number (MPN) after incubation at 37 °C, then at 44 °C. This International Standard is applicable to:
- products intended for human consumption and the feeding of animals, and
- environmental samples in the area of food production and food handling
The method is suitable for the enumeration of cells of Escherichia coli that may have been subjected to stress arising from dehydration, freezing, exposure to a saline (such as marine) environment or damage by disinfectants such as chlorine-containing products.
A limitation of the applicability of this International Standard is imposed by the susceptibility of the method to a large degree of variability.
This method has not been fully evaluated for all matrices, e.g. for milk and milk products. ISO 7251 should be used for milk and milk products.
WARNING – Strains of Escherichia coli that do not grow at 44 °C and, in particular, those that are  β glucuronidase negative, such as Escherichia coli O157 and some other strains of pathogenic E. coli, will not be detected by the method described in this International Standard.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Zählung von β-Glucuronidase-positiven Escherichia coli - Teil 3: Nachweis und Bestimmung der wahrscheinlichsten Keimzahl unter Verwendung von 5-Brom-4-Chlor-3-Indol-β-D-Glucuronid (ISO 16649-3:2015)

Dieser Teil von ISO 16649 legt ein horizontales Verfahren für den Nachweis und die Zählung von β-Glucuronidase positiven Escherichia coli durch Kultivierung im flüssigen Nährmedium und Berechnung der wahrscheinlichsten Keimzahl (en: most probable number, MPN) nach der Bebrütung bei (37 ± 1) °C und anschließend bei (44 ± 1) °C fest.
Dieser Teil von ISO 16649 ist anwendbar auf:
-   Erzeugnisse, die für den menschlichen Verzehr und als Futtermittel bestimmt sind;
-   Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln.
Das Verfahren ist geeignet für die Zählung von Zellen von Escherichia coli, die möglicherweise Stress ausgesetzt waren, bedingt durch Austrocknung, Gefrieren und dem Ausgesetztsein einer salzhaltigen (z. B. marinen) Umgebung oder Beschädigung durch Desinfektionsmittel, wie z. B. chlorhaltige Produkte.
Eine Anwendungsbeschränkung dieses Teiles der ISO 16649 wird durch die Anfälligkeit des Verfahrens für ein hohes Maß an Variabilität bedingt. Die Durchführung des Verfahrens und die Auswertung der Ergebnisse sollten unter Berücksichtigung der in Abschnitt 11 angeführten Informationen erfolgen.
Dieses Verfahren wurde noch nicht vollständig für alle Matrizes bewertet, z. B. für Milch und Milchprodukte. ISO 7251 sollte für Milch und Milchprodukte angewendet werden.

Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des Escherichia coli bêta-glucuronidase positive - Partie 3: Technique du nombre le plus probable utilisant le bromo-5-chloro-4-indolyl-3 beta-D-glucuronate (ISO 16649-3:2015)

ISO 16649-3:2015 spécifie une méthode horizontale pour la recherche et le dénombrement des Escherichia coli β-glucuronidase positives par la technique de mise en culture en milieu liquide et par le calcul du nombre le plus probable (NPP) après incubation à (37 ± 1) °C, puis à (44 ± 1) °C. La présente partie de l'ISO 16649 est applicable:
-      aux produits alimentaires destinés à la consommation humaine ou à l'alimentation des animaux;
-      aux échantillons environnementaux dans la zone de production et de manutention des produits alimentaires.
Cette méthode convient pour le dénombrement des cellules d'Escherichia coli qui ont pu souffrir de déshydratation, de congélation, d'exposition à un environnement salin (marin par exemple) ou avoir été endommagées par des désinfectants, par exemple des produits contenant du chlore.
L'applicabilité de la présente partie de l'ISO 16649 est limitée par le fait que la méthode est sujette à une importante variabilité. Il faut appliquer la méthode et interpréter les résultats à la lumière des informations fournies à l'Article 11.
Cette méthode n'a pas fait l'objet d'une évaluation complète pour toutes les matrices, par exemple pour le lait et les produits laitiers, pour lesquels il convient d'utiliser l'ISO 7251.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje števila Escherichia coli, pozitivnih na beta-glukuronidazo - 3. del: Ugotavljanje števila in metoda najverjetnejšega števila z uporabo 5-bromo-4-kloro-3-indolil-beta-D-glukuronidaze (ISO 16649-3:2015)

Ta mednarodni standard določa horizontalno metodo za ugotavljanje prisotnosti in števila Escherichia coli, pozitivnih na beta-glukuronidazo, s pomočjo tehnike kulture tekočega medija in izračuna najverjetnejšega števila (MPN) po inkubaciji pri 37 °C in nato pri 44 °C. Ta mednarodni standard velja za: – izdelke, namenjene za prehrano ljudi in krmo živali; in – okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano; Metoda je primerna za ugotavljanje števila celic Escherichia coli, ki so bile morda izpostavljene obremenitvam zaradi dehidracije, zmrzovanja, izpostavljenosti slanemu okolju (kot je morsko) ali poškodbam zaradi razkužil, kot so izdelki, ki vsebujejo klor. Omejitev uporabnosti tega mednarodnega standarda je posledica občutljivosti metode na precej spremenljivk.  Ta metoda ni bila v celoti ovrednotena za vse matrike, npr. za mleko in mlečne izdelke. Standard ISO 7251 naj bi bil uporabljen za mleko in mlečne izdelke. OPOZORILO – Sevi Escherichia coli, ki ne rastejo pri 44 °C in zlasti tisti, ki so negativni na beta-glukuronidazo, kot je Escherichia coli O157 in nekateri drugi patogenih sevi E. coli, ne bodo zaznani z metodo, opisano v tem mednarodnem standardu.

General Information

• Status: Published
• Publication Date: 04-Aug-2015
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 28-May-2015
• Due Date: 02-Aug-2015
• Completion Date: 05-Aug-2015
• Ref Project: EN ISO 16649-3:2015 - Microbiology of the food chain - Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli - Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (ISO 16649-3:2015, Corrected version 2016-12-15)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 16649-3:2015
01-september-2015
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje števila
Escherichia coli, pozitivnih na beta-glukuronidazo - 3. del: Ugotavljanje števila in
metoda najverjetnejšega števila z uporabo 5-bromo-4-kloro-3-indolil-beta-D-
glukuronidaze (ISO 16649-3:2015)
Microbiology of the food chain - Horizontal method for the enumeration of beta-
glucuronidase-positive Escherichia coli - Part 3: Detection and most probable number
technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (ISO 16649-3:2015)
0LNURELRORJLHGHU/HEHQVPLWWHONHWWH+RUL]RQWDOHV9HUIDKUHQ]XU=lKOXQJYRQȕ
*OXFXURQLGDVHSRVLWLYHQ(VFKHULFKLDFROL7HLO1DFKZHLVXQG%HVWLPPXQJGHU
ZDKUVFKHLQOLFKVWHQ.HLP]DKOXQWHU9HUZHQGXQJYRQ%URP&KORU,QGROȕ'
*OXFXURQLG,62
Microbiologie de la chaîne alimentaire - Méthode horizontale pour le dénombrement des
Escherichia coli bêta-glucuronidase positive - Partie 3: Technique du nombre le plus
probable utilisant le bromo-5-chloro-4-indolyl-3 beta-D-glucuronate (ISO 16649-3:2015)
Ta slovenski standard je istoveten z: EN ISO 16649-3:2015
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16649-3:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16649-3:2015

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SIST EN ISO 16649-3:2015

EUROPEAN STANDARD
EN ISO 16649-3

NORME EUROPÉENNE

EUROPÄISCHE NORM
May 2015
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli -
Part 3: Detection and most probable number technique using 5-
bromo-4-chloro-3-indolyl-ß-D-glucuronide (ISO 16649-3:2015)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour le dénombrement des Escherichia coli Verfahren zur Zählung von ß-Glucuronidase-positiven
bêta-glucuronidase positive - Partie 3: Recherche et Escherichia coli - Teil 3: Nachweis und Bestimmung der
technique du nombre le plus probable utilisant le bromo-5- wahrscheinlichsten Keimzahl unter Verwendung von 5-
chloro-4-indolyl-3 ß-D-glucuronate (ISO 16649-3:2015) Brom-4-Chlor-3-Indol-ß-D-Glucuronid (ISO 16649-3:2015)
This European Standard was approved by CEN on 16 April 2015.

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, 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
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16649-3:2015 E
worldwide for CEN national Members.

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SIST EN ISO 16649-3:2015
EN ISO 16649-3:2015 (E)
Contents Page
Foreword .3
2

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SIST EN ISO 16649-3:2015
EN ISO 16649-3:2015 (E)
Foreword
This document (EN ISO 16649-3:2015) 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 November 2015, and conflicting national standards shall be withdrawn
at the latest by November 2015.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16649-3:2015 has been approved by CEN as EN ISO 16649-3:2015 without any modification.
3

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SIST EN ISO 16649-3:2015

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SIST EN ISO 16649-3:2015
INTERNATIONAL ISO
STANDARD 16649-3
First edition
2015-05-15
Microbiology of the food chain —
Horizontal method for the
enumeration of beta-glucuronidase-
positive Escherichia coli —
Part 3:
Detection and most probable number
technique using 5-bromo-4-chloro-3-
indolyl-ß-D-glucuronide
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
le dénombrement des Escherichia coli bêta-glucuronidase positive —
Partie 3: Recherche et technique du nombre le plus probable utilisant
le bromo-5-chloro-4-indolyl-3 ß-D-glucuronate
Reference number
ISO 16649-3:2015(E)
©
ISO 2015

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SIST EN ISO 16649-3:2015
ISO 16649-3:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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 2015 – All rights reserved

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SIST EN ISO 16649-3:2015
ISO 16649-3:2015(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 Detection method . 2
4.2 Enumeration method . 3
4.2.1 Inoculation of three or five tubes of double strength liquid selective
enrichment medium [5.3.1.1 a)] with an equal volume of the test sample
if the initial product is liquid, or with an equal volume of the initial
suspension in the case of other products. . 3
5 Dilution fluids and culture media . 3
5.1 General . 3
5.2 Dilution fluids . 3
5.3 Culture media . 3
5.3.1 Minerals modified glutamate medium (selective enrichment medium) . 4
5.3.2 Tryptone bile glucoronide agar (second selective enrichment medium) . 5
5.3.3 Performance testing for the quality assurance of the culture media . 5
6 Apparatus and glassware . 6
7 Sampling . 6
8 Preparation of test sample . 7
9 Procedure. 7
9.1 Detection method . 7
9.1.1 Test portion, initial suspension, and dilutions . 7
9.1.2 Incubation of selective enrichment medium . 7
9.1.3 Subculturing . 7
9.1.4 Secondary incubation . 7
9.1.5 Examination of the plates . 7
9.1.6 Interpretation . 7
9.2 Enumeration method . 7
9.2.1 Test portion, initial suspension, and dilutions . 7
9.2.2 Inoculation of the selective enrichment medium . 8
9.2.3 Incubation . 8
9.2.4 Subculturing . 8
9.2.5 Second incubation . 8
9.2.6 Examination of the plates . 8
9.2.7 Interpretation . 8
10 Expression of results . 9
10.1 Detection method . 9
10.2 Enumeration method . 9
11 Precision . 9
12 Test report . 9
Bibliography .10
© ISO 2015 – All rights reserved iii

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SIST EN ISO 16649-3:2015
ISO 16649-3:2015(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: Foreword - Supplementary information.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This first edition cancels and replaces ISO/TS 16649-3:2005, which has been technically revised.
ISO 16649 consists of the following parts, under the general title Microbiology of the food chain —
Horizontal method for the enumeration of β-glucuronidase positive Escherichia coli:
— Part 1: Colony-count technique at 44 °C using membranes and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
— Part 2: Colony-count technique at 44 °C using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
— Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
iv © ISO 2015 – All rights reserved

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SIST EN ISO 16649-3:2015
ISO 16649-3:2015(E)

Introduction
Because of the large variety of food and feed products, this horizontal method might not be appropriate
in every detail for certain products. In this case, different methods which are specific to these products
might be used if absolutely necessary, for justified technical reasons. Nevertheless, every attempt will
be made to apply this horizontal method as far as possible.
When this part of ISO 16649 is next reviewed, account will be taken of all information available regarding
the extent to which this horizontal method has been followed and the reasons for deviations from this
method in the case of particular products.
The harmonization of test methods cannot be immediate and for certain groups of products, International
Standards and/or national standards might 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
part of ISO 16649 so that eventually, the only remaining departures will be those necessary for well-
established technical reasons.
© ISO 2015 – All rights reserved v

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SIST EN ISO 16649-3:2015

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SIST EN ISO 16649-3:2015
INTERNATIONAL STANDARD ISO 16649-3:2015(E)
Microbiology of the food chain — Horizontal method
for the enumeration of beta-glucuronidase-positive
Escherichia coli —
Part 3:
Detection and most probable number technique using
5-bromo-4-chloro-3-indolyl-ß-D-glucuronide
WARNING — Strains of Escherichia coli that do not grow at 44 °C and, in particular, those that are
β-glucuronidase negative, such as Escherichia coli O157 and some other strains of pathogenic
E. coli, will not be detected by the method described in this part of ISO 16649.
1 Scope
This part of ISO 16649 specifies a horizontal method for the detection and enumeration of β-glucuronidase
positive Escherichia coli, by means of the liquid-medium culture technique and calculation of the most
probable number (MPN) after incubation at (37 ± 1) °C, then at (44 ± 1) °C. This part of ISO 16649 is
applicable to the following:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling.
The method is suitable for the enumeration of cells of E. coli that might have been subjected to stress
arising from dehydration, freezing, and exposure to a saline (such as marine) environment or damage
by disinfectants such as chlorine-containing products.
A limitation of the applicability of this part of ISO 16649 is imposed by the susceptibility of the method
to a large degree of variability. The method is intended to be applied and the results interpreted in the
light of the information given in Clause 11.
This method has not been fully evaluated for all matrices (e.g. for milk and milk products). ISO 7251 is
intended to be used for milk and milk products.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation
...