EN ISO 9308-2:2014

Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method (ISO 9308-2:2012)

ISO 9308-2:2012 specifies a method for the enumeration of E. coli and coliform bacteria in water. The method is based on the growth of target organisms in a liquid medium and calculation of the "Most Probable Number" (MPN) of organisms by reference to MPN tables. This method can be applied to all types of water, including those containing an appreciable amount of suspended matter and high background counts of heterotrophic bacteria. However it must not be used for the enumeration of coliform bacteria in marine water. When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically  required, although the method has been shown to work well with some marine waters that have a lower than normal concentration of salts. In the absence of data to support the use of the method without dilution, a 1→10 dilution is used.
This method relies upon the detection of E. coli based upon expression of the enzyme b‑D‑glucuronidase and consequently does not detect many of the enterohaemorhagic strains of E. coli, which do not typically express this enzyme. Additionally, there are a small number of other E. coli strains that do not express b‑D‑glucuronidase.
The choice of tests used in the detection and confirmation of the coliform group of bacteria, including E. coli, can be regarded as part of a continuous sequence. The extent of confirmation with a particular sample depends partly on the nature of the water and partly on the reasons for the examination. The test described in ISO 9308-2:2012 provides a confirmed result with no requirement for further confirmation of positive wells.

Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen Bakterien - Teil 2: Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO 9308-2:2012)

Dieser Teil von ISO 9308 legt ein Verfahren für die Zählung von E. coli und coliformen Bakterien in Wasser fest. Das Verfahren beruht auf der Vermehrung von Zielorganismen in einem flüssigen Medium und der Berechnung der „wahrscheinlichsten Keimzahl“ (en: Most Probable Number, MPN) von Organismen unter Bezugnahme auf MPN Tabellen. Dieses Verfahren kann auf alle Arten von Wasser angewendet werden, einschließlich derer, die eine beträchtliche Menge an Schwebstoffen und eine hohe Keimzahl heterotropher Begleitbakterien enthalten. Es darf jedoch nicht zur Zählung coliformer Bakterien in Meerwasser angewendet werden. Wird das Verfahren zur Zählung von E. coli in Meerwasser eingesetzt, ist üblicherweise eine Verdünnung in sterilem Wasser von 1:10 erforderlich, obgleich das Verfahren nachweislich mit einigen Meerwässern gut funktioniert, die eine niedrigere als die übliche Salzkonzentration aufweisen. Wenn keine Daten zur Unterstützung der Anwendung des Verfahrens ohne Verdünnung vorliegen, wird eine 1:10 Verdünnung verwendet.
Dieses Verfahren stützt sich auf den Nachweis von E. coli auf der Grundlage der Expression des Enzyms  D Glucuronidase und folglich werden viele der enterohämorrhagischen Stämme von E. coli nicht nachgewiesen, die dieses Enzym üblicherweise nicht exprimieren. Außerdem gib es eine geringe Anzahl anderer E. coli Stämme, die b D Glucuronidase nicht exprimieren.
Die Auswahl der zum Nachweis und zur Bestätigung der coliformen Gruppe von Bakterien, einschließlich E. coli, angewendeten Untersuchungen kann als Teil einer kontinuierlichen Abfolge angesehen werden. Der Umfang der Bestätigung hängt bei einer bestimmten Probe teils von der Beschaffenheit des Wassers und teils von den Gründen für die Untersuchung ab. Die in diesem Teil von ISO 9308 beschriebene Untersuchung liefert ein bestätigtes Ergebnis ohne dass eine weitere Bestätigung positiver Vertiefungen erforderlich ist.
ANMERKUNG   Während in dem Verfahren die Anwendung einer handelsüblichen Zählvorrichtung beschrieben wird, kann das hier beschriebene Medium in einem Standard-MPN Verfahren verwendet werden.

Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes - Partie 2: Méthode du nombre le plus probable (ISO 9308-2:2012)

L'ISO 9308-2:2012 spécifie une méthode de dénombrement des Escherichia coli et des bactéries coliformes dans l'eau. La méthode est basée sur la croissance d'organismes cibles dans un milieu liquide et sur le calcul du «nombre le plus probable» d'organismes en fonction de tables NPP. Cette méthode peut être appliquée à tous les types d'eau, y compris ceux contenant une quantité appréciable de matière en suspension et des nombres importants de bactéries hétérotrophes. Cependant, elle ne doit pas être utilisée pour dénombrer les bactéries coliformes dans l'eau de mer. Lorsqu'elle est utilisée pour dénombrer Escherichia coli dans l'eau de mer, une dilution 1→10 dans de l'eau stérile est généralement requise, bien que la méthode ait démontré son efficacité avec certaines eaux de mer ayant une concentration en sel inférieure à la normale. En l'absence de données étayant l'utilisation de la méthode sans dilution, une dilution 1→10 est utilisée.
Cette méthode repose sur la détection des Escherichia coli en fonction de l'expression de l'enzyme β-D-glucuronidase et, par conséquent, ne permet pas de détecter les souches entérohémorragiques des Escherichia coli, qui n'expriment généralement pas cette enzyme. De plus, il existe un petit nombre d'autres souches d'Escherichia coli qui n'expriment pas la β-D-glucuronidase.
Le choix des essais utilisés lors de la détection et de la confirmation du groupe de bactéries coliformes, notamment Escherichia coli, peut être considéré comme faisant partie intégrante d'une séquence continue. Le niveau de confirmation avec un échantillon particulier dépend d'une part de la nature de l'eau et d'autre part des raisons de l'examen. L'essai décrit dans l'ISO 9308-2:2012 fournit un résultat confirmé sans avoir besoin de confirmer les puits positifs.

Kakovost vode - Ugotavljanje števila Escherichia coli in koliformnih bakterij - 2. del: Metoda najverjetnejšega števila (ISO 9308-2:2012)

EN ISO 9308-2 določa metodo za ugotavljanje števila Escherichia coli in koliformnih bakterij v vodi. Ta metoda temelji na rasti ciljnih organizmov v tekočem mediju in izračunu „najverjetnejšega števila“ (MPN) organizmov z referenco na tabele MPN. To metodo je mogoče uporabiti za vse vrste vode, vključno s tistimi, ki vsebujejo znatno količino lebdečih snovi in visoko število heterotrofnih bakterij v ozadju. Vendar se te metode ne sme uporabljati za ugotavljanje števila koliformnih bakterij v morski vodi. Pri ugotavljanju števila Escherichia coli v morski vodi je običajno treba izvesti redčenje 1 10 v sterilni vodi, čeprav ta metoda dobro deluje tudi v morski vodi z nižjo vsebnostjo koncentracije soli, kot je običajna. Ker ni podatkov, ki bi podpirali uporabo te metode brez redčenja, se uporablja razredčina v razmerju 1: 10. Ta metoda upošteva zaznavanje Escherichia coli na podlagi izražanja encima β-D-glukuronidaza, zato posledično ne zazna številnih enterohemoragičnih sevov Eschericha coli, ki tega encima običajno ne izražajo. Poleg tega obstaja majhno število drugih sevov Escherichia coli, ki ne izražajo encima β-D-glukuronidaza. Izbiro preskusov, uporabljenih za zaznavanje in potrjevanje koliformne skupine bakterij, vključno z Escherichia coli, je mogoče upoštevati kot del nepretrganega zaporedja. Obseg potrditve z določenim vzorcem je delno odvisen od lastnosti vode in delno od vzrokov za preskus. Preskus, opisan v tem delu ISO 9308, podaja potrjen rezultat brez potrebe po nadaljnjem potrjevanju pozitivnih izvorov.

General Information

• Status: Published
• Publication Date: 15-Apr-2014
• Withdrawal Date: 30-Oct-2014
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 16-Apr-2014
• Due Date: 11-Oct-2014
• Completion Date: 16-Apr-2014
• Ref Project: SIST EN ISO 9308-2:2014 - Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method (ISO 9308-2:2012)

Standards Content (Sample)

SLOVENSKI STANDARD
01-julij-2014
1DGRPHãþD
SIST ISO 9308-2:1998
Kakovost vode - Ugotavljanje števila Escherichia coli in koliformnih bakterij - 2.
del: Metoda najverjetnejšega števila (ISO 9308-2:2012)
Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most
probable number method (ISO 9308-2:2012)
Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen Organismen - Teil
2: Verfahren zur Bestimmung der Anzahl mit der höchsten Wahrscheinlichkeit (ISO 9308
-2:2012)
Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes -
Partie 2: Méthode du nombre le plus probable (ISO 9308-2:2012)
Ta slovenski standard je istoveten z: EN ISO 9308-2:2014
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.01 Kakovost vode na splošno Water quality in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 9308-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2014
ICS 07.100.20
English Version
Water quality - Enumeration of Escherichia coli and coliform
bacteria - Part 2: Most probable number method (ISO 9308-
2:2012)
Qualité de l'eau - Dénombrement des Escherichia coli et Wasserbeschaffenheit - Zählung von Escherichia coli und
des bactéries coliformes - Partie 2: Méthode du nombre le coliformen Bakterien - Teil 2: Verfahren zur Bestimmung
plus probable (ISO 9308-2:2012) der wahrscheinlichsten Keimzahl (ISO 9308-2:2012)
This European Standard was approved by CEN on 11 April 2014.

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
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9308-2:2014 E
worldwide for CEN national Members.

Contents Page
Foreword .3
Foreword
The text of ISO 9308-2:2012 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 9308-2:2014 by
Technical Committee CEN/TC 230 “Water analysis” 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 October 2014, and conflicting national standards shall be withdrawn at
the latest by October 2014.
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 9308-2:2012 has been approved by CEN as EN ISO 9308-2:2014 without any modification.
INTERNATIONAL ISO
STANDARD 9308-2
Second edition
2012-07-01
Water quality — Enumeration of
Escherichia coli and coliform bacteria —
Part 2:
Most probable number method
Qualité de l'eau — Dénombrement des Escherichia coli et des
organismes coliformes —
Partie 2: Méthode du nombre le plus probable

Reference number
ISO 9308-2:2012(E)
©
ISO 2012
ISO 9308-2:2012(E)
©  ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 9308-2:2012(E)
Contents Page
Foreword . iv
Introduction . v
1  Scope . 1
2  Normative references . 1
3  Terms and definitions . 2
4  Principle . 2
5  Apparatus and glassware . 2
6  Culture media and reagents . 3
7  Sampling . 3
8  Procedure . 3
9  Expression of results . 4
10  Test report . 4
11  Quality assurance . 4
Annex A (informative) Further microbiological information on coliform bacteria . 5
5)
Annex B (normative) The Quanti-Tray Sealer and calculation of results . 6
Annex C (informative) Composition of the Colilert-18 medium . 42
8)
Annex D (informative) Validation of Colilert-18/Quanti-Tray for the enumeration of E.coli and
coliform bacteria from water . 44
Bibliography . 46

ISO 9308-2:2012(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
ISO 9308-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This second edition cancels and replaces the first edition (ISO 9308-2:1990), which has been technically
revised.
ISO 9308 consists of the following parts, under the general title Water quality — Enumeration of Escherichia
coli and coliform bacteria:
 Part 1: Membrane filtration method for waters with low bacterial background flora
 Part 2: Most probable number method
 Part 3: Miniaturized method (Most Probable Number) for the detection and enumeration of E. coli in
surface and waste water
iv © ISO 2012 – All rights reserved

ISO 9308-2:2012(E)
Introduction
The presence and extent of faecal pollution is an important factor in assessing the quality of a body of water
and the risk to human health from infection. Examination of water samples for the presence of Escherichia coli
(E. coli), which normally inhabits the bowel of man and other warm-blooded animals, provides an indication of
such pollution. Examination for coliform bacteria can be more difficult to interpret because some coliform
bacteria live in soil and surface fresh water and are not always intestinal. Therefore, the presence of coliform
bacteria, although not a proof of faecal contamination, may indicate a failure in treatment or ingress of water
into the distribution system.
The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that
compliance with this document may involve the use of patents concerning Colilert-18 and Quanti-Tray and
Quanti-Tray 2000 given in this document.
ISO takes no position concerning the evidence, validity and scope of these patent rights.
The holder of this patent right has assured the ISO that he/she is willing to negotiate licences either free of
charge or under reasonable and non-discriminatory terms and conditions with applicants throughout the world.
In this respect, the statement of the holder of this patent right is registered with ISO. Information may be
obtained from:
IDEXX Laboratories, Inc.
One IDEXX Drive
Westbrook, Maine 04092 USA
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent
rights.
ISO (http://www.iso.org/patents) and IEC (http://patents.iec.ch) maintain on-line databases of patents relevant
to their standards. Users are encouraged to consult the databases for the most up to date information
concerning patents.
INTERNATIONAL STANDARD ISO 9308-2:2012(E)

Water quality — Enumeration of Escherichia coli and coliform
bacteria —
Part 2:
Most probable number method
WARNING – Persons using this part of ISO 9308 should be familiar with normal laboratory practice.
This International Standard does not purport to address all of the safety problems, if any, associated
with its use. It is the responsibility of the user to establish appropriate safety and health practices and
to ensure compliance with any national regulatory conditions.
IMPORTANT – It is absolutely essential that tests conducted in accordance with this part of ISO 9308
be carried out by suitably qualified staff.
1 Scope
This part of ISO 9308 specifies a method for the enumeration of E. coli and coliform bacteria in water. The
method is based on the growth of target organisms in a liquid medium and calculation of the “Most Probable
Number” (MPN) of organisms by reference to MPN tables. This method can be applied to all types of water,
including those containing an appreciable amount of suspended matter and high background counts of
heterotrophic bacteria. However, it must not be used for the enumeration of coliform bacteria in marine water.
When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically
required, although the method has been shown to work well with some marine waters that have a lower than
normal concentration of salts. In the absence of data to support the use of the method without dilution, a
1→10 dilution is used.
This method relies upon the detection of E. coli based upon expression of th
...