Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method for waters with low bacterial background flora (ISO 9308-1:2014)

ISO 9308-1:2014 specifies a method for the enumeration of Escherichia coli (E. coli) and coliform bacteria. The method is based on membrane filtration, subsequent culture on a chromogenic coliform agar medium, and calculation of the number of target organisms in the sample. Due to the low selectivity of the differential agar medium, background growth can interfere with the reliable enumeration of E. coli and coliform bacteria, for example, in surface waters or shallow well waters. This method is not suitable for these types of water.
ISO 9308-1:2014 is especially suitable for waters with low bacterial numbers that will cause less than 100 total colonies on chromogenic coliform agar (CCA). These may be drinking water, disinfected pool water, or finished water from drinking water treatment plants.
Some strains of E. coli which are β-D-glucuronidase negative, such as Escherichia coli O157, will not be detected as E. coli. As they are β-D-galactosidase positive, they will appear as coliform bacteria on this chromogenic agar.

Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen Bakterien - Teil 1: Membranfiltrationsverfahren für Wässer mit niedriger Begleitflora (ISO 9308-1:2014)

Dieser Teil von ISO 9308 beschreibt ein Verfahren für die Zählung von Escherichia coli (E. coli) und coliformen Bakterien. Das Verfahren beruht auf einer Membranfiltration, einer anschließenden Kultivierung auf einem chromogenen Nähragar und der Berechnung der Anzahl der Zielorganismen in der Probe. Wegen der geringen Selektivität des Differenzierungsagars kann die Begleitflora eine verlässliche Zählung von E. coli und coliformen Bakterien stören, z. B. in Oberflächenwässern oder oberflächennahen Brunnen. Dieses Verfahren ist für solche Wasserarten nicht geeignet.
Dieser Teil von ISO 9308 eignet sich besonders für die Untersuchung von Wässern mit niedrigen Bakterienzahlen, bei denen insgesamt weniger als 100 Kolonien auf dem Chromogenen Coliformen Agar wachsen. Dies können Trinkwässer, desinfizierte Schwimmbeckenwässer oder fertig aufbereitete Wässer aus Wasseraufbereitungsanlagen sein.
Einige Escherichia coli-Stämme, die β-Glucuronidase-negativ sind, wie z. B. Escherichia coli O157, werden nicht als E. coli bestimmt. Da die β-Galaktosidase-positiv sind, erscheinen sie als coliforme Bakterien.

Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes - Partie 1: Méthode par filtration sur membrane pour les eaux à faible teneur en bactéries (ISO 9308-1:2014)

L'ISO 9308 :2014 spécifie une méthode de dénombrement des Escherichia coli (E. coli) et des bactéries coliformes. La méthode consiste en une filtration sur membrane, suivie d'une mise en culture dans une gélose chromogène et d'un calcul du nombre des organismes cibles présents dans l'échantillon. La gélose de différenciation étant peu sélective, la flore bactérienne générale peut perturber la fiabilité du dénombrement des E. coli et des bactéries coliformes, par exemple, dans certaines eaux de surface ou certaines eaux de puits peu profonds. Cette méthode ne convient pas à ces types d'eau.
L'ISO 9308 :2014 est particulièrement adaptée aux eaux à faible teneur en bactéries qui produiront moins de 100 colonies totales sur de la gélose chromogène pour le dénombrement des bactéries coliformes. Ces eaux peuvent être l'eau potable, l'eau de piscine désinfectée ou l'eau propre sortant de stations de production d'eau potable.
Certaines souches d'E. coli négatives à la β-D-glucuronidase, telles qu'Escherichia coli O157, ne sont pas détectées en tant qu'E. coli. Comme elles sont positives à la β-D-galactosidase, elles sont considérées comme des bactéries coliformes sur la gélose chromogène pour bactéries coliformes (CCA).

Kakovost vode - Ugotavljanje števila Escherichia coli in koliformnih bakterij - 1. del: Metoda membranske filtracije za vode z majhnim številom spremljajočih bakterij (ISO 9308-1:2014)

Ta del ISO 9308 določa metodo za ugotavljanje števila Escherichia coli (E. coli) in koliformnih bakterij. Metoda temelji na membranski filtraciji, posledični kulturi na kromogenem koliformnem agarskem gojišču in izračunu števila ciljnih organizmov v vzorcu. Zaradi nizke selektivnosti diferencialnega agarskega gojišča je mogoče, da rast v ozadju vpliva na zanesljivo ugotavljanje števila E. coli in koliformnih bakterij, na primer v površinskih vodah ali plitvih vodah v vodnjakih. Ta metoda ni primerna za te vrste vod.
Ta del standarda ISO 9308 je še posebej primeren za vode z nizkim številom bakterij, ki bi povzročile manj kot 100 kolonij skupaj na kromogenem koliformnem agarju (CAA). Te so morda pitna voda, dezinficirana voda v bazenu ali končna voda v čistilnih napravah za pitno vodo.
Nekateri sevi E. coli, ki so negativni na β-D-glukuronidazo, kot so Escherichia coli O157, ne bodo
zaznani kot E. coli. Ker so pozitivni na β-D-glukuronidazo, se bodo na tem kromogenem agarju pojavili kor koliformne bakterije.

General Information

Status
Published
Publication Date
23-Sep-2014
Technical Committee
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
24-Sep-2014
Completion Date
24-Sep-2014

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SLOVENSKI STANDARD
SIST EN ISO 9308-1:2014
01-december-2014
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SIST EN ISO 9308-1:2001
SIST EN ISO 9308-1:2001/AC:2009

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Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane

filtration method for waters with low bacterial background flora (ISO 9308-1:2014)

Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen - Teil 1:

Membranfiltrationsverfahren für Wässer mit niedriger Begleitflora (ISO 9308-1:2014)

Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes -

Partie 1: Méthode par filtration sur membrane dans des eaux contenant peu de flore

bactérienne de fond (ISO 9308-1:2014)
Ta slovenski standard je istoveten z: EN ISO 9308-1:2014
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 9308-1:2014 en

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

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SIST EN ISO 9308-1:2014
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SIST EN ISO 9308-1:2014
EUROPEAN STANDARD
EN ISO 9308-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2014
ICS 07.100.20 Supersedes EN ISO 9308-1:2000
English Version
Water quality - Enumeration of Escherichia coli and coliform
bacteria - Part 1: Membrane filtration method for waters with low
bacterial background flora (ISO 9308-1:2014)

Qualité de l'eau - Dénombrement des Escherichia coli et Wasserbeschaffenheit - Zählung von Escherichia coli und

des bactéries coliformes - Partie 1: Méthode par filtration coliformen Bakterien - Teil 1: Membranfiltrationsverfahren

sur membrane pour les eaux à faible teneur en bactéries für Wässer mit niedriger Begleitflora (ISO 9308-1:2014)

(ISO 9308-1:2014)
This European Standard was approved by CEN on 24 August 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 9308-1:2014 E

worldwide for CEN national Members.
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SIST EN ISO 9308-1:2014
EN ISO 9308-1:2014 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST EN ISO 9308-1:2014
EN ISO 9308-1:2014 (E)
Foreword

This document (EN ISO 9308-1:2014) has been prepared by Technical Committee ISO/TC 147 "Water

quality" in collaboration with 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 March 2015, and conflicting national standards shall be withdrawn at

the latest by March 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.

This document supersedes EN ISO 9308-1:2000.

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 9308-1:2014 has been approved by CEN as EN ISO 9308-1:2014 without any modification.

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SIST EN ISO 9308-1:2014
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SIST EN ISO 9308-1:2014
INTERNATIONAL ISO
STANDARD 9308-1
Third edition
2014-09-15
Water quality — Enumeration
of Escherichia coli and coliform
bacteria —
Part 1:
Membrane filtration method for
waters with low bacterial background
flora
Qualité de l’eau — Dénombrement des Escherichia coli et des
bactéries coliformes —
Partie 1: Méthode par filtration sur membrane pour les eaux à faible
teneur en bactéries
Reference number
ISO 9308-1:2014(E)
ISO 2014
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SIST EN ISO 9308-1:2014
ISO 9308-1:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014

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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
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SIST EN ISO 9308-1:2014
ISO 9308-1:2014(E)
Contents Page

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

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

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

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

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

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

5 Apparatus and glassware ............................................................................................................................................................................ 2

6 Culture media and reagents ...................................................................................................................................................................... 2

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

8 Procedure..................................................................................................................................................................................................................... 3

8.1 Preparation of the sample ............................................................................................................................................................. 3

8.2 Filtration ....................................................................................................................................................................................................... 3

8.3 Incubation and differentiation .................................................................................................................................................. 3

9 Expression of results ........................................................................................................................................................................................ 4

10 Test report ................................................................................................................................................................................................................... 4

11 Quality assurance ................................................................................................................................................................................................ 4

11.1 General ........................................................................................................................................................................................................... 4

11.2 Performance testing of Chromogenic Coliform Agar (CCA) ............................................................................. 4

11.3 Performance testing of oxidase test ...................................................................................................................................... 5

Annex A (informative) Further microbiological information on coliform bacteria ............................................6

Annex B (normative) Composition and preparation of culture media and reagents ........................................7

Annex C (informative) Performance characteristics ............................................................................................................................ 9

Bibliography .............................................................................................................................................................................................................................10

© ISO 2014 – All rights reserved iii
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SIST EN ISO 9308-1:2014
ISO 9308-1:2014(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 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This third edition cancels and replaces the second edition (ISO 9308-1:2000), which has been technically

revised.
It also incorporates the Corrigendum ISO 9308-1:2000/Cor.1:2007.

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 2014 – All rights reserved
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SIST EN ISO 9308-1:2014
ISO 9308-1:2014(E)
Introduction

The presence and extent of faecal pollution is an important factor in assessing the quality 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 failure in

treatment, storage, or distribution.
© ISO 2014 – All rights reserved v
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SIST EN ISO 9308-1:2014
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SIST EN ISO 9308-1:2014
INTERNATIONAL STANDARD ISO 9308-1:2014(E)
Water quality — Enumeration of Escherichia coli and
coliform bacteria —
Part 1:
Membrane filtration method for waters with low bacterial
background flora

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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 document

be carried out by suitably qualified staff.
1 Scope

This part of ISO 9308 specifies a method for the enumeration of Escherichia coli (E. coli) and coliform

bacteria. The method is based on membrane filtration, subsequent culture on a chromogenic coliform

agar medium, and calculation of the number of target organisms in the sample. Due to the low selectivity

of the differential agar medium, background growth can interfere with the reliable enumeration of

E. coli and coliform bacteria, for example, in surface waters or shallow well waters. This method is not

suitable for these types of water.

This part of ISO 9308 is especially suitable for waters with low bacterial numbers that will cause less

than 100 total colonies on chromogenic coliform agar (CCA). These may be drinking water, disinfected

pool water, or finished water from drinking water treatment plants.

Some strains of E. coli which are β-D-glucuronidase negative, such as Escherichia coli O157, will not be

detected as E. coli. As they are β-D-galactosidase positive, they will appear as coliform bacteria on this

chromogenic agar.
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 3696, Water for analytical laboratory use — Specification and test methods

ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses

ISO 8199, Water quality — General guidance on the enumer
...

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