EN ISO 9308-1:2014

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
• Withdrawal Date: 30-Mar-2015
• ICS: 07.100.20 - Microbiology of water
• ICS: 13.060.30 - Sewage 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: 24-Sep-2014
• Completion Date: 24-Sep-2014
• Ref Project: SIST EN ISO 9308-1:2014 - 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)

Relations

• Replaces: EN ISO 9308-1:2000 - Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method (ISO 9308-1:2000)
• Effective Date: 01-Oct-2014
• Replaces: EN ISO 9308-1:2000/AC:2008 - Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method (ISO 9308-1:2000/Cor 1:2007)
• Effective Date: 01-Oct-2014
• Amended By: EN ISO 9308-1:2014/A1:2017 - 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/Amd 1:2016)
• Effective Date: 11-Nov-2015

Overview

EN ISO 9308-1:2014 (CEN adoption of ISO 9308-1:2014) defines a standardized membrane filtration method for the enumeration of Escherichia coli (E. coli) and coliform bacteria in waters that have a low bacterial background. The procedure uses filtration onto a membrane, culture on a chromogenic coliform agar (CCA), and calculation of organism counts. It is intended for low-burden waters such as drinking water, disinfected pool water, and finished water from treatment plants.

Key topics and technical requirements

• Scope & suitability
  • Designed for waters that yield less than 100 total colonies on CCA (low bacterial background).
  • Not suitable for surface waters or shallow wells where background flora interfere with reliable counts.
• Principle
  • Filtration through a membrane filter that retains microorganisms, placement on CCA, incubation and colony differentiation.
• Incubation conditions
  • Incubate at (36 ± 2) °C for (21 ± 3) hours.
• Colony interpretation
  • β-D-galactosidase positive (pink to red) colonies = presumptive coliform bacteria (must be oxidase negative to confirm; this avoids false positives from oxidase-positive genera such as Aeromonas).
  • β-D-galactosidase and β-D-glucuronidase positive (dark-blue to violet) colonies = presumptive E. coli.
  • Note: Some E. coli strains (e.g., β-D-glucuronidase negative strains such as E. coli O157) will not appear as E. coli on CCA but will appear as coliforms.
• Apparatus & media
  • Typical equipment: autoclave-capable apparatus, incubator, pH meter, membrane filtration apparatus.
  • Membrane filters: cellulose ester or equivalent, nominal pore size 0.45 µm, typically 47 mm or 50 mm diameter.
• Quality and reporting
  • Includes guidance on expression of results, test reporting and quality assurance, and performance testing of CCA and oxidase test.

Applications and users

• Practical uses:
  • Routine drinking water monitoring, finished water quality control, pool water testing, and verification after water treatment/disinfection.
• Primary users:
  • Water testing laboratories, public health agencies, water utilities, environmental consultants, and regulatory compliance teams performing microbiological analysis of low-background waters.

Related standards

• EN/ISO 9308 series:
  • Part 2: Most probable number (MPN) method.
  • Part 3: Miniaturized MPN method for surface and waste waters.
• Normative references cited in EN ISO 9308-1:2014 include ISO 3696, ISO 7704, ISO 8199, ISO 11133, ISO 19458.

Keywords: EN ISO 9308-1:2014, membrane filtration, E. coli enumeration, chromogenic coliform agar, water quality testing, drinking water microbiology, coliform bacteria.

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2014
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SIST EN ISO 9308-1:2001
SIST EN ISO 9308-1:2001/AC:2009
.DNRYRVWYRGH8JRWDYOMDQMHãWHYLOD(VFKHULFKLDFROLLQNROLIRUPQLKEDNWHULM
GHO0HWRGDPHPEUDQVNHILOWUDFLMH]DYRGH]PDMKQLPãWHYLORPVSUHPOMDMRþLK
EDNWHULM,62
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
Foreword .3
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.
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
ISO 9308-1:2014(E)
© 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
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Tel. + 41 22 749 01 11
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Published in Switzerland
ii © ISO 2014 – All rights reserved

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 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

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.
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
...