oSIST prEN ISO 9308-2:2026

Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method (ISO/DIS 9308-2:2026)

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/DIS 9308-2:2026)

Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes - Partie 2: Méthode du nombre le plus probable (ISO/DIS 9308-2:2026)

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/DIS 9308-2:2026)

ISO 9308-2:2012 določa metodo za določanje števila E. coli in koliformnih bakterij v vodi. Metoda temelji na rasti ciljnih organizmov v tekočem gojišču in izračunu "najverjetnejšega števila" (MPN - Most Probable Number) organizmov z uporabo MPN tabel. Ta metoda je uporabna za vse vrste vode, vključno s tistimi, ki vsebujejo znatno količino suspendiranih snovi in visoko število heterotrofnih bakterij. Vendar pa se ne sme uporabljati za določanje števila koliformnih bakterij v morski vodi. Pri uporabi za določanje števila E. coli v morskih vodah je običajno potrebna 1→10 redčitev v sterilni vodi, čeprav je bilo dokazano, da metoda dobro deluje z nekaterimi morskimi vodami, ki imajo nižjo od običajne koncentracijo soli. V odsotnosti podatkov, ki bi podpirali uporabo metode brez redčenja, se uporablja 1→10 redčitev.
Ta metoda se opira na zaznavanje E. coli na podlagi izražanja encima b-D-glukuronidaza in zato ne zazna mnogih enterohemoragičnih sevov E. coli, ki tega encima običajno ne izražajo. Poleg tega obstaja majhno število drugih sevov E. coli, ki ne izražajo b-D-glukuronidaze.
Izbor testov, uporabljenih pri zaznavanju in potrjevanju skupine koliformnih bakterij, vključno z E. coli, se lahko obravnava kot del neprekinjenega zaporedja. Obseg potrditve pri določenem vzorcu je deloma odvisen od narave vode in deloma od razlogov za preiskavo. Test, opisan v ISO 9308-2:2012, zagotavlja potrjen rezultat brez potrebe po nadaljnji potrditvi pozitivnih vzorcev.

General Information

• Status: Not Published
• Public Enquiry End Date: 01-Jun-2026
• ICS: 07.100.20 - Microbiology of water
• ICS: 13.060.01 - Water quality in general
• Technical Committee: KAV - Water quality
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 08-Apr-2026
• Due Date: 26-Aug-2026
• Ref Project: prEN ISO 9308-2 - Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method (ISO/DIS 9308-2:2026)

Relations

• Revises: 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)
• Effective Date: 01-May-2026

Overview

oSIST prEN ISO 9308-2:2026:2026 (Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method) is a draft European standard developed by CEN, harmonizing with ISO. This standard describes a microbiological method for detecting and quantifying Escherichia coli (E. coli) and coliform bacteria in water using the Most Probable Number (MPN) method. The procedure involves incubating water samples with selective liquid media and determining bacterial counts by referencing MPN tables.

The method is suitable for a range of water types-including drinking water, groundwater, surface water, recycled, and wastewater-even when high background levels of heterotrophic bacteria or suspended solids are present. However, it is not intended for direct enumeration of coliforms in marine waters unless a 1:10 dilution with sterile water is applied. The standard emphasizes express laboratory assessment for public health, environmental monitoring, and compliance with regulatory requirements.

Key Topics

• Most Probable Number (MPN) Method: The process utilizes selective liquid media to culture target organisms (E. coli and coliforms). After incubation, positive wells or tubes are assessed for color change and fluorescence. Results are compared with MPN tables to estimate bacterial concentrations.
• Sample Types: Applicable to various water categories such as drinking water, surface water, and wastewater. Special instructions are provided for samples with high turbidity or salt content.
• Detection Principles: Relies on the presence of β-D-glucuronidase in E. coli and β-D-galactosidase in coliforms, using colorimetric and fluorometric responses for identification.
• Limitations: This method may not detect certain E. coli strains (including some enterohaemorrhagic types) that do not express β-D-glucuronidase.
• Quality Assurance: Laboratories must use validated media, maintain rigorous quality controls, and follow standard procedures for sample transport, incubation, and reporting.

Applications

• Drinking Water Monitoring: Ensures compliance with national and international water quality standards by quantifying coliforms and E. coli as indicators of faecal contamination.
• Environmental Surveillance: Assesses surface and recreational water for microbial contamination, supporting public health protection and regulatory oversight.
• Wastewater Testing: Provides quantifiable data for treated and untreated wastewater to evaluate treatment efficacy and prevent environmental pollution.
• Emergency Water Quality Assessments: Facilitates rapid microbial testing in disaster or outbreak situations where waterborne pathogens pose a risk.

Why Adopt the Standard?

• International Consistency: Aligns testing protocols across laboratories, supporting reliable results and data comparability.
• Robust for Varied Water Quality: Methods are validated for water with suspended matter and high background microbial counts.
• Regulatory Compliance: Provides methods widely referenced in legal and public health frameworks for water quality.

Related Standards

Organizations relying on oSIST prEN ISO 9308-2:2026:2026 may also reference:

• ISO 8199 - General requirements and guidance for microbiological examinations by culture.
• ISO 11133 - Preparation, production, storage, and performance testing of culture media.
• ISO 19458 - Guidelines for sampling for microbiological analysis.
• ISO 13843 - Performance characterization of microbiological methods.

By adopting this standard, water laboratories, utilities, and environmental agencies ensure their testing aligns with internationally recognized methods for E. coli and coliform enumeration, promoting safe water and public health protection.

Keywords: water quality, E. coli enumeration, coliform bacteria, MPN method, ISO 9308-2, microbial water testing, CEN standard, microbiology of water, regulatory compliance.

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2026
Kakovost vode - Ugotavljanje števila Escherichia coli in koliformnih bakterij - 2.
del: Metoda najverjetnejšega števila (ISO/DIS 9308-2:2026)
Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most
probable number method (ISO/DIS 9308-2:2026)
Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen Bakterien - Teil 2:
Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO/DIS 9308-2:2026)
Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes -
Partie 2: Méthode du nombre le plus probable (ISO/DIS 9308-2:2026)
Ta slovenski standard je istoveten z: prEN ISO 9308-2
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.

DRAFT
International
Standard
ISO/DIS 9308-2
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of Escherichia coli and coliform
Voting begins on:
bacteria —
2026-03-31
Part 2:
Voting terminates on:
2026-06-23
Most probable number method
Qualité de l'eau — Dénombrement des Escherichia coli et des
bactéries coliformes —
Partie 2: Méthode du nombre le plus probable
ICS: 07.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
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NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 9308-2:2026(en)
DRAFT
ISO/DIS 9308-2:2026(en)
International
Standard
ISO/DIS 9308-2
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of Escherichia coli and coliform
Voting begins on:
bacteria —
Part 2:
Voting terminates on:
Most probable number method
Qualité de l'eau — Dénombrement des Escherichia coli et des
bactéries coliformes —
Partie 2: Méthode du nombre le plus probable
ICS: 07.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2026
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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BE CONSIDERED IN THE LIGHT OF THEIR
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or ISO’s member body in the country of the requester.
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TO SUBMIT, WITH THEIR COMMENTS,
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Published in Switzerland Reference number
ISO/DIS 9308-2:2026(en)
ii
ISO/DIS 9308-2:2026(en)
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 medium and reagents . 3
6.1 General .3
6.2 Culture medium .3
6.3 Diluent .3
6.4 Antifoam Reagent .3
6.5 Colour/fluorescence control .3
7 Procedure . 3
7.1 Preparation of the sample .3
7.2 Inoculation of media .4
7.3 Incubation and differentiation .4
7.3.1 Examination of results .4
8 Expression of results . 4
9 Test report . 4
10 Quality assurance . 5
Annex A (informative) Further microbiological information on coliform bacteria . 6
Annex B (normative) The Tray Sealer and calculation of results. 7
Annex C (normative) Composition of the medium .11
Annex D (informative) Performance characteristics of the method for the detection of E .coli
and coliform bacteria from water generated in two laboratories .13
Bibliography . 14

iii
ISO/DIS 9308-2:2026(en)
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 of the voluntary nature of standards, 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 www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This third edition cancels and replaces the second edition (ISO 9308-2:2012), which has been technically
revised.
The main changes compared to the previous edition are as follows:
— Removal of specific trademarks and names of manufacturers to generic terms where appropriate.
— Change to quality control cultures
— In Clause 11, criteria for the performance testing of culture media have been added.
— Change to the definitions of “coliform bacterium” and E. coli
A list of all parts in the ISO 9308 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/DIS 9308-2:2026(en)
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 gastrointestinal tract of warm-blooded animals, including humans,
provides an indication of such pollution. Examination for coliform bacteria can be more difficult to interpret
because some strains of coliform bacteria live in soil and fresh surface water and are not always intestinal.
The presence of coliform bacteria, although not a proof of faecal contamination, may indicate a failure in
treatment, regrowth or ingress of water into the distribution system.

v
DRAFT International Standard ISO/DIS 9308-2:2026(en)
Water quality — Enumeration of Escherichia coli and coliform
bacteria —
Part 2:
Most probable number method
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 document 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 or by use of appropriate formulae 1]. This method
can be applied to a range of types of water (for example drinking water, groundwater, surface water,
[2-5]
recreational water and wastewater), including those containing a considerable amount of suspended
matter and high background counts of heterotrophic bacteria. Users should satisfy themselves that any
product used has been validated for its intended use (e.g. chlorinated drinking water) and should generate
[6]
performance characteristics using ISO13843. Performance characteristic data generated prior to 2017
[7]
using ISO TR 13843 is also acceptable. If a water sample has some background colour, the incubated
sample should be compared to a blank control of the same water sample
This method relies upon the detection of E. coli based upon expression of the enzyme β-D-glucuronidase and
consequently does not detect some enteropathogenic 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 β-D-glucuronidase.
As they are β-D-galactosidase positive, they will appear as coliform bacteria.
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 8199, Water quality — General requirements and guidance for microbiological examinations by culture
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purpose of this document, the following terms and definitions apply. It is recognised that other
definitions for “coliform bacterium” are in common use, specifically that these organisms are Gram negative
and do not produce cytochrome oxidase. The method described within the Standard includes specific

ISO/DIS 9308-2:2026(en)
inhibitors to prevent the growth of oxidase positive organisms such as Aeromonas spp. and Gram positive
bacteria that may also possess galactosidase.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org ./ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
coliform bacterium
organism that possesses β-D-galactosidase and cleaves ortho-Nitrophenyl-β-galactoside to produce a yellow
colour within 20 (±2) h
3.2
Escherichia coli
coliform bacterium that expresses both β-D-galactosidase and β-D-glucuronidase producing a yellow colour
and fluorescence within 20 (±2) h
4 Principle
Dehydrated medium which has been appropriately sterilised, for example by gamma irradiation, is added to
a sample of water (100 ml), or a dilution of a sample made up to 100 ml. Sample plus medium is gently shaken
to ensure adequate mixing and to facilitate dissolution of the medium. The sample plus medium is then
aseptically poured into an MPN-tray (e.g. Quanti-Tray). Alternatively, the solution (sample plus medium) can
[8]
be dispensed into sterile tubes to facilitate a multiple tube MPN sequence (e.g. 1 × 50 ml and 5 × 10 ml) .
After incubation, sample wells (or tubes) that have a yellow colour of equal or greater intensity to that of
the colour/fluorescence control are considered positive for coliform bacteria. Yellow wells that also exhibit
a degree of fluorescence greater or equal to that of the colour/fluorescence control are considered positive
for E. coli.
When using for the enumeration of E. coli in marine waters, a tenfold dilution in sterile water is typically
required because elevated levels of salts can slow the growth of the target bacteria.
By means of statistical tables, or a simple computer program, the most probable number (MPN) of coliform
bacteria and E. coli in 100 ml of the sample can be determined.
When using commercially available materials, all components (medium, trays, MPN tables and sealer) shall
be obtained from the same manufacturer unless cross-manufacturer comparability has been proven by a
validation study.
5 Apparatus and glassware
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 8199) and, in particular, the following:
5.1 Incubator, thermostatically controlled at (36 ± 2) °C
5.2 Tray sealer. See annex B if an MPN tray format is used.
5.3 Sterile wide mouthed vessels, of at least 110 ml capacity.
5.4 Sterile MPN trays if an MPN tray format is being used.
5.5 MPN trays containing the colour/fluorescence control, if an MPN tray format is being used.
5.6 Ultraviolet lamp, 365 + 20 nm.

ISO/DIS 9308-2:2026(en)
5.7 Sterile tubes, if a multiple tube MPN format is being used.
5.8 Tubes containing the colour/fluorescence control, if a tube format is being used
6 Culture medium and reagents
6.1 General
For the preparation of culture media and reagents, use ingredients of uniform quality and chemicals of
analytical grade (see ISO 8199 and ISO 11133). The use of chemicals of other grades is possible, providing
they are shown to be of equal performance in the test. Follow the instructions given in Annex C. It should be
noted that due to the extremely small quantities of some components of the medium, it may be difficult to
ensure an even distribution of the components and therefore the reliability of laboratory-prepared medium
may be questionable. Preferably, use commercially available media and reagents which comply with the
compositions given in Annex C and strictly follow the manufacturer's instructions.
The medium deteriorates with time, particularly in a humid atmosphere. Adherence to the manufacturer’s
expiry date is essential.
6.2 Culture medium
The method utilizes a medium based on defined substrate technology available for a 100 ml sample as a
ready to use powder. Each pack contains sufficient medium for a single test. Medium shall be stored under
ambient conditions (2 °C to 25 °C) out of direc
...