prEN ISO 9308-4

Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 4: Membrane filtration method for Escherichia coli in water with high levels of background bacteria (ISO/DIS 9308-4:2025)

This document describes a highly specific method for the enumeration of Escherichia coli (E. coli) in water. The method is based on membrane filtration, subsequent culture at 44 °C on a chromogenic agar medium containing a chromogenic ingredient for the detection of the enzyme ß-glucuronidase, and calculation of the number of target organisms in the sample.
Because of the high specificity of the method, this document is suitable for waters with high levels of background bacteria, such as surface waters including bathing water and wastewater. E. coli strains which do not grow at 44 °C and those that are β-glucuronidase negative, such as E. coli O157, will not be detected as E. coli by this method.

Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen Bakterien - Teil 4: Membranfiltrationsverfahren für Wässer mit hoher Begleitflora (ISO/DIS 9308‑4:2025)

Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes - Partie 4 : Méthode par filtration sur membrane pour Escherichia coli pour les eaux à teneurs élevées en bactéries (ISO/DIS 9308-4:2025)

[Not translated]

General Information

• Status: Not Published
• Publication Date: 14-Jun-2027
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 4020 - Submission to enquiry - Enquiry
• Start Date: 20-Nov-2025
• Due Date: 12-Jul-2024
• Completion Date: 20-Nov-2025
• Ref Project: oSIST prEN ISO 9308-4:2026 - Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 4: Membrane filtration method for Escherichia coli in water with high levels of background bacteria (ISO/DIS 9308-4:2025)

Standards Content (Sample)

SLOVENSKI STANDARD
01-januar-2026
[Not translated]
Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 4: Membrane
filtration method for Escherichia coli in water with high levels of background bacteria
(ISO/DIS 9308-4:2025)
Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen
Bakterien - Teil 4: Membranfiltrationsverfahren für Wässer mit hoher Begleitflora
(ISO/DIS 9308-4:2025)
Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes -
Partie 4 : Méthode par filtration sur membrane pour Escherichia coli pour les eaux à
teneurs élevées en bactéries (ISO/DIS 9308-4:2025)
Ta slovenski standard je istoveten z: prEN ISO 9308-4
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.

DRAFT
International
Standard
ISO/DIS 9308-4
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of Escherichia coli and coliform
Voting begins on:
bacteria —
2025-11-18
Part 4:
Voting terminates on:
2026-02-10
Membrane filtration method for
Escherichia coli in water with high
levels of background bacteria
Qualité de l'eau - Dénombrement des Escherichia coli et des
bactéries coliformes —
Partie 4: Méthode par filtration sur membrane pour Escherichia
coli pour les eaux à teneurs élevées en bactéries
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
WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED
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NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 9308-4:2025(en)
DRAFT
ISO/DIS 9308-4:2025(en)
International
Standard
ISO/DIS 9308-4
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of Escherichia coli and coliform
Voting begins on:
bacteria —
Part 4:
Voting terminates on:
Membrane filtration method for
Escherichia coli in water with high
levels of background bacteria
Qualité de l'eau - Dénombrement des Escherichia coli et des
bactéries coliformes —
Partie 4: Méthode par filtration sur membrane pour Escherichia
coli pour les eaux à teneurs élevées en bactéries
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 2025
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
ISO/CEN PARALLEL PROCESSING
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BE CONSIDERED IN THE LIGHT OF THEIR
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
CH-1214 Vernier, Geneva
NOTIFICATION OF ANY RELEVANT PATENT
Phone: [phone]
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 9308-4:2025(en)
ii
ISO/DIS 9308-4:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Culture media and reagents . 2
6 Equipment and consumables . 2
7 Sampling, transportation and storage of samples . 3
8 Procedure . 3
8.1 General .3
8.2 Preparation of the sample .4
8.3 Membrane filtration.4
8.4 Incubation .4
8.4.1 Procedure A .4
8.4.2 Procedure B .4
8.5 Counting of colonies .4
9 Expression of results . 4
10 Performance characteristics . 5
11 Test report . 5
12 Quality assurance . 6
Annex A (informative) Flow diagram of the main steps of the procedures . 7
Annex B (normative) Culture media and reagent . 8
Annex C (informative) Performance characteristics of the method .12
Bibliography . 14

iii
ISO/DIS 9308-4:2025(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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.
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 https://www.iso.org/members.html.

iv
ISO/DIS 9308-4:2025(en)
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.
EXAMPLE 1 In European Union Member States the monitoring of levels of E. coli is mandatory for official bathing
waters, as indicated by the Directive 2006/7/EC of the European Parliament and of the Council of 15 February 2006
[9]
concerning the management of bathing water quality .
EXAMPLE 2 According to the United States Environmental Protection Agency, states may require the measurement
of E. coli to determine whether water quality criteria have been met for fresh water that is designated for primary
[10]
contact recreation, including swimming and bathing .
As surface waters often contain other bacteria than E. coli, either of environmental origin or through
contamination with faeces from animal or human origin, a method with high specificity is essential for the
enumeration of E. coli in surface water. The aim of this method is to detect thermotolerant E. coli as these are
most representative indicators for recent faecal contamination.

v
DRAFT International Standard ISO/DIS 9308-4:2025(en)
Water quality — Enumeration of Escherichia coli and coliform
bacteria —
Part 4:
Membrane filtration method for Escherichia coli in water with
high levels of background bacteria
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
microbiological analysis are only undertaken in properly equipped laboratories, under the control
of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials.
Persons using this document should be familiar with good laboratory practice. This document does
not purport to address all of the safety aspects, if any, associated with its use. It is the responsibility
of the user to establish appropriate safety and health practices.
1 Scope
This document describes a highly specific method for the enumeration of Escherichia coli (E. coli) in water.
The method is based on membrane filtration followed by culture at 44 °C on chromogenic agar medium
containing an ingredient for the detection of the enzyme β-glucuronidase, and calculation of the number of
[5,6,13]
target organisms in the sample .
Because of the high specificity of the method, this document is suitable for waters with high levels of
background bacteria, such as surface waters including bathing water and wastewater provided that they
contain little particulate or colloidal matter relative to the E. coli concentration. The method can be used for
fresh water and, with an adaptation, for marine water.
E. coli strains which do not grow at 44 °C and those that are β-glucuronidase negative, such as E. coli O157,
will not be detected as E. coli by this method.
This method is not applicable to the enumeration and detection of coliform bacteria other than E. coli.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 7704, Water quality — Requirements for the performance testing of membrane filters used for direct
enumeration of microorganisms by culture methods
ISO 8199:2018, 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 purposes of this document, the following terms and definitions apply.

ISO/DIS 9308-4:2025(en)
ISO and IEC maintain terminology 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
Escherichia coli
E. coli
member of the Enterobacteriaceae that expresses β-D-glucuronidase and is capable of growing at 44 °C in
the presence of bile salts
4 Principle
The detection and enumeration of E. coli consists of the following steps:
— Filtration of test portion(s) of the sample and/or dilutions through a membrane filter, which retains the
organisms.
— Depending on the type of sample, two different procedures for the detection and enumeration of E. coli
are used:
— Procedure A: detection and enumeration of E. coli in fresh water by placement of the membrane filter
on tryptone bile-X-glucuronide (TBX) agar, placing the TBX agar at 36 °C for 4 h to 5 h to resuscitate
stressed cells, followed by incubation at 44 °C for 21 h.
— Procedure B: detection and enumeration of E. coli in marine water and brackish water by placement
of the membrane filter on minerals-modified glutamate agar (MMGA), placing the MMGA at 36 °C for
4 h to 5 h to resuscitate stressed cells, followed by incubation on TBX at 44 °C for 21 h.
— Counting of β-D-glucuronidase positive colonies (blue or blue green) as E. coli.
— Calculation of the colony forming units (cfu) per millilitre (ml) of the sample from the number of colonies
counted.
The flow diagram of the main steps of the procedures is given in Annex A.
NOTE 1 Recovery of E. coli from marine water appears to be suboptimal on TBX while limited data is available for
brackish water (see https:// standards .iso .org/ iso/ 9308/ -4/ ed -1/ en). Pre-incubation on MMGA enhances resuscitation
[11,12]
of damaged cells and optimizes recovery .
NOTE 2 The colour of β-D-glucuronidase positive colonies can vary on TBX agar from different manufacturers (see
https:// standards .iso .org/ iso/ 9308/ -4/ ed -1/ en): follow the manufacturer’s instructions for the specific colour of β-D-
glucuronidase positive colonies.
5 Culture media and reagents
Follow good laboratory practices in accordance with ISO 8199. Use peptone saline solution for dilutions or,
alternatively, one of the other diluents presented in ISO 8199.
The composition of the culture media (TBX and MMGA) and their preparation are specified in Annex B. For
performance testing of the culture media, follow the procedures in accordance with Annex B and ISO 11133.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable items if these have the suitable specifications.
The usual microbiological laboratory equipment (see ISO 8199) and, in particular, the following shall be used.

ISO/DIS 9308-4:2025(en)
6.1 Apparatus for sterilization by steam (autoclave) capable of operating at (121 ± 3) °C, as specified in
ISO 8199. In case the procedure for pre-incubation on MMGA (Procedure B) is used, the autoclave shall also
be capable of operating at (115 ± 3) °C.
6.2 Disinfected or sterilized forceps, for handling of membrane filters.
6.3 Equipment for membrane filtration, with funnels that can be disinfected or sterilized.
6.4 Incubators, one capable of operating at (36 ± 2) °C and one capable of operating at (44 ± 0,5) °C or,
alternatively a cycling temperature incubator programmed to switch from (36 ± 2) °C to (44 ± 0,5) °C.
6.5 Membrane filters, composed of cellulose esters or other suitable material, usually 47 mm or 50 mm
in diameter, with filtration characteristics equivalent to a rated nominal pore diameter of 0,45 μm and,
preferentially, with grid lines.
6.6 pH meter, with an accuracy of ±0,1 at 20 °C to 25 °C.
6.7 Refrigerator (e.g. for storage of samples or media), capable of operating at (5 ± 3) °C.
6.8 Sterile Petri dishes with vents, with a diameter of an appropriate diameter.
6.9 Sterile pipettes and micropipettes with sterile tips.
6.10 Water bath (optional), capable of operating at 47 °C to 50 °C (if culture medium is prepared from
dehydrated media).
7 Sampling, transportation and storage of samples
Take the samples and deliver them to the laboratory in accordance with ISO 19458.
Keep the time between sampling and analysis as short as possible in accordance with ISO 19458, and
maximally for 24 h. Samples can be kept at 5 °C (6.7) up to 24 h prior to analysis.
NOTE The maximum sample storage time of 24 h is above the acceptable maximum sample storage time for E.
coli as given in ISO 19458 (which is 18 h). The deviation from ISO 19458 is based on experimental data (see https://
standards .iso .org/ iso/ 9308/ -4/ ed -1/ en).
8 Procedure
8.1 General
Depending on the type of sample, two different procedures for the detection and enumeration of E. coli are used:
— Procedure A: detection and enumeration of E. coli in fresh water.
— Procedure B: detection and enumeration of E. coli in marine water and brackish water.
For brackish water with salinity of maximal 6 ppt, data indicated an equally efficient recovery on TBX
as fresh water. The amount of data on brackish water is very limited however, and the level of salinity at
which recovery becomes significantly inhibited is unknown. For brackish water it is, therefore, required
to use Procedure B or test the relative recovery with Procedure A versus Procedure B (following the
method described in ISO 17994) to determine the suitability of Procedure A for brackish water prior to
implementation of the method.
ISO/DIS 9308-4:2025(en)
8.2 Preparation of the sample
For preparation and diluting of the sample (6.9), follow the instructions given in ISO 8199:2018, 6.3 and 8.2.1.
8.3 Membrane filtration
Filter appropriate test portions using membrane filtration in accordance with the instructions in
ISO 8199:2018, 9.1.4. Appropriate test portions are volumes or dilutions where growth of at least 10 specific
colonies and a maximum of 150 target colonies are expected. If the content of the water is unknown, a range
of four-fold dilutions is recommended.
NOTE 1 The test-volumes or levels of dilution depend on the presumed level of faecal contamination of the water to
2 5
be tested: “as a g
...