Iso 7704 2023

INTERNATIONAL ISO
STANDARD 7704
Second edition
2023-01
Water quality — Requirements for

the performance testing of membrane
filters used for direct enumeration of
microorganisms by culture methods
Qualité de l'eau — Exigences relatives aux essais de performance
des membranes filtrantes utilisées pour le dénombrement direct des
iTeh STANDARD PREVIEW
micro-organismes par des méthodes de culture
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ISO 7704:2023
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7704-2023
Reference number
ISO 7704:2023(E)
© ISO 2023
ISO 7704:2023(E)

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ISO 7704:2023
7704-2023
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ISO 7704:2023(E)
Contents Page
Foreword...........................................................................................................................................................................................................................................v
Introduction..............................................................................................................................................................................................................................vii
1 Scope.................................................................................................................................................................................................................................. 1
2 Normative references...................................................................................................................................................................................... 1
3 Terms and definitions..................................................................................................................................................................................... 1
3.1 General terminology........................................................................................................................................................................... 2
3.2 Terminology of performance testing................................................................................................................................... 3
3.3 Terminology for test microorganisms................................................................................................................................ 4
4 Principle......................................................................................................................................................................................................................... 5
4.1 General............................................................................................................................................................................................................ 5
4.1.1 Introduction............................................................................................................................................................................ 5
4.1.2 Batch testing........................................................................................................................................................................... 6
4.1.3 Supplementary testing................................................................................................................................................... 6
4.2 Performance testing........................................................................................................................................................................... 7
4.2.1 Modules for batch and supplementary testing......................................................................................... 7
4.2.2 Absence of microbial contamination................................................................................................................. 7
5 Apparatus and glassware............................................................................................................................................................................ 8
6 Culture media and diluents....................................................................................................................................................................... 8
7 Preparation of microorganisms for performance testing........................................................................................ 8
7.1 General............................................................................................................................................................................................................ 8
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7.2 Reference count...................................................................................................................................................................................... 8
7.2.1 Quantitative productivity testing........................................................................................................................ 8
7.2.2 Qualitative selectivity testing................................................................................................................................. 9
7.2.3 Qualitative specificity ISOtesting 7704:2023 ................................................................................................................................. 9
7.3 Preparation of a standardized test suspension using a working culture........................................... 9
7704-2023
7.3.1 General......................................................................................................................................................................................... 9
7.3.2 Preparation of the working culture................................................................................................................... 9
7.3.3 Preparation of a standardized test suspension (inoculum) for the test......................... 10
7.4 Preparation of a test suspension using reference material.......................................................................... 10
8 Sampling of membrane filters for testing............................................................................................................................... 10
9 Procedure.................................................................................................................................................................................................................. 11
9.1 General......................................................................................................................................................................................................... 11
9.2 Inoculation by spread plate technique............................................................................................................................ 11
9.2.1 General...................................................................................................................................................................................... 11
9.2.2 Inoculation............................................................................................................................................................................. 12
9.3 Inoculation by membrane filtration technique....................................................................................................... 12
9.3.1 General...................................................................................................................................................................................... 12
9.3.2 Inoculation............................................................................................................................................................................. 12
9.4 Incubation and counting.............................................................................................................................................................. 13
9.5 Test for absence of microbial contamination............................................................................................................ 13
10 Calculation, expression and interpretation of results.............................................................................................. 13
10.1 General......................................................................................................................................................................................................... 13
10.2 Productivity testing......................................................................................................................................................................... 13
10.3 Selectivity testing.............................................................................................................................................................................. 14
10.4 Specificity testing.............................................................................................................................................................................. 14
11 Documentation of test results............................................................................................................................................................. 14
11.1 Test report................................................................................................................................................................................................ 14
11.2 Information provided by the manufacturer............................................................................................................... 15
11.3 Traceability.............................................................................................................................................................................................. 15
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ISO 7704:2023(E)
Annex A (normative) Diagram of the procedure for batch testing.................................................................................... 16

Annex B (informative) Example of a card for recording test results from batch testing.......................... 17
Annex C (informative) Quantitative supplementary testing of membrane filters........................................... 18
Annex D (informative) Qualitative supplementary testing of membrane filters.............................................. 21
Annex E (informative) Practical example of quantitative batch testing and quantitative
supplementary testing by the end user..................................................................................................................................... 25
Bibliography.............................................................................................................................................................................................................................. 35

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ISO 7704:2023
7704-2023
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ISO 7704:2023(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 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
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 230, Water ISOanalysis,
7704:2023
in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
7704-2023
This second edition cancels and replaces the first edition (ISO 7704:1985), which has been technically
revised.
The main changes are as follows:
— the scope has been changed to cover the requirements for the performance testing of membrane
filters used for retention and direct enumeration;
— clauses have been added for terms and definitions, microorganisms, sampling and replicates,
procedure, inoculation and incubation, counting, calculation and documentation;
— the clauses referencing to culture media and diluents, test strain preparation, performance testing
and procedure have been revised to align with ISO 8199 and ISO 11133;
— Annex A has been added with a diagram of the batch testing;
— Annex B has been added to give an example of a card to record the test results from batch testing
and supplementary testing of membrane filters;
— Annex C has been added to describe the quantitative additional testing of membrane filters including
a diagram of the procedure;
— Annex D has been added to describe the qualitative supplementary testing of membrane filters;
— Annex E has been added to give a practical example of batch testing and quantitative additional
testing by the end user including a diagram of the procedure;
— the Bibliography has been added.
© ISO 2023 – All rights reserved v

ISO 7704:2023(E)
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.

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ISO 7704:2023
7704-2023
vi © ISO 2023 – All rights reserved

ISO 7704:2023(E)
Introduction
In laboratories carrying out microbiological examinations, the main objectives are to either capture,
resuscitate, grow, detect or enumerate, or all, a wide variety of microorganisms. Membrane filters are
used in many traditional microbiological culture techniques and are commercially available in various
brands and types. Many comparison studies of membrane filters which have been reported in the
literature show differences in their ability to recover bacteria from water samples, see References [22],
[23], [28], [30], [31], [32], [33] and [34]. The complex manufacturing process means that the chemical
composition, pore size and pore structure can vary, depending on the brands, and even on the lot of
material. Furthermore, the manufacturing process can also release leachables that can potentially
interfere with the recovery of microorganisms.
Thus, it is very important to standardize the performance testing of membrane filters as much as
possible, not only to provide consistent results, but also to enable the development of standardized
procedures for enumerating specific microorganisms.

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ISO 7704:2023
7704-2023
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ISO 7704:2023
7704-2023
INTERNATIONAL STANDARD ISO 7704:2023(E)
Water quality — Requirements for the performance

testing of membrane filters used for direct enumeration of
microorganisms by culture methods
1 Scope
This document specifies the requirements for the performance testing of membrane filters used for the
retention followed by direct enumeration of microorganisms by culture methods.
This document is applicable to membrane filters which are used for retention followed by direct
enumeration of specific microorganisms on solid media or on other devices containing media, like
absorbent pads[19].
This document is not applicable for membrane filters used for concentration and elution or for
qualitative methods.
These tests are applicable to the membrane filters intended for the microbiological analysis of different
types of water, such as:
— drinking water, bottled water and other types of water with expected low numbers of
microorganisms;
— (standards.iteh.ai)
water with expected higher
water.
numbers of microorganisms, for example, surface water and process
These tests are intended to demonstrateISO the 7704:2023

suitability of the whole system (membrane filter together
with the culture medium including the filtration step) required for the specific tests described in
References [3], [6], [8], [10], [12] and [13].
7704-2023
This document applies to:
— manufacturers producing membrane filters;
— microbiological laboratories using membrane filters for their own testing or providing these to
other end users.
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 8199:2018, Water quality — General requirements and guidance for microbiological examinations by
culture
ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 11133:2014/Amd1:2018, Microbiology of food, animal feed and water — Preparation, production,
storage and performance testing of culture media
3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
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ISO 7704:2023(E)
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://w ww.iso.org/obp
— IEC Electropedia: available at https://w ww.electropedia.org/
3.1 General terminology

3.1.1
membrane filter
porous hydrophilic filtration matrix, composed of different polymers with filtration characteristics
equivalent to its/their rated nominal pore sizes, typically ranging from 0,1 µm to 1,2 µm, which is
intended to be used for the retention of microorganisms
Note 1 to entry: The membrane filter ensures the effective retention of microorganisms depending on the
membrane pore size when a differential (positive or negative) pressure is applied.
Note 2 to entry: The type of membrane filter to be used for a certain microbiological method is described in the
corresponding specific standard (3.1.7).
3.1.2
culture medium
formulation of substances, in liquid, semi-solid or solid form, which contain either natural and/
or synthetic constituents intended to support the multiplication (with or without constituents for
inhibition of certain microorganisms), identification or preservation of viability of microorganisms
Note 1 to entry: The culture medium to be used for a certain microbiological method is described in the
corresponding specific standard (3.1.7), see specific culture medium (3.1.3).
(standards.iteh.ai)
[SOURCE: ISO 11133:2014, 3.3.1, modified — “constituents for” has been included and Note 1 to entry
has been replaced.]
ISO 7704:2023
3.1.3 https://standards.iteh.ai/catalog/standards/sist/93de3bf6-2ee0-40fd-824a-882ad8da410a/iso-
specific culture medium
7704-2023
culture medium (3.1.2), usually selective, as designated in a specific standard (3.1.7) for use with
membrane filters (3.1.1)
EXAMPLE 1 Chromogenic coliform agar (CCA) used with membrane filters in accordance with ISO 9308-1[8].
EXAMPLE 2 Slanetz and Bartley medium used with membrane filters in accordance with ISO 7899-2[6].
3.1.4
reference medium
culture medium (3.1.2), usually non-selective, for determination of the reference count (3.3.8) and
supplementary quantitative testing (3.2.2)
Note 1 to entry: The reference medium is usually a non-selective culture medium (3.1.2), which is different to the
culture medium under test and has been demonstrated to be suitable for use in the performance testing.
EXAMPLE Tryptone soya agar (TSA) is in accordance with ISO 11133.
[SOURCE: ISO 11133:2014, 3.3.4.13, modified — Note 1 to entry has been added and "is in accordance
with ISO 11133" has been added in the example.]
3.1.5
batch of membrane filter
lot of membrane filter
homogeneous and fully traceable units of membrane filter (3.1.1) referring to a defined amount of bulk,
semi-finished product or end product, which is consistent in type and quality, and which has been
produced within one defined production period, having been assigned the same batch (or lot) number
2 © ISO 2023 – All rights reserved

ISO 7704:2023(E)
3.1.6
batch of culture medium
lot of culture medium
homogeneous and fully traceable units of a culture medium (3.1.2) referring to a defined amount of
bulk, semi-finished product or end product, which is consistent in type and quality, and which has been
produced within one defined production period, having been assigned the same batch (or lot) number
Note 1 to entry: If the user does not define the batch or lot of culture medium used for performance testing of
membrane filters as the “end product”, it is important to demonstrate that the production process is sufficiently
controlled to produce culture media of consistent quality (e.g. by monitoring culture media performance testing
results).
[SOURCE: ISO 11133:2014, 3.1.2, modified — "medium" has been changed to "culture medium" and
Note 1 to entry has been added.]
3.1.7
specific standard
International Standard or guidance document describing the microbiological analysis of different types
of water for the detection or enumeration of a specific microorganism (or group of microorganisms)
3.2 Terminology of performance testing

3.2.1
batch testing
test of units of membrane filters (3.1.1) from a batch of membrane filter (3.1.5) for its/their intended use
with the specific culture medium (3.1.3) and determination of reference count (3.3.6)
3.2.2
(standards.iteh.ai)
supplementary quantitative testing
test of units of membrane filters (3.1.1) from a batch of membrane filter (3.1.5) for its/their intended use
with the specific culture medium (3.1.3), ISO 7704:2023of reference count (3.3.6), test of the membrane
determination
filters on a non-selective culture medium and test of the culture medium without membrane filter
7704-2023
Note 1 to entry: Annex C gives detailed description of the supplementary quantitative testing.
Note 2 to entry: Supplementary quantitative testing is used when a new type of a membrane filter(s) (3.1.1), or a
new manufacturer is tested initially, or when a problem in the day-to-day use or batch testing (3.2.1) of membrane
filters is noticed.
3.2.3
performance of the membrane filter
response of a membrane filter (3.1.1) challenged by a test organism (3.3.3) under defined conditions
Note 1 to entry: The defined conditions are described in the specific standard (3.1.7) for the intended use of
the membrane filter (3.1.1), like: test organism (3.3.3), specific culture medium (3.1.3), reference medium (3.1.4),
productivity (3.2.5), selectivity (3.2.6), specificity (3.3.7), incubation time and temperature.
3.2.4
performance of the culture medium
response of a culture medium (3.1.2) to challenge by test organisms (3.3.3) under defined conditions
[SOURCE: ISO 11133:2014, 3.2.1]
3.2.5
productivity
level of recovery of a target microorganism (3.3.1) from the culture medium (3.1.2) with the membrane
filter (3.1.1) under defined conditions
[SOURCE: ISO 11133:2014, 3.2.4, modified — "with the membrane filter" has been added to the term
and the definition.]

ISO 7704:2023(E)
3.2.6
selectivity
degree of inhibition of a non-target microorganism (3.3.2) on or in a selective culture medium (3.1.2)
with the membrane filter (3.1.1) under defined conditions
[SOURCE: ISO 11133:2014, 3.2.5, modified — "with the membrane filter" has been added to the term
and the definition.]
3.2.7
specificity
demonstration, under defined conditions, that non-target microorganisms (3.3.2), if able to grow on the
medium, do not show the same visual characteristics as the target microorganisms (3.3.1) on the culture
medium (3.1.2) with membrane filter (3.1.1) under defined conditions
3.3 Terminology for test microorganisms

3.3.1
target microorganism
microorganism or group of microorganisms to be detected or enumerated which can be expected to
grow under defined conditions
[SOURCE: ISO 11133:2014, 3.2.2, modified — "which can be expected to grow under defined conditions"
has been added to the definition.]
3.3.2
non-target microorganism iTeh STANDARD PREVIEW
microorganism that is suppressed by the medium and/or conditions of incubation or does not show
(standards.iteh.ai)
expected characteristics of the target microorganism
[SOURCE: ISO 11133:2014, 3.2.3]
ISO 7704:2023
3.3.3
test microorganism
control strain 7704-2023
microorganism generally used for performance testing of membrane filters (3.1.1) and/or culture media
(3.1.2)
Note 1 to entry: Test microorganisms/control strains are further defined according to their source (see
3.3.4 to 3.3.10).
3.3.4
reference strain
microorganism obtained directly from a reference culture collection and defined to at least the species
level
Note 1 to entry: A reference culture collection is a culture collection which is a member of the World Federation
of Culture Collections (WFCC) or the European Culture Collections’ Organization (ECCO).
Note 2 to entry: A reference strain is catalogued and described according to its characteristics and preferably
originating from water as applicable.
3.3.5
reference stock
set of separate identical cultures obtained by a single subculture from the reference strain (3.3.4) either
in the laboratory or from a supplier
[SOURCE: ISO 11133:2014, 3.4.3]
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ISO 7704:2023(E)
3.3.6
stock culture
primary subculture from a reference stock (3.3.5)
[SOURCE: ISO 11133:2014, 3.4.4]
3.3.7
working culture
subculture from a reference stock (3.3.5) or stock culture (3.3.6) or a reference material (3.3.9) or a
certified reference material (3.3.10)
Note 1 to entry: Multi-strain reference material (3.3.9) and multi-strain certified reference material (3.3.10) can
also be used for the preparation of working cultures.
[SOURCE: ISO 11133:2014, 3.4.4, modified — "certified or not" has been replaced with " or a certified
reference material" Note 1 to entry has been added.]
3.3.8
reference count
inoculum level
total count of colonies on a reference medium (3.1.4) obtained without usage of a membrane filter (3.1.1)
3.3.9
reference material
RM
microbiological material containing a quantity of revivable microorganisms, sufficiently homogenous
and stable with respect to the quantity of revivable microorganisms, which has been established to be
fit for its intended use in a measurement process
(standards.iteh.ai)
Note 1 to entry: See ISO Guide 30 .[15]
Note 2 to entry: For in-house prepared quality control reference materials (QRMs), often so-called “in-house
ISO 7704:2023
reference materials” or “internal RM”, see ISO Guide 80[18] and References [23], [25], [27] and [28].
[SOURCE: ISO 11133:2014, 3.4.6, modified7704-2023
— the reference in Note 1 to entry has been updated and
Note 2 to entry has been added.]
3.3.10
certified reference material
CRM
microbiological reference material (3.3.9) characterized by a metrologically valid procedure for the
quantity of revivable microorganisms
Note 1 to entry: See ISO Guide 30[15].
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs are given in, among
others, ISO 17034,[14] ISO Guide 31[16] and ISO Guide 35[17].
Note 3 to entry: A microbiological CRM is accompanied by a certificate that provides the value of the specified
quantity of revivable microorganisms, its associated uncertainty and a statement of metrological traceability.
A CRM is certified only for the method/methods and media that were included in the certification process,
otherwise its function is like an (ordinary) reference material (3.3.9).
4 Principle
4.1 General
4.1.1 Introduction
Membrane filters are used for retention followed by direct enumeration of microorganisms in water
samples. Many materials, pore sizes and brands are commercially available and designed for specific

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INTERNATIONAL ISO

Water quality — Requirements for

iTeh STANDARD PREVIEW

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Annex A (normative) Diagram of the procedure for batch testing.................................................................................... 16

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iTeh STANDARD PREVIEW

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Water quality — Requirements for the performance

These tests are intended to demonstrateISO the 7704:2023

3 ​Terms and definitions

© ISO 2023 – All rights reserved ﻿ 1

3.1 ​General terminology

EXAMPLE Tryptone soya agar (TSA) is in accordance with ISO 11133.

2 ﻿ © ISO 2023 – All rights reserved

3.2 ​Terminology of performance testing

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3.3 ​Terminology for test microorganisms

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