ISO 7704 - 2023 Hieu Nang Mang Loc

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
micro-organismes par des méthodes de culture
Reference number
ISO 7704:2023(E)
© ISO 2023
Copyright ISO Sold to: Công ty Cổ Phần Chăn nuôi C.P. Việt Nam
Provided by Vietnam ISMQ – STAMEQ under license with ISO Not for resale, 2023-05-05; End user: Phòng Thí nghiệm
No production or networking permitted without license from Vietnam ISMQ – STAMEQ Email: [email protected]
ISO 7704:2023(E)
COPYRIGHT PROTECTED DOCUMENT

© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: [email protected]
Website: www.iso.org
Published in Switzerland
ii © ISO 2023 – All rights reserved


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

.
7.2 Reference count ..................................................................................................................................................................................... 8

.
7.2.1 Quantitative productivity testing ....................................................................................................................... 8

.
7.2.2 Qualitative selectivity testing ................................................................................................................................ 9

.
7.2.3 Qualitative specificity testing ................................................................................................................................ 9

.
7.3 Preparation of a standardized test suspension using a working culture .......................................... 9

.
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

.
© ISO 2023 – All rights reserved iii

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
.
iv © ISO 2023 – All rights reserved


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
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 analysis, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
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.
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.
© ISO 2023 – All rights reserved vii

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;
— water with expected higher numbers of microorganisms, for example, surface water and process
water.
These tests are intended to demonstrate the 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].
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.
© ISO 2023 – All rights reserved 1

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://www.iso.org/obp

— IEC Electropedia: available at https://www.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).
[SOURCE: ISO 11133:2014, 3.3.1, modified — “constituents for” has been included and Note 1 to entry
has been replaced.]
3.1.3
specific culture medium
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
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), determination of reference count (3.3.6), test of the membrane
filters on a non-selective culture medium and test of the culture medium without membrane filter
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
microorganism that is suppressed by the medium and/or conditions of incubation or does not show
expected characteristics of the target microorganism
[SOURCE: ISO 11133:2014, 3.2.3]
3.3.3
test microorganism
control strain
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]


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
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
reference materials” or “internal RM”, see ISO Guide 80[18] and References [23], [25], [27] and [28].
[SOURCE: ISO 11133:2014, 3.4.6, modified — 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


ISO 7704:2023(E)
growth purposes. In laboratories carrying out the microbiological examination of water, the main
objectives are to capture and retain, resuscitate, grow, detect and/or enumerate a wide variety of
microorganisms. Membrane filters which meet the required performance criteria are therefore a
prerequisite for any reliable microbiological work.
Direct enumeration includes all colony-count methods following retention of the microorganisms on the
membrane filters and placement of the membrane filters on solid media or on other devices containing
culture media, see ISO 6461-2[3], ISO 7899-2[6], ISO 9308-1[8], ISO 11731[10], ISO 14189[12], ISO 16266[13]
and Reference [19]. The direct enumeration is a quantitative method.
Sufficient performance testing should be carried out to demonstrate that a batch is ‘fit for purpose’
and that the filter can produce consistent results for the specific analysis. The quality of the membrane
filters shall be tested together with the intended specific culture medium to demonstrate their
suitability for use in a so-called “microbiological function test”.
This implies that batch testing should demonstrate the suitability of the whole system (membrane
filter in combination with the specific culture medium including the filtration step). It is not required to
evaluate the performance of the membrane filter separately to the performance of the culture medium.
It is expected that suitable media, incubation temperature, incubation duration, incubation atmosphere
and controls will be used for the specific applications. Results obtained from one species or group of
microorganisms may not be valid for other groups.
Suitable specification and acceptance criteria are given in the corresponding specific standard, or
ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F.

The testing for meeting the performance criteria is an essential part of the quality assurance procedure
and appropriate documentation is necessary.
It is the responsibility of the end user to ensure all required combinations of test strains, microbiological
culture media, and membrane filters have been tested before use. If testing before use is not possible
due to the lability of the culture medium, parallel performance testing alongside the sample testing
shall be performed.
NOTE Annex E gives a practicable example for the testing of the membrane filters by the end user.
4.1.2 Batch testing

The batch testing is required when a different batch or lot of a membrane filter is tested.
The batch testing needs to be performed for each batch of membrane filters with each batch of culture
medium that is used in the quantitative analysis within the procedure of the enumeration method
required. This shall be done before usage of the combination membrane filter/culture medium by the
manufacturer or by the user.
Therefore, the batch testing needs to be adjusted to the procedure of the applicable standard in terms of
culture media, incubation temperature and time as well as suitable test microorganism. The procedure
for batch testing is described in Clause 9.
If the criteria for the batch testing of membrane filters given in the corresponding specific standard,
or ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F are not achieved, the laboratory

should assess the discrepancies between the results by supplementary testing given in 4.1.3.
4.1.3 Supplementary testing

Supplementary quantitative testing can be used when a new type of a membrane filter or a new
manufacturer is initially tested, or when a problem in the day-to-day use or batch testing is noticed.
The procedure for supplementary quantitative testing is described in Annex C. Supplementary
qualitative testing for a membrane filter is appropriate to consider when there are discrepancies in
colony appearance between different membrane filter brands, types or lots. Some qualitative aspects


ISO 7704:2023(E)
for a membrane filter used in combination with the culture medium (e.g. colony characteristics and
morphology) and how they can be checked by use of scores are described in Annex D.
NOTE For the supplementary testing on homogeneity based on a microbiological culture method, see
Reference [29].
4.2 Performance testing

4.2.1 Modules for batch and supplementary testing

The quantitative testing procedure of the membrane filters consists of four different modules, see
Table 1. Depending upon the purpose of the testing, different modules are required:
— for batch testing of the membrane filters, modules 1 and 2 shall be tested simultaneously;
— for supplementary quantitative testing of the membrane filters, the modules 1, 2, 3 and 4 shall be
tested simultaneously.
From the counts achieved in modules 1 and 2, the productivity ratio can be calculated for the membrane
filter used in combination with the specific culture medium. The productivity shall reach a defined
minimum limit, in accordance with the corresponding standard, or ISO 11133:2014, Annex F and
ISO 11133:2014/Amd1:2018, Annex F.

Information on sampling of membrane filters for the testing is given in Clause 8.
Table 1 — Modules for batch and supplementary quantitative testing

Required for
Module
Testing purpose Testing procedure Batch Supplementary
no.
testing quantitative testing
Count obtained on a non-selective
Determination of the reference culture medium using
1 x x
reference count spread plate technique without a
membrane filter
Productivity,
Membrane filter used with the
selectivitya, specificitya
2 specific culture medium using x x
of the membrane filter in
membrane filtration technique
its intended use
Membrane filter used with a
Detection of inhibition of
non-selective culture medium
3 target organisms due to — x
using membrane filtration tech-
the membrane filter
nique
Detection of inhibition
Specific culture medium using
of target organisms due
4 spread plate technique without a — x
to the specific (selective)
membrane filter
culture medium
a If applicable.
4.2.2 Absence of microbial contamination

For testing the absence of microbial contamination, an appropriate quantity of membrane filters shall
be tested by incubation under appropriate conditions.
If a batch of membrane filters is specified by the supplier as sterilized in a validated sterilization cycle,
a test for absence of microbial contamination by the end user is not required.


ISO 7704:2023(E)
5 Apparatus and glassware

Appropriate apparatus and glassware are stated in the corresponding specific standards or in ISO 8199.
6 Culture media and diluents

Appropriate culture media and diluents are stated in the corresponding specific standards or in
ISO 8199.
For the preparation, production, storage and performance testing of culture media and diluents, follow
the procedures given in the corresponding specific standards, ISO 11133 or ISO 8199. Before use, allow
the agar plates to equilibrate at room temperature if stored at a lower temperature.
Appropriate culture media for estimation of the reference count are stated in the corresponding specific
standards, or ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F.

In order to ensure the reliability of results of performance testing, the reference medium used shall be
of consistent high quality. Examples of aspects to be considered by the user are given in ISO 11133:2014,
6.3.2.
7 Preparation of microorganisms for performance testing

7.1 General

This protocol requires the use of a quantified bacterial suspension (which can be a quantitative
reference material) with an appropriate colony count of a target strain.
Guidance for procedures suitable for preservation and maintenance of microorganisms is stated in
ISO 11133.
Appropriate microorganisms for performance testing are stated in corresponding standards, or
ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F. Where the standards list more than

one control strain for each aspect of performance testing (e.g. productivity, selectivity, specificity),
the minimum number of strains to be used is indicated. Usually at least one or two strains that are
considered typical target or non-target microorganisms are used in the performance testing.
A complete list of culture media and reagents used in Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 4, Microbiological methods, with their names of control strains and their World Data
Centre for Microorganisms (WDCM) collection numbers that should be used for testing the performance
of culture media and reagents can be found in Reference [35].
NOTE 1 Well-characterized strains isolated by the laboratory can be included additionally.
NOTE 2 ISO 11133:2014, Annex J and ISO 11133:2014/Amd1:2018, Annex J provides instructions for the

creation of new microbiological performance criteria, methods and control strains if these are not given in a
specific standard, or ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F.

7.2 Reference count

7.2.1 Quantitative productivity testing

For all quantitative tests, the use of a quantified inoculum of the specified microorganisms is needed.
Follow the procedures in accordance with ISO 11133:2014, 5.4.2.5 and ISO 11133:2014/Amd1:2018,

5.4.2.5. The inoculum can be prepared by the laboratory or from a RM or a CRM.
NOTE 1 For the laboratory-prepared inoculum, additional information on “Quality Control Materials” (QCMs)
used for specific in-house quality control applications is given in ISO Guide 80[17].


ISO 7704:2023(E)
The reference count of the laboratory-prepared inoculum or RM is determined in relation to recovery on

a non-selective reference culture medium. Follow the procedures in accordance with ISO 11133:2014,
7.2.2. Appropriate reference media are given in the corresponding specific standard or ISO 11133.
The quantitative enumeration test using membrane filters on productivity requires a level of
approximately 100 colony forming units (cfu) to achieve sufficient precision, see ISO 11133:2014,
Table 1. A practicable range to be used will be 80 cfu to 120 cfu. If more colonies are counted, the
precision is increased.
NOTE 2 By assuming Poisson distribution in a sample, which is usually relevant, colony numbers on membrane
filters from several aliquots from the sample can be added, see ISO 13843:2017, Clause A.2[11].
NOTE 3 ISO 11133:2014, Table 1 shows the 95 % confidence intervals associated with colony counts.
As guidance, the general upper limit for counting on membrane filters with a diameter of 47 mm to
50 mm can be regarded as 80 cfu, see ISO 8199:2018, 9.1.4.2.
However, for certain parameters lower, or even higher, upper limits are more appropriate, see the
specific standards and NOTE 4.
Thus, more than one membrane filter can be required to reach a total of approximately 100 cfu for
counting. Two membrane filters are enough with approximately 50 target colonies on each. Three and
four membrane filters are necessary when about 35 and 25 target colonies, respectively, appear on
each.
NOTE 4 It can be difficult to count a minimum of 50 cfu per membrane filter for bacteria that form large
colonies, such as Pseudomonas aeruginosa (P. aeruginosa). Their performance tests are easier to read with
three plates with approximately 30 cfu per membrane filter or four plates with approximately 25 cfu per
membrane filter to reach a total of approximately 100 cfu. The opposite applies to enterococci: due to their small
and defined colonies, these can often be easily counted when approximately 100 cfu or greater are present on a
single membrane filter.
7.2.2 Qualitative selectivity testing

For the qualitative selectivity testing using membrane filters in combination with the specific culture
medium, a suspension of the non-target microorganism containing at least 104 cfu is inoculated
by membrane filtration. Follow the procedures in accordance with ISO 11133:2014, 5.4.2.5.1.2 and
ISO 11133:2014/Amd1:2018, 5.4.2.5.1.2.

7.2.3 Qualitative specificity testing

For the qualitative specificity testing using membrane filters together with the specific culture medium,
a suspension of the non-target microorganism containing at least 103 cfu is inoculated by membrane
filtration. Follow the procedures in accordance with ISO 11133:2014, 5.4.2.5.1.3.
7.3 Preparation of a standardized test suspension using a working culture

7.3.1 General

The following guidance is given as an example of a procedure suitable for producing standardized test
suspensions for the performance testing of membrane filters. These procedures are generally applicable
but some microorganisms can require special conditions. For example, the culture of anaerobes may
require special conditions (e.g. anaerobic atmosphere, nutritional requirements).
7.3.2 Preparation of the working culture

The test microorganism is obtained as a reference strain and stored as a reference stock. From the
reference stock, a stock culture by using a solid medium is prepared and used for the preparation of
a working culture. A working culture shall be prepared from the reference stock or from the stock
culture as a pure stationary phase culture in a non-selective broth.


ISO 7704:2023(E)
All details for the steps of preparation and maintenance of the test microorganisms including different
methods are described in detail in ISO 11133:2014, 5.4.2 and Annex B and ISO 11133:2014/Amd1:2018,

5.4.2. For Legionella spp. working culture and test suspension, follow the procedures in accordance
with ISO 11731:2017, 11.3[10].
Although various techniques can be used, they shall be applied in a standardized way to guarantee
both purity and known inoculum level.
NOTE Annex E gives an example of the preparation of the working cultures for the performance testing in
accordance with ISO 9308-1[8].
For the preparation of a test suspension using reference material, see 7.4.
7.3.3 Preparation of a standardized test suspension (inoculum) for the test

From the working culture, prepare serial dilutions in a suitable diluent, for example, quarter-strength
Ringer’s solution, peptone salt solution, see ISO 8199:2018, Annex D. Use the most suitable dilution step
for the target level of microorganisms (cfu) in a specified volume or refer to the supplier.
For every test microorganism, the suitable dilution to use as a test suspension (inoculum) should be
determined from previous tests conducted under strictly standardized conditions for all steps.
Use the test suspension (inoculum) within a specified time, for example, up to 2 h at room temperature
or within 24 h if stored at (5 ± 3) °C. Longer storage periods can be acceptable if the stability of the
stored culture has been verified[24].
Frozen inocula can be used if it can be shown that the microorganism can survive for the chosen period.
NOTE Annex E gives an example of the preparation of the test suspensions (inocula) for the performance
testing according to ISO 9308-1[8].
7.4 Preparation of a test suspension using reference material

This protocol uses RMs or CRMs to provide a stable bacterial suspension containing a known number of
cfu of the target or non-target strain.
The recovery from the new batch of membrane filters used together with the specific culture medium
is compared to the expected number of cfu from the RM or CRM.
The quality of the RM (including the quantity of cfu) shall be verified on the reference medium.
NOTE Reference material verification is done preferably by the manufacturer.
8 Sampling of membrane filters for testing

An appropriate quantity of membrane filters shall be tested under conditions described in the specific
standard.
Manufacturers of membrane filters shall set specifications using appropriate acceptable quality limits
for batch control.
NOTE 1 Information about sampling is given in the ISO 2859 series[1], the ISO/TR 8550 series[7] or other
specific standards. ISO 2859-1[1] contains an introduction to the ISO 2859 series[1] attribute sampling system and
provides a brief summary of the attribute sampling schemes and plans used in the different parts of the ISO 2859
series[1].
Laboratories (either those using membrane filters for their own testing or those providing these filters
to other end users) shall perform the quantitative productivity testing (and either qualitative selectivity


ISO 7704:2023(E)
or specificity testing, or both, if applicable) each time the batch of membrane filters changes. For every
intended use, the batch of membrane filters shall be tested with the specific batch of culture medium.
NOTE 2 The ISO 2859 series[1] specifies sampling plans with a controlled statistical power according to the
size of the batch. The manufacturer's risk of being refused a valid lot according to its compliance criteria as well
as the laboratory's risk of accepting an occasional poor lot are assessed in the standard.
The minimum number of membrane filters to be tested is determined by the requirements for the
performance test together with the specific culture medium in accordance with corresponding specific
standards, or ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F.

NOTE 3 Annex E gives a practical example for the testing of the membrane filters by the end user.
9 Procedure

9.1 General

The batch testing of membrane filters shall be carried out as specified in Annex A.
For the quantitative productivity testing during the batch testing, modules 1 and 2 shall be tested
simultaneously; see 4.2 and Table 1.
The quantitative productivity testing requires a test suspension (inoculum) as specified in 7.2.1. The
same volume should preferably be used as for the determination of the reference count by spread
plating (see 9.2) and for the inoculation by membrane filtration (see 9.3). A qualitative selectivity and a
qualitative specificity testing shall be included in the batch testing, if required in accordance with the
requirements for the performance testing given by the corresponding specific standard or ISO 11133.
NOTE Examples of suitable reference media are given in the corresponding specific standard, or

Module 1 is not needed when using a CRM which is certified for its number of cfu with the specified
method and culture medium; see 3.3.10, Note 3 to entry.
For the qualitative testing on selectivity and specificity during the batch testing, module 2 is tested.
Module 1 is not required as these are qualitative tests. The qualitative tests on selectivity and specificity
require test suspensions (inocula) as respectively specified in 7.2.2 and. 7.2.3.
For the test on absence of microbial contamination, see 9.5.
9.2 Inoculation by spread plate technique

9.2.1 General

The test portion (inoculum) containing the reference count and obtained as indicated in 7.3 or 7.4 is
spread over the dry surface of a solid medium with a sterile implement. Colonies that develop on the
surface after incubation are counted.
Spread-plate technique is used for modules 1 and 4; see Table 1.
Avoid any mechanical damage to the bacteria by surface-plating using the spread-plate technique with
Drigalski spatula, see Reference [26].
The test portion containing the reference count should be chosen so that the expected number of
colonies is achieved; see 7.2.
For a Petri dish of 90 mm diameter, the volume of the test portion containing the reference count should
be from 0,1 ml to a maximum of 0,5 ml portion of the inoculum.


ISO 7704:2023(E)
Spiral platers use smaller volumes and the limit of determination will consequently be raised. Follow
the manufacturer’s guidance for use of spiral platers. Spiral platers may not be suitable for fungi. For
more information, see ISO 7218[5].
9.2.2 Inoculation

Prepare and mark the plates required, each containing (18 ± 2) ml of culture medium for 90 mm Petri
dishes. For longer incubation periods, larger volumes of culture media can be required. In this case,
refer to ISO 11133 for guidance on culture medium volumes.
If necessary, dry the surface of the medium before use, as described in ISO 11133:2014, 4.5.5, and
ISO 8199:2018, 9.1.3.3.
Pipette the test portion onto the surface of the medium and spread over the surface with a sterile
implement, or mechanical device such as a spiral plater, avoiding the edges of the agar. Leave the plates
on the bench until the inoculum is absorbed (maximum time is 15 min), then incubate the plates in
accordance with 9.4.
9.3 Inoculation by membrane filtration technique

9.3.1 General

For the test on productivity, selectivity and specificity (if applicable), filter the test portion (inoculum)
containing the reference count and obtained as described in 7.3 or 7.4, according to the requirements of
the specific standard, using standardized procedures described in the specific standard or in ISO 8199.
Use equipment for membrane filtration and handle the filtration and placement of the membrane filter
as described in the specific standard or in ISO 8199.
Membrane filtration technique is used for modules 2 and 3, see Table 1.
9.3.2 Inoculation

The test portion containing the reference count should be chosen so that the expected number of
colonies will be achieved, see 7.2.
After placing the membrane filter under test and positioning the funnel on the filter base, pipette or
pour the following after each other into the funnel (with the vacuum stopcock turned off) in three
subsequent steps:
a) a volume of at least 10 ml of the sterile water or a suitable sterile diluent;
b) the test suspension containing the reference count, preferably the same volume as used for the
determination of the reference count by surface plating;
c) approximately 10 ml of sterile water or a sterile diluent.
Open the stopcock and apply the vacuum to filter the water through the membrane filter. Close the
stopcock as soon as the sample has been filtered. The funnel should be rinsed by filtering one to three
portions of 10 ml to 30 ml of sterile water or diluent, while the filter is still in place, to remove organisms
adhering to the funnel.
Alternatively, the inoculum can be added to a volume of at least 20 ml of the sterile water or a suitable
sterile diluent. Mix well and filter the whole volume as described above.
After filtration, place the membrane filter on the culture medium, ensuring that there is no air trapped
underneath and invert the Petri dish.
For membrane transfer techniques using liquid media or diluents, refer to the specific standard or
ISO 8199:2018, 9.1.4.6.


ISO 7704:2023(E)
9.4 Incubation and counting

According to the method, choose the duration, temperature and special conditions (if needed) of the
incubation for the plates with and without membrane filters, as given by the relevant specific standard
or in ISO 8199.
Counting should be done as specified in the relevant specific standard or in ISO 8199.
9.5 Test for absence of microbial contamination

An appropriate quantity, depending on the size of the batch of membrane filters, shall be tested for
absence of microbial contamination (sterility) by incubation under appropriate conditions. This can be
performed by placing the membrane filter on the specific culture medium. This may be the selective
culture medium used for the test or a non-selective culture medium.
NOTE For more information, see Reference [20].
Information on sampling of membrane filters for the testing is given in Clause 8.
10 Calculation, expression and interpretation of results

10.1 General

In the following subclauses, the calculation, expression and interpretation of the results are described;
see productivity in 10.2, selectivity in 10.3 and specificity in 10.4 testing.
A batch of membrane filters performs satisfactorily if all the test microorganisms used perform
according to the given specifications. It shall be accepted if both general and microbiological quality
criteria are met.
Acceptance criteria are given in the corresponding specific standard, or ISO 11133:2014, Annex F and
ISO 11133:2014/Amd1:2018, Annex F.

Either supplementary quantitative or qualitative testing, or both, can be used when a problem in the
testing is noticed, see 4.1.2.
NOTE The procedure for supplementary quantitative testing is described in Annex C. The procedure for
supplementary qualitative testing is described in Annex D.
10.2 Productivity testing

From the result of this quantitative testing, the productivity ratio, PR , is determined.
PR shall reach a defined minimum limit, see the corresponding specific standard, or ISO 11133:2014,
Annex F and ISO 11133:2014/Amd1:2018, Annex F.

The productivity ratio PR[24] is determined using Formula (1):
Ns
PR = (1)
No
where
Ns is the total count of colonies obtained from one (or more) membrane filter(s) in intended use
with the specific culture medium (see module 2, productivity of the membrane filter, in Table 1);
No is the total count of colonies obtained from one (or more) plate(s) of non-selective reference
culture medium by direct inoculation without a membrane filter (see module 1, determination
of the reference count, in Table 1).


ISO 7704:2023(E)
The results are accepted as valid if the following conditions are satisfied:
— each replicate shall give a positive quantitative result (target bacterial growth);
— each single reported result is included in the practicable range of analysis, see 7.2.
If the PR exceeds 1,4, the cause shall be traced.
For using recovery from CRMs, critical difference can be used for the calculation of tolerance limits, see
ISO 5725-6[2] and ISO 11133:2014, Table 1.
10.3 Selectivity testing

For the interpretation of results obtained in selectivity testing of membrane filters together with
the specific culture medium, the amount of growth of non-target microorganisms after incubation is
assessed as follows:
— 0 corresponds to no growth;
— 1 corresponds to weak growth (either reduction in amount of growth or colony size);
— 2 corresponds to good growth.
For selectivity tests, the degree of inhibition depends on the type of medium. The growth of non-
target microorganisms shall be partly or completely inhibited; see corresponding specific standard, or

10.4 Specificity testing

The specificity of using membrane filters in combination with the specific culture medium is given by
essential physiological characteristics to differentiate related organisms by the presence, absence and/
or grade of expression of biochemical responses, by colony sizes and by morphology, see corresponding
specific standard, or ISO 11133:2014, Annex F and ISO 11133:2014/Amd1:2018, Annex F.

11 Documentation of test results

11.1 Test report

The test report of the performance testing of membrane filters shall contain at least the following
information:
a) the test method used, together with a reference to this document, i.e. ISO 7704:2023;
b) product name, product reference and batch number, manufacturer, material and pore size;
c) product name, product reference and batch number, manufacturer of all used media including
diluents;
d) date of testing;
e) test organism(s) with identification code, for example, culture collection number or internal
laboratory identification code of the test strain(s);
f) any particular occurrence(s) observed during the supplementary testing analysis and any
operation(s) not specified in this document, which can have an influence on the results;
g) the results of the quantitative and, if applicable, of the qualitative testing (selectivity and specificity)
and supplementary testing, see Annexes C and D;
h) reference standards and specifications / limits.


ISO 7704:2023(E)
11.2 Information provided by the manufacturer

The manufacturer or supplier of the membrane filters shall provide, on request, the microbiological
growth characteristics for specific microorganisms and general information relating to the specific
batch of culture medium used. Furthermore, information on ensuring homogeneity of the batch shall be
provided, on request.
Manufacturers shall show that the membrane filters meet the requirements for use for quantitative
enumeration as given in relevant specific standards.
11.3 Traceability

All the data from performance testing should be documented in an appropriate way and kept for
a sufficient period of time according to the quality system in use. The use of control sheets for
documenting and evaluating the results of the tests is recommended; see Annex B.


ISO 7704:2023(E)
Annex A
(normative)
Diagram of the procedure for batch testing
Figure A.1 shows the diagram of the procedure for the batch testing of membrane filters.
Figure A.1 — Diagram of the procedure for the batch testing


ISO 7704:2023(E)
Annex B
(informative)
Example of a card for recording test results from batch testing
Table B.1 shows an example of a card for recording test results from batch testing of membrane filters.
Table B.1 — Example of a card for recording test results from batch testing
Name/manufacturer of the Batch no.:
membrane filter:
Non-selective reference culture Batch no.:
medium:
Selective culture medium: Batch no.:
Test organism/reference Batch no.:
material (RM):
Number of test organisms in the cfu (µl):
used sample (dilution):
Dilutions used for the test: Volume used for
inoculation:
Incubation temperature (°C): Incubation time (h):
Test started (date): Test finished (date):
Results
Dilution of inoculum Replicate Module 1 Module 2
Determination of the Productivity of the
reference count membrane filters
10 - 1
2
No: Ns:
Specification productivity
ratio P R a
Test results productivity
ratio P R a
Remarks:
Batch of membrane filters: Released for use with Batch:
culture medium:
( ) yes ( ) no Date: Signature:
a Requirement: The productivity ratio (PR) of the membrane filtration on selective culture medium shall be in
accordance with the corresponding specific standard, or ISO 11133:2014, Annex F and ISO 11333:2014/Amd1:2018,
Annex F.


ISO 7704:2023(E)
Annex C
(informative)
Quantitative supplementary testing of membrane filters
C.1 Introduction

For the quantitative supplementary testing, the modules 1, 2, 3 and 4 are tested simultaneously, see 4.2
and Table 1.
It is preferred to use the same non-selective culture medium for modules 1 and 3.
C.2 Method for supplementary testing

Module 1 is not needed when using CRM, see 7.4.
NOTE 1 Examples of suitable reference media are given in the specific standard, or ISO 11133:2014, Annex F
and ISO 11133:2014/Amd1:2018, Annex F.

NOTE 2 For the performance testing of the culture medium without membrane filtration use a method as
described in ISO 11133.
A diagram of the procedure of supplementary testing is given in Figure C.1.
C.3 Diagram of the procedure for supplementary testing

Figure C.1 — Diagram of the procedure for supplementary testing


ISO 7704:2023(E)
C.4 Calculation and interpretation of the test results

For the calculation and interpretation of results of the quantitative supplementary testing of membrane
filters, the following points can be considered:
— If PR on non-selective culture medium with membrane filter (Module 3) is not lower than PR on
selective culture medium with membrane filter (Module 2), the batch of membrane filters does not
seem to have a pronounced inhibitory effect.
— If PR on non-selective culture medium with membrane filter (Module 3) is <0,70, the batch of
membrane filters can be a possible cause of growth inhibition of the control strains.
— If PR on selective culture medium with membrane filter (Module 2) of the batch testing does not
meet the specifications and productivity on non-selective culture medium with membrane filter
(Module 3) shows also low PR , the inhibitory effects of the membrane filters can be a possible cause.
— If PR is <0,70 on non-selective culture medium with membrane filters (Module 3), this points towards
inhibitory effects of the membrane filters.
— If PR is low (<0,70 respectively <0,50, see ISO 11133:2014, 7.2.2.1.2) on selective culture medium
without membrane filter (Module 4), this can also cause low PR in the batch testing. But it is also
possible that this selective culture medium tested with membrane filters shows PR as specified
and not the same degree on inhibition, see below for possible damage of the control strains using
spread-plating.
— If PR on non-selective culture medium with membrane filter (Module 3) is >1,40, inhibitory effects
from damaging the test microorganism during spread-plating can be considered, see Reference
[26]. For points to consider for reference culture medium, see ISO 11133:2014, 6.3.2.
Table C.1 shows examples for interpretation of the test results for one control strain according to
ISO 9308-1:2014/Amd1:2016, Table 1[9].



ISO 7704:2023(E)
Table C.1 — Examples for interpretation of the test results for one control strain according to
ISO 9308-1:2014/Amd1:2016, Table 1[9]

Module no.
Comment
1 2 3 4
Testing Determination of Productivity of the Detection of Detection of inhibition
purpose the reference count membrane filter in inhibition of target of target organisms
its intended use organisms due to due to the specific —
the membrane filter (selective) culture
medium
Testing Non-selective Specific culture Non-selective cul- Specific culture According to ISO 9308-1[8],
procedure reference culture medium using ture medium using medium using spread Escherichia coli (E. coli)
medium using membrane membrane plate technique WDCM 00013 can be used for
spread plate filtration filtration quantitative productivity testing
technique technique technique with P R ≥ 0,7 as test criterium.
E. coli spread plate E. coli membrane E. coli membrane E. coli spread plate on
Example —
on TSA filtration on CCA filtration on TSA CCA
138 cfu
143 cfu P R = 1,22 No major inhibitory effect of
1 113 cfu —
P R = 1,27 membrane filter.
P R about the same
as in module 2
Membrane filter can have inhibi-
72 cfu
2 113 cfu — — tory effects on growth of microor-
P R = 0,64 (<0,70)
ganisms.
72 cfu 68 cfu Inhibitory effects of the membrane
3 113 cfu —
P R = 0,64 (<0,70) P R = 0,60 (<0,70) filters can be a possible cause.
Inhibitory effects from damaging
165 cfu 98 cfu
4 113 cfu — the test microorganism during
P R = 1,46 (>1,40) P R = 0,87
spread-plating can be considered.
Inhibitory effects of medium can
72 cfu 68 cfu
5 113 cfu — be considered as a possible cause
P R = 0,64 (<0,70) P R = 0,60 (<0,70)
for low P R in batch testing.
Membrane filter in combination
105 cfu with selective culture medium
57 cfu 68 cfu
6 113 cfu P R = 0,93 can work well although both com-
P R = 0,50 (<0,70) P R = 0,60 (<0,70)
(≥0,70, < 1,40) pounds tested separately show
inhibition of the target strains.
NOTE This table does not list counts that are irrelevant for the listed interpretation.
As long as productivity, selectivity and specificity tests are within the specifications given in the
specific standard, a low productivity in the supplementary testing does not disqualify the combination
of membrane filters and culture medium. Batch testing (Module 2) can meet the specifications, although
PR of the testing of Module 3 and Module 4 are <0,7 respectively <0,5.


ISO 7704:2023(E)
Annex D
(informative)
Qualitative supplementary testing of membrane filters
D.1 Introduction

In the testing of membrane filter suitability for retention followed by direct enumeration, the recovery
of the challenged microorganisms is of highest importance. The qualitative supplementary testing
of membrane filters is appropriate to consider when there are discrepancies in colony appearance
between different membrane filter brands/types/lots.
Some examples of the qualitative characteristics and how they can be checked by the use of scores
are described. Categorical scores need to be given to each property to be able to sum up those that
are appropriate for a filter for comparison against a certain reference value or against values for other
filters. Ranks based on the sums can be used for an easier comparison when several filters are studied
simultaneously.
D.2 Global properties

The global properties are:
a) hydrophobicity of the membrane filters, see D.4.2;
b) grid line growth inhibition, see D.4.3;
c) grid line colony proliferation, see D.4.4.
D.3 Colony type specific properties

The colony type specific properties are:
a) colony colour, see D.4.5;
b) colony irregularity, see D.4.6;
c) colony convexity, see D.4.7;
d) colony size, see D.4.8.
D.4 Categorical scores

D.4.1 General

Example scores are given below. If necessary, there can be more categories than those indicated. Most
of them intrinsically are more or less subjective. To make it manageable not too many categories should
be used.
Although some of the listed properties have no direct impact either on colony recovery or on the
suitability of a particular membrane filter for quantitative purposes, they can be useful for the ease of
reading the plates.


ISO 7704:2023(E)
D.4.2 Hydrophobicity of the membrane filters

Hydrophobic parts of the filters will not attach to the media surface, leading to reduced transport of
nutrients from the medium to the filter and the possibility of reduced growth.
The scores are:
— 0 = None;
— −1 = Hydrophobic spots (small, ≤ 10 % of the filter area);
— −2 = Hydrophobic spots (large, > 10 %).
The filters are checked by the naked eye regarding the presence of hydrophobic spots.
NOTE 1 As hydrophobic spots can be wet due to capillary force, it is useful to check the hydrophobicity after
approximately 10 % of the incubation time at the given incubation temperature.
NOTE 2 For more information, see Reference [21].
D.4.3 Grid line growth inhibition

The grid lines simplify the counting of colonies but their ink composition should not restrain bacterial
growth or impart hydrophobicity to the membrane. An inhibiting grid line can lead to irregular colony
presentation and potentially colonies almost forming two parts to avoid contact with a line, affecting
the ease of counting or even leading to a too high recovery. The inhibition can even lead to deteriorated
growth and a lower recovery. There shall be complete growth of the colonies over the grid lines without
changes in the typical shape of the colony.
The scores are:
— 0 = None;
— −1 = Small (1 % to 10 % of colonies on lines affected);
— −2 = Large (>10 % of colonies on lines affected).
Filters are generally checked by the naked eye.
NOTE The grid line inhibition can be easier assessed by use of a binocular stereo microscope with
4× to 10× magnification.
D.4.4 Grid line colony proliferation

The grid line (imprint on membrane filters) can cause a channel in which water collects. Colonies can
proliferate along the grid lines giving irregular shaped, smeared or overlapping colonies which cannot
be counted correctly.
The scores are:
— 0 = None;
— −1 = Small (1 % to 10 % of colonies on lines affected);
— −2 = Large (>10 % of colonies on lines affected).
NOTE 1 Grid line growth promotion can either be an effect of the dye of the line itself but probably rather an
effect of surface tension properties of the lines compared to the rest of the filter. Colonies can proliferate along
the grid lines. Sometimes, it can look as if there are several colonies that will give a too high count.
NOTE 2 The grid line inhibition can be easier assessed by use of a binocular stereo microscope with 4× to
10× magnification.


ISO 7704:2023(E)
D.4.5 Colony colour

The colony colour is specified in the relevant standards or by ISO 11133:2014/Amd1:2018, Annex F.

The scores are:
— 0 = Atypical colour;
— 1 = Weak intensity of typical colour;
— 2 = Anticipated typical colour.
NOTE Colour and colour zones are not always well defined but a bit variable. Therefore, they need to be
evaluated with care in relation to prior experiences and relevant definitions, for example, the basic colour itself, a
colour zone or a metallic sheen.
D.4.6 Colony irregularity

Some bacterial species usually have more or less irregular colonies, while others normally are smooth.
The scores are:
— 0 = Smooth;
— −1 = Somewhat irregular;
— −2 = Very irregular.
NOTE Irregularity of colonies is somewhat arbitrary and can be caused by dust or other particles on
the surface of membrane filters. Hence, interpretation of irregularity needs to be taken with care and prior
experience, and the typical appearance needs to be taken into consideration.
D.4.7 Colony convexity

Usually colony convexity does not affect the recovery of colonies but needs to be considered when the
colony appearance is important.
The scores are:
— 0 = Flat;
— 1 = Somewhat convex;
— 2 = Very convex.
NOTE The convexity of the colony is normally typical for a strain on a medium. However, it depends strongly
on the age of the colonies and needs to be studied only within a defined time span.
D.4.8 Colony size

Colony size varies between different groups, genera, species and strains of microorganisms. The size
can also vary within a bacterial strain, partly depending on when the growth of the particular colony
started and on the density of colonies on the filters.
The scores (e.g. depending on strain and media) are:
— 0 = 0 mm to 1 mm;
— 1 = 1 mm to 2 mm;
— 2 = >2 mm.

NOTE The size classes need to be decided for the particular strain used and care needs to be taken when
interpreting the results. Prior experience and knowledge of the strains needs to be taken into consideration.


ISO 7704:2023(E)
D.5 Combining scores

D.5.1 General

Only scores for properties showing differences between filter “types” (brands, lots etc.) need to be used
in the final assessment.
The scores from all properties retained are summed up for each analytical parameter, using the
scores from all properties/strains/samples used and also from each person reading a particular type
of membrane filters. The average over strains/samples and persons is then calculated giving the
characteristic over-all score of that filter type.
If interesting or necessary for an over-all choice of a filter type, scores can even be averaged over target
organisms for several analytical parameters.
D.5.2 Comparison of filters

If several filters are compared simultaneously in one study, ranks from 1 to the number of filter
types compared can be given to the filter types in accordance with the average scores obtained. The
highest (most positive) score is yielding rank 1, the second highest rank 2, etc. If two or more types of
membrane filters have the same score, they need to be given the same and lowest rank number in turn.
The following rank needs then be the one following the number of filters that have already obtained
ranks.
EXAMPLE Five types of membrane filters are studied simultaneously. One of them has the highest average
score and obtains rank 1. Two types have the same, second highest score. Both of them obtain rank 2. After these
follow types with second lowest and lowest score, they obtain ranks 4 and 5, respectively.
D.6 Assessment

For the assessment of the suitability of certain membrane filters, a criterion to compare against is
appropriate. The criterion can be a minimum value for summarized and averaged scores both when
only one membrane filter or several are examined. The criterion will depend on the particular study
design, i.e. how many and which properties are studied and what scores are given for the various
properties.
When several membrane filters are compared, ranks can be an alternative to select one or more suitable
membrane filters.


ISO 7704:2023(E)
Annex E
(informative)
Practical example of quantitative batch testing and quantitative

supplementary testing by the end user
E.1 Introduction

Performance testing for membrane filters used for a quantitative microbiological test method needs to
be conducted in its intended use. For ISO 9308-1[8], this translates into testing plates of CCA with the
membrane filter that is chosen to be used for the testing. Performance tests on productivity, selectivity
and specificity are necessary for every combination of batches of CCA and membranes that are used
together.
In this example of the performance testing of membrane filters, the test procedure does not need any
confirmation step, as a working culture of a known pure single test strain is used. If different colony
morphologies are observed on the plates, confirmation steps can be taken in order to ensure that there
is no contamination.
ISO 9308-1:2014/Amd1:2016, Table 1[9] specifies the control strains to be used for the productivity

testing. According to this Reference [8], productivity needs to be PR ≥ 0,7. As the method combines the
enumeration of E. coli and other coliform bacteria, one strain for each group needs to be tested.
In this example, the performance test on productivity requires quantitative testing while qualitative
testing is required for selectivity and specificity. The control strains E. coli WDCM 00013 and
Citrobacter freundii (C. freundii) WDCM 00006 are used for the quantitative testing on productivity,
Enterococcus faecalis (E. faecalis) WDCM 00009 for the qualitative testing on selectivity and P. aeruginosa
WDCM 00024 for the qualitative testing on specificity, see Table E.1.
For a flow diagram of batch testing and supplementary testing of this example, see Clause E.9.
Table E.1 — Choice of control strains and criteria used in this example for performance testing
of CCA according to ISO 9308-1:2014/Amd1:2016, Table 1[9]

Method of Characteristic
Function Control strainsa Criteriab
control reactionsb
Dark-blue to violet
E. coli WDCM 00013 Quantitative P R ≥ 0,7
Productivity colonies
C. freundii WDCM 00006 Quantitative P R ≥ 0,7 Pink to red colonies
Total or partial
Selectivity E. faecalis WDCM 00009 Qualitative —
inhibition
Specificity P. aeruginosa WDCM 00024 Qualitative — Colourless colonies
a Refer to Reference [36] for the reference strain catalogue on culture collection strain numbers and contact details.
b See ISO 9308-1:2014/Amd1:2016, Table 1[9].

A practical example for the preparation of bacterial suspensions with known quantity of the control
strains is described in Clause E.2. This can be used as a batch testing in lab routine functioning as daily
control measures. Adaptation for a one-time batch testing procedure is also possible, for example, as an
incoming control of a new batch of membrane filters to be tested with a batch of culture medium, see
4.1.1.
For each control strain, a test suspension with a known quantity of cfu has to be prepared. This is best
achieved by following a strictly standardized protocol.


ISO 7704:2023(E)
The outcome of the performance testing is closely related to the physiological state of the bacteria in
the test portion as well as on the suitability of the materials used.
All volumes described in this annex can be adjusted, if needed, depending on the exact protocol used
(time, temperature, culture medium). Nevertheless, it is important that all plates in the different
modules conducted at the same time are inoculated with the exact same volume of the test portion. This
standardized performance testing allows a reliable detection of significant differences in the recovery.
For each control strain and performance test, the liquid medium and diluent need to be considered
carefully. The material should be tested to give reliable results before being used in regular batch
testing.
E.2 Preparation of a standardized test suspension

E.2.1 Working cultures

Take a single well separated colony of E. coli WDCM 00013 from the plate with the stock culture (3.3.4),
for example, from TSA or blood TSA, and transfer this into 10 ml of tryptone soya broth (TSB). Using
a separate tube for each test strain, carry out the same with C. freundii WDCM 00006, E. faecalis
WDCM 00009 and P. aeruginosa WDCM 00024. Mix the tubes carefully to avoid large cell-aggregates in
the broth.
Incubate for 16 h to 18 h with shaking at (36 ± 2) °C to avoid anoxic zones in the medium.
The strains will grow to stationary phase during this time. By strictly following the above protocol, the
cultures will repeatedly grow to certain number of cells that is specific to each strain. Therefore, the
number of dilution steps necessary for the preparation of test portions does not need to be determined
for every single test, but may need to be verified for a new batch of the broth. For example, if incubation
of the working cultures starts at 3 p.m. on the day of inoculation (working day 1), the cultures can
be used for the preparation of the test suspension (inoculum) at 8.00 a.m. the next day (working day
2). For achieving best consistency when preparing the test suspension from the working cultures, the
variation in the length of incubation time should be minimized, for example, to ±30 min.
For the preparation of the working cultures, other non-selective liquid media than TSB can be used. For
each broth and control strain used, the adequate dilutions for the appropriate bacterial counts need to
be determined upfront in pre-trials.
E.2.2 Test suspensions — Preparation of inoculum

E.2.2.1 For quantitative testing on productivity according to the given example, a reference count
(inoculum level) of approximately 100 cfu (in total) on two plates of the reference medium (50 cfu/
plate) are required for each control strain. Therefore, the test suspension should contain approximately
500 cfu/ml or 50 cfu/0,1 ml, respectively.
E. coli WDCM 00013 and C. freundii WDCM 00006 are tested separately.
Dilute the working cultures obtained from E.2.1 with sterile phosphate buffer solution (PBS) (e.g.
8.00 a.m. at working day 2) by following 3 steps to achieve the required reference count:
— Step 1: 0,1 ml working culture + 10 ml PBS (dilution: 1:100, dilution step: 10−2);
— Step 2: 0,1 ml from 10−2 + 10 ml PBS (dilution: 1:10 000, dilution step: 10−4);
— Step 3: 0,1 ml from 10−4 + 20 ml PBS (dilution: 1:2 000 000, dilution step: approximately10−6).
Use 0,1 ml of the last suspension resulting from dilution step 3 as a test portion for the testing on
productivity.
NOTE For the composition, preparation and performance testing of phosphate buffer solution, see
ISO 8199:2018, Annex D.


ISO 7704:2023(E)
For the preparation of the test suspensions, other diluents than PBS can be used, see ISO 8199. For each
diluent and control strain used, the adequate dilutions for the appropriate bacterial counts need to be
determined upfront in pre-trials.
E.2.2.2 For the qualitative testing on selectivity according to the given example, a reference count
(inoculum level) of at least 104 cfu is needed for each control strain. Therefore, the test suspension
should contain at least 105 cfu/ml or 104/0,1 ml, respectively. For E. faecalis WDCM 00009, use 0,1 ml of
the suspension resulting from dilution step 1 as a test portion for the testing on productivity.
E.2.2.3 For the qualitative testing on specificity according to the given example, a reference count
(inoculum level) of at least 103 cfu is needed for each control strain. Therefore, the test suspension
should contain at least 104 cfu/ml or 103/0,1 ml, respectively. For P. aeruginosa WDCM 00024, use
0,1 ml of the suspension resulting from dilution step 2 as a test portion for the testing on productivity.
Best standardized results can be achieved for most control strains if the test suspensions are freshly
prepared from a freshly prepared working culture at each day of testing.
E.3 Performance testing for batch testing

For the performance testing of a batch of membrane filters with a batch of CCA, a batch testing is
needed, see 4.1.1. It has to reach the required criteria, see Table E.1 and ISO 9308-1[8].
From the result of quantitative testing on productivity, the productivity ratio (PR) is determined. PR is
calculated by using the count of cfu obtained with the membrane filter/culture medium combination
under test (Ns obtained by using module 2) and the reference count (No obtained by using module 1),
see 10.2.
From the result of qualitative testing on selectivity, the degree of inhibition is determined, see 10.3.
From the result of qualitative testing on specificity, it is determined that a non-target microorganism
does not show the same visual characteristics as the target microorganism, see 10.4.
E.4 Performance testing for supplementary quantitative testing

For the need of supplementary testing, see 4.1.2.
Modules 1, 2, 3 and 4 are used in parallel for supplementary testing, see Table 1 and Annex B.
Modules 3 and 4 are used to collect further information on the materials used or in search for problems
with the materials whenever low or unsatisfactory productivity is observed in the batch testing
procedure.
Module 3 is used to detect inhibitory effects mainly due to membrane filters.
Module 4 is used to detect inhibitory effects mainly due to specific (selective) culture medium.
E.5 Practical testing of the modules

E.5.1 General

The practical testing by using the modules is described in the following clauses.
In this example, CCA filled in plates with a diameter of 90 mm and membrane filters with a diameter of
47 mm are used.
Table E.2 summarizes the control strains, plates and their codes as described in E.5.2 to E.5.4.


ISO 7704:2023(E)
E.5.2 Module 1: Determination of the reference count from TSA by direct inoculation

without membrane filter
Use 0,1 ml of the suspension resulting from step 3 for E. coli (see E.2.2) for the inoculation of a plate of
TSA by using spread plate technique, see 9.2. Repeat this step for the inoculation of a second plate of
TSA. It results in two replicates; label the plates ‘A’ and ‘B’.
Repeat the same procedure using the suspension from step 3 of C. freundii and label the replicate plates
‘a’ and b’.
For either the qualitative selectivity or specificity testing, or both, the determination of the reference
count is not required.
E.5.3 Module 2: Productivity of the membrane filters by using the membrane filters in

combination with CCA
Transfer 0,1 ml of the suspension from step 3 of E. coli (see E.2.2) to a minimum of 10 ml of sterile saline
solution as diluent and mix gently. A minimum of 10 ml sterile saline solution has to be used for an even
distribution of the test microorganisms during the filtration. The volume can be higher and there is no
need to measure it exactly, see 9.3.
NOTE For the composition, preparation and performance testing of saline solution, see ISO 8199:2018,
Annex D. Other diluents can also be used.
Perform membrane filtration on the whole volume. Transfer the membrane filter to CCA directly
afterwards. Use the same pipette to measure the volume, if possible, and repeat this step using a second
membrane filter. It results in two replicates, label the plates 'C' and 'D'.
Repeat the same procedure using the suspension from step 3 of C. freundii (see E.2.2) and label the
replicate plates ‘c’ and ‘d’.
For the qualitative testing on selectivity, repeat the same procedure with only one plate using the
suspension from step 1 of E. faecalis (see E.2.2) and label the plate ‘I’.
For the qualitative testing on specificity, repeat the same procedure with only one plate using the
suspension from step 2 of P. aeruginosa (see E.2.2) and label the plate ‘i'.
E.5.4 Module 3: Detection of inhibition of target organisms due to the membrane filters

using the membrane filters on TSA
Transfer 0,1 ml of the suspension from step 3 of E. coli (see E.2.2) to a minimum of 10 ml of sterile saline
solution as diluent and mix gently. A minimum of 10 ml sterile saline solution has to be used for an even
distribution of the test microorganisms during the filtration. The volume can be higher and there is no
need to measure it exactly.
Perform membrane filtration on the whole volume. Transfer the membrane to the same culture
medium as used for the determination of the reference count, in this example TSA. Use the same pipette
to measure the volume, if possible, and repeat this step using a second membrane filter. It results in
two replicates; label the plates 'E' and 'F'.
Repeat the same procedure with the suspension from step 3 of C. freundii (see E.2.2) and label the
replicate plates ‘e’ and ‘f’.
Repeat the same procedure with only one plate using the suspension from step 1 of E. faecalis (see
E.2.2) and label the plate ‘J’.
Repeat the same procedure with only one plate using the suspension from step 2 of P. aeruginosa
(see E.2.2) and label the plate ‘j’.


ISO 7704:2023(E)
E.5.5 Module 4: Detection of inhibition of target organisms due to the specific

(selective) culture medium using CCA by direct inoculation without membrane filters
Transfer 0,1 ml of the suspension from step 3 of E. coli (see E.2.2) to a plate of CCA using spread plate
technique. Repeat this step for inoculation of a second plate of CCA. It results in two replicates; label the
plates ‘G’ and ‘H’.
Repeat the same procedure with the suspension from step 3 of C. freundii and label the replicate plates
‘g’ and ‘h’.
Repeat the same procedure with only one plate using the suspension from step 1 of E. faecalis (see
E.2.2) and label the plate ‘K’.
Repeat the same procedure with only one plate using the suspension from step 2 of P. aeruginosa
(see E.2.2) and label the plate ‘k’.
Table E.2 — Control strains, plates and their codes as used in the examples of Annex E
Module no.
1: Determination of 2: Productivity 3: Detection of 4: Detection of inhibi-
the reference count of the membrane inhibition of target tion of target organisms
Control strain from TSA with direct filter in its intended organisms due to the due to the specific (se-
inoculation without use from CCA with membrane filter from lective) culture medium
membrane filter membrane filter TSA with membrane from CCA with direct
filter inoculation without
membrane filter
E. coli
A, B C, D E, F G, H
WDCM 00013
C. freundii
a, b c, d e, f g, h
WDCM 00006
E. faecalis
— I J K
WDCM 00009
P. aeruginosa
— i j k
WDCM 00024
Number of plates 4 plates of TSA 6 plates of CCA 6 plates of TSA 6 plates of CCA
Number of
— 6 6 —
membrane filters
NOTE 1 E. coli WDCM 00013 and WDCM 00006 are used for quantitative testing on productivity.
NOTE 2 E. faecalis WDCM 00009 is used for qualitative testing on selectivity.
NOTE 3 P. aeruginosa WDCM 00024 is used for qualitative testing on specificity.
E.6 Incubation

Incubate all plates at (36 ± 2) °C for 21 h to 24 h, as given in ISO 9308-1[8].
E.7 Counting and expression of results

E.7.1 Module 1

Count all colonies as cfu on plates A and B as E. coli. Calculate the sum and use as reference count for
E. coli WDCM 00013.
Count all colonies as cfu on plates a and b as C. freundii. Calculate the sum and use as reference count for
C. freundii WDCM 00006.


ISO 7704:2023(E)
The counts on the two plates (A and B, and a and b respectively) for the reference count should read
between 20 cfu and 100 cfu each so that for a suitable combination of membrane filter with CCA, the
counts are still in the linear range of ISO 9308-1[8].
If the total number of colonies counted on the two plates for the reference counts is significantly less
than 100 cfu, the performance testing has not passed and needs to be repeated until that number is
reached.
E.7.2 Module 2

Count all dark-blue to violet colonies as cfu on membrane filters or plates ‘C’ and ‘D’ as E. coli. Calculate
the total count.
Count all pink to red colonies as cfu on membrane filters or plates ‘c’ and ‘d’ as C. freundii. Calculate the
total count.
Check the growth on membrane filter/plate ‘I’ as E. faecalis and assess the growth or inhibition,
respectively.
Check the growth of colonies on membrane filter or plate ‘i’ as P. aeruginosa and assess the colour of the
colonies.
E.7.3 Module 3

Count all colonies as cfu on membrane filters or plates ‘E’ and ‘F’ as E. coli. Calculate the total count.
Count all colonies as cfu on membrane filters or plates ‘e’ and ‘f’ as C. freundii. Calculate the total count.
Check the growth on membrane filters or plate ‘J’ as E. faecalis and assess the growth or inhibition,
respectively.
Check the growth of colonies on membrane filter or plate ‘j’ as P. aeruginosa and assess the colour of the
colonies.
E.7.4 Module 4

Count all dark-blue to violet colonies as cfu on plates ‘G’ and ‘H’ as E. coli. Calculate the total count.
Count all pink to red colonies as cfu on plates ‘g’ and ‘h’ as C. freundii. Calculate the total count.
Check the growth on plate ‘K’ as E. faecalis and asses the growth or inhibition, respectively.
Check the growth of colonies on plate ‘k’ as P. aeruginosa and assess the colour of the colonies.
E.8 Calculation and interpretation of the test results

E.8.1 Productivity

Calculate the productivity ratio of the combination of tested membrane filters and CCA for each of the
control strains E. coli and C. freundii using Formulae (E.1) and (E.2), equivalent to Formula (1) in 10.2.
— E. coli WDCM 00013
Ns ( counts from plates C+D)
PR = (E.1)
No ( counts from plates A +B)
where


ISO 7704:2023(E)
Ns(counts from plates C + D) is the total count of the E. coli colonies from both replicate plates of Module 2,
see E.5.2;
No(counts from plates A + B) is the total count of the E. coli colonies from both replicate plates of Module 1,
see E.5.1.
— C. freundii WDCM 00006
Ns ( counts from plates c+d )
PR = (E.2)
No ( counts from plates a + b)
where
Ns(counts from plates c + d) is the total count of the C. freundii colonies from both replicate plates of
Module 2, see E.5.2;
No(counts from plates a + b) is the total count of the C. freundii colonies from both replicate plates of
Module 1, see E.5.1.
If PR ≥ 0,7, for each of the control strains E. coli WDCM 00013 and C. freundii WDCM 00006, the
combination of the batch of membrane filters with the batch of CCA has passed the test on productivity.
If PR < 0,7 or if PR ≥ 1,4, the combination has not passed the test, either the performance of the batch
testing itself or the combination of the batches of membrane filters and CCA. The results of testing
modules 3 and 4 can be considered in order to find possible cause for the low productivity, see E.8.4.
E.8.2 Selectivity

If plate I (inoculated with E. faecalis WDCM 00009 by membrane filtration) shows total or partial
inhibition, the combination of the batch of membrane filters with the batch of CCA has passed the test
on selectivity.
NOTE The amount of growth or degree of the inhibition is described in ISO 11133:2014, 7.4.1.2 by the
following: 0 corresponds to no growth (= total inhibition); 1 corresponds to weak growth (= partly inhibition;
either reduction in amount of growth or colony size); 2 corresponds to good growth.
E.8.3 Specificity

If plate i (inoculated with P. aeruginosa WDCM 00024 by membrane filtration) shows colourless
colonies, the combination of the batch of membrane filters with the batch of CCA has passed the test on
specificity.
NOTE The amount of growth is not specified for specificity testing.
E.8.4 Interpretation of results from batch testing

A batch of membrane filters performs satisfactorily if all the test microorganisms used perform
according to the given specification, see Table E.1 for the example. It shall be accepted if both general
and microbiological quality criteria are met.
NOTE For the general quality criteria such as absence of microbial contamination, see 9.5.
E.8.5 Interpretation of results from supplementary testing

Calculate the productivity ratio of the combination of tested membrane filters and in combination
with the non-selective reference medium TSA to each of the control strains E. coli and C. freundii using
Formulae (E.3) and (E.4), equivalent to Formula (1) in 10.2.
— E. coli WDCM 00013


ISO 7704:2023(E)
Ns ( counts from plates E+F )

PR = (E.3)
No ( counts from plates A +B)
where
Ns(counts from plates E + F) is the total count of the E. coli colonies from both replicate plates of Module 3,
see E.5.3;
No (counts from plates A + B) is the total count of the E. coli colonies from both replicate plates of Module 1,
see E.5.1.
— C. freundii WDCM 00006
Ns ( counts from plates e+ f )
PR = (E.4)
No ( counts from plates a + b)
where
Ns (counts from plates e + f) is the total count of the C. freundii colonies from both replicate plates of
Module 3, see E.5.3;
No (counts from plates a + b) is the total count of the C. freundii colonies from both replicate plates of
Module 1, see E.5.1.
For Module 4, the counts from plates ‘G’, ‘H’, ‘g’ and ‘h’ are used instead of ‘E’, ‘F’, ‘e’ and ‘f’ in the example
above to calculate productivity ratio on the selective agar without membrane filters.
If PR on TSA (with membrane filter) is not lower than PR on CCA (with membrane filter), the batch of
membrane filters does not seem to have a significant inhibitory effect.
If PR on TSA (with membrane filter) is <0,7, the batch of membrane filters can be a possible cause of
growth inhibition of the control strains.
If PR on CCA (with membrane filter) of the batch testing is unsatisfactory and productivity on TSA (with
membrane filters) shows also low, inhibitory effects of the membrane filters can be a possible cause.
If productivity is low on CCA (without membrane filter) (Module 4), this can also cause low productivity
in the batch testing. However, it is also possible that this selective culture medium tested with
membrane filters shows PR as specified and not the same degree on inhibition, see below for possible
damage of the control strains using spread-plating, see Reference [26].
In both cases, productivity can be calculated in relation to the reference count from Module 1.
As long as productivity, selectivity and specificity tests are within the specifications given in the
specific standard, a low productivity in the supplementary testing does not disqualify the combination
of membrane filters and culture medium.
It is also possible that membranes with low productivity on non-selective culture medium (Module 3)
in combination with selective culture medium with membrane filter (Module 2) work well although
both compounds tested separately show inhibition of the target strains.
For this example, colony counts and productivity ratio calculated for the test strains are listed in
Table E.3.


ISO 7704:2023(E)
Table E.3 — Colony counts for this example (codes of the plates as given in Table E.2)
Module no.
4: Detection of
1: Determination of 3: Detection of inhibition of target
2: Productivity of
the reference count inhibition of target organisms due to the
Control strain the membrane fil-
from TSA with organisms due to specific (selective)
ter in its intended
direct inoculation the membrane filter culture medium
use from CCA with
without membrane from TSA with from CCA with direct
membrane filter
filter membrane filter inoculation without
membrane filter
E. coli WDCM 00013
98 / 87 80 / 90 77 / 88 105 / 115
[cfu per plate]
Productivity ratio
— 0,92 0,88 1,19
P R E. coli WDCM 00013
C. freundii
WDCM 00006 66 / 68 56 / 59 62 / 73 55 / 64
[cfu per plate]
Productivity ratio
— 0,86 1,01 0,89
P R C. freundii WDCM 00006
Selectivity
E. faecalis — Partly inhibition Good growth Total inhibition
WDCM 00009
Specificity
P. aeruginosa — Colourless colonies Colourless colonies Colourless colonies
WDCM 00024


ISO 7704:2023(E)
E.9 Diagram of the procedures for the practical example

Figure E.1 — Diagram of the procedure for batch testing on quantitative productivity testing
and quantitative supplementary testing by the end user on the example of the control strain
E. coli WDCM 00013


ISO 7704:2023(E)
Bibliography
[1] ISO 2859 (all parts), Sampling procedures for inspection by attributes
[2] ISO 5725-6, Accuracy (trueness and precision) of measurement methods and results — Part 6: Use
in practice of accuracy values
[3] ISO 6461-2, Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes
(clostridia) — Part 2: Method by membrane filtration
[4] ISO 6887-1:2017, Microbiology of the food chain — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation
of the initial suspension and decimal dilutions
[5] ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
[6] ISO 7899-2, Water quality — Detection and enumeration of intestinal enterococci — Part 2:
Membrane filtration method
[7] ISO/TR 8550 (all parts), Guidance on the selection and usage of acceptance sampling systems for
inspection of discrete items in lots
[8] ISO 9308-1, Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 1:
Membrane filtration method for waters with low bacterial background flora
[9] ISO 9308-1:2014/Amd1:2016, Water quality — Enumeration of Escherichia coli and coliform

bacteria — Part 1: Membrane filtration method for waters with low bacterial background flora —
Amendment 1
[10] ISO 11731:2017, Water quality — Enumeration of Legionella
[11] ISO 13843:2017, Water quality — Requirements for establishing performance characteristics of
quantitative microbiological methods
[12] ISO 14189, Water quality — Enumeration of Clostridium perfringens — Method using membrane
filtration
[13] ISO 16266, Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by
membrane filtration
[14] ISO 17034, General requirements for the competence of reference material producers
[15] ISO Guide 30, Reference materials — Selected terms and definitions
[16] ISO Guide 31, Reference materials — Contents of certificates, labels and accompanying
documentation
[17] ISO Guide 35, Reference materials — Guidance for characterization and assessment of homogeneity
and stability
[18] ISO Guide 80, Guidance for the in-house preparation of quality control materials (QCMs)
[19] SCA (2016). The Microbiology of Drinking Water (2016) – Part 4 - Methods for the isolation and
enumeration of coliform bacteria and Escherichia coli (including E. coli O157:H7) — Method A: The
enumeration of coliform bacteria and Escherichia coli by a two membrane filtration technique using
membrane lauryl sulphate broth incubated at 37 °C and 44 °C — Method B: The enumeration of
coliform bacteria and Escherichia coli by a single membrane filtration technique using membrane
lactose glucuronide agar incubated at 37 °C. Methods for the Examination of Waters and
Associated Materials. Standing Committee of Analysts, Environment Agency, Nottingham, UK.


ISO 7704:2023(E)
[20] ASTM D4196, Standard Test Method for Confirming the Sterility of Membrane Filters
[21] ASTM D4200-82, Standard Test Method for Evaluating Inhibitory Effects of Ink Grids on Membrane
Filters
[22] Brenner K.P., Rankin C.C., New screening test to determine the acceptability of 0,45 µm
membrane filters for analysis of water. Appl. Environ. Microbiol. 1990, 56(1), pp. 54-64
[23] Brodsky M.H., Schiemann D.A., Influence of coliform source on evaluation of membrane filters.
J. App. Microbiol. 1975, 30(5), pp. 727-730
[24] Corry J.E.L., Curtis G. D.W., Baird R.M., eds. Handbook of Culture Media for Food and Water
Microbiology. Royal Society of Chemistry, UK, Third Edition, 2012
[25] Havelaar A.H., Heisterkamp S.H., Hoekstra J.A., Mooijman K.A., Performance characteristics
of methods for the bacteriological examination of water. Wat. Sci. Tech. 1993, 27(3-4), pp. 1-13
[26] Hedderich R., Muller, R. Greulich, Y. Bannert, N. Holland, G., Kaiser, P., Reissbrodt, R.
Mechanical damage to Gram-negative bacteria by surface plating with the Drigalksi-spatula
technique. Int J Food Microbiol. 2011, 146(1), pp. 105-107
[27] Jarvis B., Statistical aspects of the microbiological examination of foods. Academic Press,
London, Second Edition, 2008
[28] McFeters G.A., Cameron S.C., LeChevallier M.W., Influence of diluents, media, and membrane
filters on detection of injured waterborne coliform bacteria. Appl. Environ. Microbiol. 1982,
43(1), pp. 97-103
[29] Schijven J.F., Havelaar A.H., Bahar M., A simple and widely applicable method for preparing
homogeneous and stable quality control samples in water microbiology. Appl. Environ. Microbiol.
1994, 60(11), pp. 4160-4162
[30] Schijven J.F., Drost Y.C., CASTELLJNS H., van der List C. Quality control of membrane filters
for bacteriological examination of water. J. Appl. Microbiol. 1995, 79(3), pp. 308-313
[31] Smith L., Carroll K., Mottice S., Comparison of membrane filters for recovery of Legionellae
from water samples. Appl. Environ. Microbiol. 1993, 59(1), pp. 344-346
[32] Tobin R.S., Dutka B.J., Comparison of the surface structure, metal binding, and fecal coliform
recoveries of nine membrane filters. Appl. Environ. Microbiol. 1977, 34(1), pp. 69-79
[33] Tobin R.S., Lomax P., Kushner D.J., Comparison of nine brands of membrane filter and the Most-
Probable-Number methods for total coliform enumeration in sewage-contaminated drinking
water. Appl. Environ. Microbiol. 1980, 40(2), pp. 186-191
[34] US Environmental Protection Agency. Proceedings of the Symposium on the recovery of
indicator organisms employing membrane filters. Publication EPA-600/9-77-024 National
Technical Information Service, Springfield, Virginia 22161, 1977
[35] https://committee .iso .org/sites/tc34sc9/ home/general-standards/content-left-area/culture

-media/iso-11133-on-culture-media.html (viewed 2022-12-21)

[36] https://refs.wdcm.org/ (viewed 2022-12-21)



ISO 7704:2023(E)
ICS 07.100.20
Price based on 36 pages
© ISO 2023 – All rights reserved


ISO 7704 - 2023 Hieu Nang Mang Loc

Uploaded by

Copyright:

Available Formats

ISO 7704 - 2023 Hieu Nang Mang Loc

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

ISO 7704 - 2023 Hieu Nang Mang Loc

Uploaded by

Copyright:

Available Formats

INTERNATIONAL ISO

Water quality — Requirements for

COPYRIGHT PROTECTED DOCUMENT

ii © ISO 2023 – All rights reserved

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

© ISO 2023 – All rights reserved v

vi © ISO 2023 – All rights reserved

© ISO 2023 – All rights reserved vii

Water quality — Requirements for the performance

3 Terms and definitions

© ISO 2023 – All rights reserved 1

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

2 © ISO 2023 – All rights reserved

3.2 Terminology of performance testing

© ISO 2023 – All rights reserved 3

3.3 Terminology for test microorganisms

4 © ISO 2023 – All rights reserved

© ISO 2023 – All rights reserved 5

4.1.2 Batch testing

4.1.3 Supplementary testing

6 © ISO 2023 – All rights reserved

4.2 Performance testing

Table 1 — Modules for batch and supplementary quantitative testing

4.2.2 Absence of microbial contamination

© ISO 2023 – All rights reserved 7

5 Apparatus and glassware

6 Culture media and diluents

7 Preparation of microorganisms for performance testing

8 © ISO 2023 – All rights reserved

The reference count of the laboratory-prepared inoculum or RM is determined in relation to recovery on

7.2.2 Qualitative selectivity testing

7.3 Preparation of a standardized test suspension using a working culture

7.3.2 Preparation of the working culture

© ISO 2023 – All rights reserved 9

7.3.3 Preparation of a standardized test suspension (inoculum) for the test

7.4 Preparation of a test suspension using reference material

8 Sampling of membrane filters for testing

10 © ISO 2023 – All rights reserved

9.2 Inoculation by spread plate technique

© ISO 2023 – All rights reserved 11

9.3 Inoculation by membrane filtration technique

12 © ISO 2023 – All rights reserved

9.4 Incubation and counting

9.5 Test for absence of microbial contamination

10 Calculation, expression and interpretation of results

10.2 Productivity testing

© ISO 2023 – All rights reserved 13

10.3 Selectivity testing

14 © ISO 2023 – All rights reserved

11.2 Information provided by the manufacturer

© ISO 2023 – All rights reserved 15

Diagram of the procedure for batch testing

Figure A.1 — Diagram of the procedure for the batch testing

16 © ISO 2023 – All rights reserved

Example of a card for recording test results from batch testing

© ISO 2023 – All rights reserved 17

Quantitative supplementary testing of membrane filters

C.2 Method for supplementary testing

A diagram of the procedure of supplementary testing is given in Figure C.1.