EN ISO 7704:2023

Water quality - Requirements for the performance testing of membrane filters used for direct enumeration of microorganisms by culture methods (ISO 7704:2023)

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.

Wasserbeschaffenheit - Anforderungen für die Bewertung von Membranfiltern zur direkten Zählung mittels mikrobiologischer Kulturverfahren (ISO 7704:2023)

Dieses Dokument legt die Anforderungen an die Leistungsprüfung von Membranfiltern fest, die für die Rückhaltung und die anschließende direkte Zählung von Mikroorganismen durch Verfahren zur Kultivierung verwendet werden.
Dieses Dokument ist anwendbar für Membranfilter, die für die Rückhaltung und anschließende direkte Zählung bestimmter Mikroorganismen auf festen Medien oder auf anderen Vorrichtungen, die Medien enthalten, wie absorbierendes Trägermaterial, verwendet werden [19].
Dieses Dokument ist nicht anwendbar für Membranfilter, die zur Aufkonzentrierung und Elution oder für qualitative Verfahren verwendet werden.
Die Prüfungen sind anwendbar für Membranfilter, die für die mikrobiologische Analyse verschiedener Arten von Wasser bestimmt sind, wie:
   Trinkwasser, in Flaschen abgefülltes Wasser und andere Arten von Wasser mit voraussichtlich geringer Anzahl von Mikroorganismen;
   Wasser mit einer erwarteten höheren Anzahl von Mikroorganismen, z. B. Oberflächenwasser und Prozesswasser.
Die Prüfungen sollen die Eignung des gesamten Systems (Membranfilter zusammen mit dem Nährmedium einschließlich der Filtrationsstufe) nachweisen, die für die in den Literaturhinweisen [3] [6] [8] [10] [12] und [13] beschriebenen spezifischen Prüfungen erforderlich ist.
Dieses Dokument gilt für:
   Hersteller, die Membranfilter produzieren;
   Laboratorien für Mikrobiologie, die Membranfilter für ihre eigenen Prüfungen verwenden oder diese an andere Endanwender liefern.

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 (ISO 7704:2023)

Le présent document spécifie les exigences pour les essais de performance des membranes filtrantes utilisées pour la rétention suivie d'un dénombrement direct des micro-organismes par des méthodes de culture.
Le présent document s'applique aux membranes filtrantes qui sont utilisées pour la rétention suivie d'un dénombrement direct de micro-organismes spécifiques sur des milieux solides ou sur d'autres dispositifs contenant des milieux, comme des pads en papier absorbants [19].
Le présent document ne s'applique pas aux membranes filtrantes utilisées pour la concentration et l'élution ou pour des méthodes qualitatives.
Les essais s'appliquent aux membranes filtrantes destinées à l'analyse microbiologique de différents types d'eau, tels que:
—    l'eau potable, l'eau embouteillée et d'autres types d'eau dans laquelle un faible nombre de micro-organismes est attendu;
—    l'eau dans laquelle un plus grand nombre de micro-organismes est attendu, par exemple l'eau de surface et l'eau de process.
Ces essais sont destinés à démontrer l'adéquation de l'ensemble du système (membrane filtrante et milieu de culture, y compris l'étape de filtration) requis pour les essais spécifiques décrits dans les Références [3], [6], [8], [10], [12] et [13].
Le présent document s'applique:
—    aux fabricants produisant des membranes filtrantes;
—    aux laboratoires de microbiologie utilisant des membranes filtrantes pour leurs propres essais ou les fournissant à d'autres utilisateurs finaux.

Kakovost vode - Zahteve za preskušanje lastnosti membranskih filtrov, ki se uporabljajo za štetje mikroorganizmov z metodami kultivacije (ISO 7704:2023)

Ta mednarodni standard določa zahteve za oceno membranskih filtrov za koncentriranje za neposredno štetje specifičnih mikroorganizmov in mešanih populacij. Te zahteve veljajo za vse membranske filtre, namenjene za mikrobiološko analizo vseh vrst vzorcev vode in drugih tekočin. Te zahteve so namenjene za preskus, s katerim se dokaže ustreznost celotnega sistema – membranski filter z medijem za gojenje kulture, vključno s fazo filtriranja – potrebnega za določene preskuse, opisane v mednarodnih standardih.  Mehanski filtri, zahtevani za uporabo, so opisani v posameznih standardih. Ta mednarodni standard se uporablja za proizvajalce in uporabnike, kot so: – komercialni organi, ki proizvajajo in/ali distribuirajo membranske filtre;
– nekomercialni organi, ki membranske filtre dobavljajo tretjim strankam;
– mikrobiološki laboratoriji, ki uporabljajo membranske filtre za lastno preskušanje ali jih zagotavljajo drugim uporabnikom.

General Information

• Status: Published
• Publication Date: 24-Jan-2023
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 25-Jan-2023
• Completion Date: 25-Jan-2023
• Ref Project: SIST EN ISO 7704:2023 - Water quality - Requirements for the performance testing of membrane filters used for direct enumeration of microorganisms by culture methods (ISO 7704:2023)

Standards Content (Sample)

SLOVENSKI STANDARD
01-junij-2023
Kakovost vode - Zahteve za preskušanje lastnosti membranskih filtrov, ki se
uporabljajo za štetje mikroorganizmov z metodami kultivacije (ISO 7704:2023)
Water quality - Requirements for the performance testing of membrane filters used for
direct enumeration of microorganisms by culture methods (ISO 7704:2023)
Wasserbeschaffenheit - Anforderungen für die Bewertung von Membranfiltern zur
direkten Zählung mittels mikrobiologischer Kulturverfahren (ISO 7704:2023)
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 (ISO 7704:2023)
Ta slovenski standard je istoveten z: EN ISO 7704:2023
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 7704
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2023
EUROPÄISCHE NORM
ICS 07.100.20
English Version
Water quality - Requirements for the performance testing
of membrane filters used for direct enumeration of
microorganisms by culture methods (ISO 7704:2023)
Qualité de l'eau - Exigences relatives aux essais de Wasserbeschaffenheit - Anforderungen für die
performance des membranes filtrantes utilisées pour Bewertung von Membranfiltern zur direkten Zählung
le dénombrement direct des micro-organismes par des mittels mikrobiologischer Kulturverfahren (ISO
méthodes de culture (ISO 7704:2023) 7704:2023)
This European Standard was approved by CEN on 19 November 2022.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 7704:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 7704:2023) has been prepared by Technical Committee ISO/TC 147 "Water
quality" in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of
which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by July 2023, and conflicting national standards shall be
withdrawn at the latest by July 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 7704:2023 has been approved by CEN as EN ISO 7704:2023 without any modification.

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 7704:2023(E)
© 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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO 7704:2023(E)
Contents Page
Foreword .v
Introduction .vii
1 S c op e . 1
2 Nor m at i ve r ef er enc e s . 1
3 T erms and definitions . 1
3.1 G eneral terminology . 2
3.2 T erminology of performance testing . 3
3.3 T erminology for test microorganisms . 4
4 P r i nc iple . 5
4.1 General . 5
4 .1.1 I nt r o duc t ion . 5
4.1.2 Batch testing . 6
4.1.3 Supplementary testing . 6
4.2 P erformance testing . 7
4.2.1 M odules for batch and supplementary testing . 7
4.2.2 A bsence of microbial contamination . 7
5 A pparatus and glassware .8
6 C ulture media and diluents .8
7 P reparation of microorganisms for performance testing . 8
7.1 G eneral . 8
7. 2 R ef er enc e c ou nt . 8
7.2.1 Q uantitative productivity testing . 8
7.2.2 Q ualitative selectivity testing . 9
7.2.3 Qualitative specificity testing . 9
7.3 P reparation 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 P reparation of a test suspension using reference material . 10
8 S ampling of membrane filters for testing .10
9 P rocedure .11
9.1 G eneral . 11
9.2 I noculation by spread plate technique . 11
9.2.1 G eneral . 11
9.2.2 Inoculation .12
9.3 I noculation by membrane filtration technique .12
9.3.1 General .12
9.3.2 Inoculation .12
9.4 I ncubation and counting . 13
9.5 T est for absence of microbial contamination . 13
10 C alculation, expression and interpretation of results .13
10.1 G eneral .13
10.2 P roductivity testing . 13
10.3 S electivity testing . 14
10.4 S pecificity testing . 14
11 Documentation of test results .14
11.1 Te s t r ep or t . 14
11.2 I nformation provided by the manufacturer . 15
11.3 T raceability . 15
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 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.
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 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.
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 S cope
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
[19]
absorbent pads .
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 Normat ive 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 T erms and definitions
For the purposes of this document, the following terms and definitions apply.
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 terminolo gy
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)
[8]
EXAMPLE 1 Chromogenic coliform agar (CCA) used with membrane filters in accordance with ISO 9308-1 .
[6]
EXAMPLE 2 Slanetz and Bartley medium used with membrane filters in accordance with ISO 7899-2 .
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
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 T erminology 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 T erminology 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
[15]
Note 1 to entry: See ISO Guide 30 .
Note 2 to entry: For in-house prepared quality control reference materials (QRMs), often so-called “in-house
[18]
reference materials” or “internal RM”, see ISO Guide 80 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
[15]
Note 1 to entry: See ISO Guide 30 .
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs are given in, among
[14] [16] [17]
others, ISO 17034, ISO Guide 31 and ISO Guide 35 .
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
[3] [6] [8] [10] [12] [13]
culture media, see ISO 6461-2 , ISO 7899-2 , ISO 9308-1 , ISO 11731 , ISO 14189 , ISO 16266
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
a a
selectivity , specificity
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 A pparatus and glassware
Appropriate apparatus and glassware are stated in the corresponding specific standards or in ISO 8199.
6 C ulture 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 Pr eparation 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,
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