SIST EN ISO 11731:2017

SLOVENSKI STANDARD
SIST EN ISO 11731:2017
01-december-2017
1DGRPHãþD
SIST EN ISO 11731-2:2008
Kakovost vode - Ugotavljanje števila legionel (ISO 11731:2017)
Water quality - Enumeration of Legionella (ISO 11731:2017)
Wasserbeschaffenheit - Zählung von Legionellen (ISO 11731:2017)
Qualité de l'eau - Dénombrement des Legionella (ISO 11731:2017)
Ta slovenski standard je istoveten z: EN ISO 11731:2017
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 11731:2017 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11731:2017

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SIST EN ISO 11731:2017


EN ISO 11731
EUROPEAN STANDARD

NORME EUROPÉENNE

June 2017
EUROPÄISCHE NORM
ICS 07.100.20 Supersedes EN ISO 11731-2:2008
English Version

Water quality - Enumeration of Legionella (ISO
11731:2017)
Qualité de l'eau - Dénombrement des Legionella (ISO Wasserbeschaffenheit - Zählung von Legionellen (ISO
11731:2017) 11731:2017)
This European Standard was approved by CEN on 12 February 2017.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11731:2017 E
worldwide for CEN national Members.

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SIST EN ISO 11731:2017
EN ISO 11731:2017 (E)
Contents Page
European foreword . 3

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SIST EN ISO 11731:2017
EN ISO 11731:2017 (E)
European foreword
This document (EN ISO 11731:2017) 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 December 2017, and conflicting national standards
shall be withdrawn at the latest by December 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 11731-2:2008.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11731:2017 has been approved by CEN as EN ISO 11731:2017 without any modification.

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SIST EN ISO 11731:2017

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SIST EN ISO 11731:2017
INTERNATIONAL ISO
STANDARD 11731
Second edition
2017-05
Water quality — Enumeration of
Legionella
Qualité de l’eau — Dénombrement des Legionella
Reference number
ISO 11731:2017(E)
©
ISO 2017

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SIST EN ISO 11731:2017
ISO 11731:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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SIST EN ISO 11731:2017
ISO 11731:2017(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Examination. 2
4.3 Confirmation . 2
5 Apparatus and glassware . 2
6 Culture media and reagents . 3
7 Sampling . 4
8 Procedure. 4
8.1 Samples . 4
8.2 Concentration of water samples . 5
8.2.1 General. 5
8.2.2 Membrane filtration and direct placing of the membrane filter on culture media 5
8.2.3 Membrane filtration followed by a washing procedure . 5
8.3 Sample pre-treatment . 6
8.3.1 Heat treatment. 6
8.3.2 Acid treatment . 6
8.4 Culture . 6
8.4.1 General. 6
8.4.2 Samples with a high concentration of Legionella species and a low
concentration of interfering microorganisms . 6
8.4.3 Samples with a low concentration of Legionella species and a low
concentration of interfering microorganisms . 6
8.4.4 Samples with a high concentration of interfering microorganisms . 7
8.4.5 Samples with an extremely high concentration of interfering microorganisms . 7
8.4.6 Incubation . 7
8.4.7 Examination of the plates . 7
8.5 Confirmation of presumptive Legionella colonies on culture media: BCYE agar and
BCYE–cys agar . 8
9 Expression of results . 8
10 Test report . 9
11 Quality assurance .10
11.1 General .10
11.2 Performance testing of Legionella culture media .10
11.3 Preparing working culture and test suspension for performance testing .10
Annex A (informative) Legionella species .12
Annex B (normative) Culture media .14
Annex C (normative) Diluents .20
Annex D (normative) Acid solution.21
Annex E (informative) Scraping or rubbing the bacteria from membrane filters .22
Annex F (informative) Centrifugation technique .23
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SIST EN ISO 11731:2017
ISO 11731:2017(E)

Annex G (informative) Indirect immunofluorescent antibody assay for the identification of
Legionella species .24
Annex H (informative) Performance data .27
Annex I (informative) Pre-treatment of water related matrices .31
Annex J (normative) Decision matrix .32
Bibliography .38
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SIST EN ISO 11731:2017
ISO 11731:2017(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 on 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 the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This second edition of ISO 11731 cancels and replaces ISO 11731:1998 and ISO 11731-2:2004, which
have been technically revised.
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ISO 11731:2017(E)

Introduction
After the first recognized outbreak of Legionnaires’ disease in 1976, the isolated bacterium was named
Legionella pneumophila. Legionellae are widely found in natural and artificial aquatic environments,
soils, composts and can cause legionellosis. Legionellae can grow intracellularly in protozoa like
Acanthamoeba castellanii, Hartmannella species or Naegleria species. At least 61 different Legionella
species have been described. In 26 of these species, some strains infecting humans have been reported.
Legionella pneumophila can be subtyped into at least 15 different serogroups; nine other species also
can be subtyped into at least two separate serogroups. Monitoring for legionellae is important for
public health reasons to identify environmental sources which can pose a risk of legionellosis, such
as evaporative cooling towers, hot- and cold-water distribution systems in buildings and associated
equipment such as spa pools, dental units, air conditioning units, etc. Monitoring is also important for
validation of control measures and ongoing verification that controls remain effective.
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SIST EN ISO 11731:2017
INTERNATIONAL STANDARD ISO 11731:2017(E)
Water quality — Enumeration of Legionella
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user of this document to establish appropriate safety and
health practices and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified and competent staff.
1 Scope
This document specifies culture methods for the isolation of Legionella and estimation of their numbers
in water samples.
These methods are applicable to all kinds of water samples including potable, industrial, waste and
natural waters. These methods can be used for water related matrices, e.g. biofilms, sediments, etc.
Not all Legionella species are culturable; therefore, the methods described in this document do not
recover all species of Legionella.
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 3696, Water for analytical laboratory use — Specification and test methods
ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses
ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
Legionella
genus of microorganisms normally capable of growth on buffered charcoal yeast extract (BCYE) agar
containing L-cysteine and iron(III) salts
Note 1 to entry: For a more detailed description of Legionella species, see Annex A.
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4 Principle
4.1 General
Legionellae in the water sample are concentrated by membrane filtration, diluted or directly plated
depending on the origin/characteristics of the sample. The desired level of detection can vary depending
on (inter)national legislation and the reason for sampling or investigation. Samples expected to contain
high numbers of legionellae, such as those obtained during outbreak investigations, can be processed
with and/or without the concentration steps. To reduce the growth of the concentrated non-target
bacteria, which can interfere with the recovery of the target legionellae, portions of the water samples
are also subjected to heat treatment, acid treatment or a combination of both treatments.
Dilution is necessary when high concentrations of Legionella and/or other bacteria are expected.
Separate portions of the diluted sample should be pre-treated; one with heat and a second with acid
solution or, in case of extremely contaminated samples, with a combination of acid solution and heat
before culturing on selective media.
Treated and/or untreated portions of the sample are transferred onto plates of the chosen culture
medium selective for Legionella and incubated.
NOTE Mechanical treatment of the sample can enhance the recovery of Legionella.
4.2 Examination
After incubation, morphologically characteristic colonies on the selective culture media are regarded
as presumptive Legionella.
4.3 Confirmation
Presumptive colonies are confirmed as Legionella by subculture to demonstrate their growth
requirement for L-cysteine and iron(III).
NOTE If species and serotype identification are requested, further tests are needed (see Annex G). These
tests are not part of the standardized methods described in this document.
5 Apparatus and glassware
Usual laboratory equipment and in particular:
5.1 Sterile Petri dishes.
5.2 Incubator, capable of being maintained at (36 ± 2) °C.
5.3 Ultraviolet lamp, emitting light of wavelength (360 ± 20) nm.
5.4 Membrane filtration equipment, suitable for filtering water volumes of 10 ml up to 1 000 ml.
5.5 Membrane filter.
5.5.1 Membrane filter for concentration and elution, polycarbonate or polyethersulfone membrane
filters, diameter 47 mm to 142 mm with rated pore sizes of 0,2 µm; see Reference [6]. These types of
membrane filters are used for concentration followed by a washing procedure.
5.5.2 Membrane filter for direct placing on culture media, membrane filters from cellulose nitrate
or mixed cellulose esters, diameter 47 mm to 50 mm with rated pore sizes of 0,2 µm or 0,45 µm. These
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SIST EN ISO 11731:2017
ISO 11731:2017(E)

types of membrane filters are used for direct placing onto the culture media after filtration. Filters shall
be evaluated prior to use in accordance with ISO 7704.
NOTE Black membrane filters contrast better with the white Legionella colonies than light-coloured
membrane filters.
5.6 pH meter, with an accuracy of ± 0,1 at 20 °C to 25 °C.
5.7 Vortex mixer.
5.8 Ultrasonic water bath, suitable for ensuring that the level of diluent covering the membrane filter
is below the level of water in the water bath.
5.9 Water bath, capable of being maintained at (50 ± 1) °C.
5.10 Glassware, sterilized according to ISO 8199.
5.11 Dissection microscope, stereoscopic, with magnification of at least 4× and with oblique incident
illumination.
NOTE Also, a hand lens (magnification at least 4×) can be used.
5.12 Disinfected forceps, for handling of membrane filters.
NOTE Forceps with round ends are generally used in order not to damage the membrane during handling.
5.13 Screw cap sterile container, with or without sterile glass beads. To ensure maximum removal of
the legionellae from the membrane filter, sterile glass beads (diameter 2 mm to 3 mm) can be added to
the sterile container. Add sufficient glass beads to the sterile container just enough to cover the bottom of
the container.
6 Culture media and reagents
Use chemicals of analytical grade in the preparation of culture media and reagents unless otherwise
stated (see the Note). Prepare the culture media and reagents according to the instructions given in
Annexes B, C and D. Prepare culture media using distilled or demineralized water, which is free from
substances that might affect growth of microorganisms under the test conditions. The water shall
comply with the requirements of ISO 3696, grade 3.
Alternatively, use commercially available culture media and reagents prepared and used according to
the manufacturer’s instructions.
NOTE Chemicals of other grades can be used, providing they are shown to be of equal performance in the test.
6.1 Culture media.
See Annex B.
6.1.1 Buffered charcoal yeast extract (BCYE) agar.
See B.1.
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6.1.2 Buffered charcoal yeast extract agar without L-cysteine (BCYE–cys).
See B.2.
NOTE Blood agar (see B.6), nutrient agar (see B.7) or tryptone soy agar (see B.8) can be used instead of
BCYE–cys agar.
6.1.3 Buffered charcoal yeast extract agar with selective supplements (BCYE+AB).
See B.3.
6.1.4 Glycine vancomycin polymyxin B cycloheximide (GVPC) agar.
See B.4.
6.1.5 Modified Wadowsky Yee (MWY) agar.
See B.5.
6.2 Diluents.
See Annex C.
6.2.1 Page’s saline.
See C.1.
6.2.2 Diluted Ringer’s solution.
See C.2.
6.3 Acid solution.
See Annex D.
7 Sampling
Carry out sampling, transport and storage of the samples in accordance with ISO 19458. Take care not
to expose the samples to adverse temperature conditions (e.g. freezing or overheating).
NOTE The use of insulated containers is helpful in this regard.
8 Procedure
8.1 Samples
Due to the complex nature of different sample matrices, the laboratory shall determine the appropriate
method for each sample type. The decision matrix is provided in Annex J to determine which appropriate
method shall be undertaken. Annex J describes the requirements and provides additional options.
In order to ensure the detection of legionellae from water samples, a concentration technique by
membrane filtration (see 8.2.2 or 8.2.3) will be required in most cases. Where the concentration of
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legionellae is expected to be greater than 10 colony forming units per litre (cfu/l), direct plating of
the unconcentrated sample can also be carried out. For highly contaminated samples, dilute (refer
to Annex C for suitable diluents) and use direct plating before and after the pre-treatment (see 8.3).
Record volumes of sample diluted or processed and which pre-treatment(s) has (have) been applied.
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When the number of legionellae in any given sample is not known, concentration techniques are usually
performed. Therefore, follow the procedure described in 8.2.2 or 8.2.3.
8.2 Concentration of water samples
8.2.1 General
For a general description of the membrane filtration technique, see ISO 8199. Filtration can be done by
vacuum filtration or positive pressure filtration.
The flow rate should be adjusted so as not to exceed the maximum specified by the manufacturer for
the filter size or type.
NOTE The procedure for water related matrices (swabs, sediment, etc.) is described in Annex I.
8.2.2 Membrane filtration and direct placing of the membrane filter on culture media
Filter the water sample (without treatment, after acid treatment and, if required, after heat treatment)
through a cellulose nitrate or mixed cellulose esters membrane filter (5.5.2). The acid treatment can
also be done directly on the membrane filter in the funnel (see 8.3.2). The volume filtered depends on
the particulate content of the water or the desired detection level. The filtered volume of the sample
shall be recorded. Carefully remove the membrane filter from the stand with disinfected forceps
(5.12) and place it (right-side up) directly on the culture media, ensuring that no air bubble is trapped
underneath.
NOTE Where concentration by filtration is not possible (e.g. due to a high level of deposit), the sample can be
concentrated by centrifugation (see Annex F).
8.2.3 Membrane filtration followed by a washing procedure
Filter the water sample through a polycarbonate or polyethersulfone membrane filter (5.5.1). The
volume filtered depends on the particulate content of the water or the desired detection level. The
filtered volume of the sample shall be recorded. Remove the membrane filter from the stand w
...