Water quality - Enumeration of Legionella (ISO 11731:2017)

ISO 11731:2017 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.

Wasserbeschaffenheit - Zählung von Legionellen (ISO 11731:2017)

Dieses Dokument legt Kultivierungsverfahren zur Isolierung von Legionella und Abschätzung ihrer Anzahl in Wasserproben fest.
Diese Verfahren sind für alle Arten von Wasser, einschließlich Trinkwasser, Brauchwasser, Abwasser und natürliche Gewässer, anwendbar. Diese Verfahren können für wasserhaltige Matrices, z. B. Biofilme, Sedimente usw., verwendet werden.
Nicht alle Legionella-Spezies sind kultivierbar; deshalb werden mit den in dem vorliegenden Dokument beschriebenen Verfahren nicht alle Spezies von Legionella gefunden.

Qualité de l'eau - Dénombrement des Legionella (ISO 11731:2017)

ISO 11731:2017 spécifie des méthodes de culture pour l'isolement des Legionella et leur dénombrement dans des échantillons d'eau.
Ces méthodes sont applicables à tous les types d'échantillons d'eau, y compris les eaux potables, industrielles, usées et naturelles. Elles peuvent également être utilisées pour les matrices liées à l'eau (biofilms, sédiments, etc.).
Toutes les espèces de Legionella n'étant pas cultivables, les méthodes décrites dans le présent document peuvent ne pas retrouver la totalité des espèces de Legionella.

Kakovost vode - Ugotavljanje števila legionel (ISO 11731:2017)

Ta dokument določa metode gojenja za izolacijo legionele in oceno njenega števila v vzorcih vode.
Te metode se uporabljajo za vse vrste vzorcev vode, vključno s pitnimi, industrijskimi, odpadnimi in
naravnimi vodami. Te metode se lahko uporabljajo za matrice, povezane z vodo, npr. biofilme, sedimente itd.
Nekaterih vrst legionele ni mogoče gojiti, tako da metode, opisane v tem dokumentu, ne
zajamejo vseh vrst legionele.

General Information

Status
Published
Publication Date
06-Jun-2017
Technical Committee
Drafting Committee
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
07-Jun-2017
Completion Date
07-Jun-2017

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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

© ISO 2017 – All rights reserved iii
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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

iv © ISO 2017 – All rights reserved
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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.
© ISO 2017 – All rights reserved v
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SIST EN ISO 11731:2017
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.

vi © ISO 2017 – All rights reserved
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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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SIST EN ISO 11731:2017
ISO 11731:2017(E)
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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SIST EN ISO 11731:2017
ISO 11731:2017(E)
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

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

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

...

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