SIST EN ISO 11731-2:2008
(Main)Water quality - Detection and enumeration of Legionella - Part 2: Direct membrane filtration method for waters with low bacterial counts (ISO 11731-2:2004)
Water quality - Detection and enumeration of Legionella - Part 2: Direct membrane filtration method for waters with low bacterial counts (ISO 11731-2:2004)
This International Standard describes a monitoring method for the isolation and enumeration of Legionella organisms in water intended for human use (e.g. not and cold water, water used for washing), for human consumption and for treated bathing waters (e.g. swimming pools). It is escpecially suitable for waters expected to contain low number of Legionella. As the growth of Legionella may be inhibited by overgrowth of other bacterial colonies on the membrane, the method is only suitable for waters containing low bacterial counts.
Wasserbeschaffenheit - Nachweis und Zählung von Legionellen - Teil 2: Direktes Membranfiltrationsverfahren mit niedriger Bakterienzahl (ISO 11731-2:2004)
Dieser Teil von ISO 11731 beschreibt ein Kontrollverfahren zur Isolierung und Zählung von Legionella in Wasser für den menschlichen Gebrauch (z. B. Heiß und Kaltwasser, Waschwasser), Trinkwasser und aufbereitetem Badebeckenwasser (z. B. Schwimmbeckenwasser).
Das Verfahren ist besonders für Wasser mit einer voraussichtlich geringen Anzahl von Legionella geeignet. Da das Wachstum von Legionella auf der Membran durch Überwachsen mit anderen Bakterien beeinträchtigt sein kann, ist das Verfahren nur für Wasser mit niedriger Bakterienzahl geeignet.
Qualité de l'eau - Recherche et dénombrement des Legionella - Partie 2: Méthode par filtration directe sur membrane pour les eaux à faible teneur en bactéries (ISO 11731-2:2004)
L'ISO 11731:2004 décrit une méthode de contrôle pour l'isolation et le dénombrement des Legionella dans l'eau destinée à des usages (par exemple eau chaude ou froide, eau utilisée pour le lavage), dans l'eau destinée à la consommation humaine et dans les eaux de baignade traitées (par exemple eaux de piscines). Cette méthode s'applique en particulier aux eaux à faible teneur en Legionella. La croissance des Legionella peut être inhibée par la croissance excessive d'autres colonies bactériennes sur la membrane; par conséquent, la méthode n'est applicable qu'aux eaux à faible teneur en bactéries.
Kakovost vode - Ugotavljanje prisotnosti in števila legionel - 2. del: Metoda neposredne membranske filtracije za vode z majhnim številom bakterij (ISO 11731-2:2004)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11731-2:2008
01-julij-2008
Kakovost vode - Ugotavljanje prisotnosti in števila legionel - 2. del: Metoda
neposredne membranske filtracije za vode z majhnim številom bakterij (ISO 11731-
2:2004)
Water quality - Detection and enumeration of Legionella - Part 2: Direct membrane
filtration method for waters with low bacterial counts (ISO 11731-2:2004)
Wasserbeschaffenheit - Nachweis und Zählung von Legionellen - Teil 2: Direktes
Membranfiltrationsverfahren mit niedriger Bakterienzahl (ISO 11731-2:2004)
Qualité de l'eau - Recherche et dénombrement des Legionella - Partie 2: Méthode par
filtration directe sur membrane pour les eaux à faible teneur en bactéries (ISO 11731-
2:2004)
Ta slovenski standard je istoveten z: EN ISO 11731-2:2008
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 11731-2:2008 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 11731-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2008
ICS 07.100.20
English Version
Water quality - Detection and enumeration of Legionella - Part 2:
Direct membrane filtration method for waters with low bacterial
counts (ISO 11731-2:2004)
Qualité de l'eau - Recherche et dénombrement des Wasserbeschaffenheit - Nachweis und Zählung von
Legionella - Partie 2: Méthode par filtration directe sur Legionellen - Teil 2: Direktes Membranfiltrationsverfahren
membrane pour les eaux à faible teneur en bactéries (ISO mit niedriger Bakterienzahl (ISO 11731-2:2004)
11731-2:2004)
This European Standard was approved by CEN on 24 February 2008.
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 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11731-2:2008: E
worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 11731-2:2008 (E)
Contents Page
Foreword.3
Annex ZA (informative) A-deviations.4
2
---------------------- Page: 3 ----------------------
EN ISO 11731-2:2008 (E)
Foreword
The text of ISO 11731-2:2004 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 11731-2:2008 by
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 September 2008, and conflicting national standards shall be
withdrawn at the latest by September 2008.
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.
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, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 11731-2:2004 has been approved by CEN as a EN ISO 11731-2:2008 without any
modification.
3
---------------------- Page: 4 ----------------------
EN ISO 11731-2:2008 (E)
Annex ZA
(informative)
A-deviations
A-deviation: National deviation due to regulations, the alteration of which is for the time being outside the
competence of the CEN/CENELEC national member.
This European Standard does not fall under any Directive of the EC.
In the relevant CEN/CENELEC countries these A-deviations are valid instead of the provisions of the
European Standard until they have been removed.
Deviation
Country National Regulation
Netherlands Waterleidingbesluit, 7 June 1960. Amended at 28
December 2004.
Besluit hygiëne en veiligheid badinrichtingen en
zwemgelegenheden, 6 October 1984. Amended at 28
December 2004.
The Dutch regulation for detection of Legionella Waterleidingbesluit, Article 17p,
requires the use of the Dutch Standard NEN 6265 as
reference method. Criterion of number of colony
forming units in water samples is based on the results
obtained with this Standard Method.
Besluit hygiëne en veiligheid badinrichtingen en
zwemgelegenheden, Appendix IV.
Analysevoorschriften
4
---------------------- Page: 5 ----------------------
INTERNATIONAL ISO
STANDARD 11731-2
First edition
2004-05-01
Water quality — Detection and
enumeration of Legionella —
Part 2:
Direct membrane filtration method for
waters with low bacterial counts
Qualité de l'eau — Recherche et dénombrement des Legionella —
Partie 2: Méthode par filtration directe sur membrane pour les eaux à
faible teneur en bactéries
Reference number
ISO 11731-2:2004(E)
©
ISO 2004
---------------------- Page: 6 ----------------------
ISO 11731-2:2004(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2004
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2004 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 11731-2:2004(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Safety. 2
5 Principle . 2
6 Culture media and reagents. 2
7 Apparatus . 6
8 Sampling . 7
9 Procedure . 7
10 Expression of results. 9
11 Test report . 9
© ISO 2004 – All rights reserved iii
---------------------- Page: 8 ----------------------
ISO 11731-2:2004(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 11731-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
ISO 11731 consists of the following parts, under the general title Water quality — Detection and enumeration
of Legionella:
Part 2: Direct membrane filtration method for waters with low bacterial counts
The general method will be the subject of a future Part 1 of ISO 11731.
iv © ISO 2004 – All rights reserved
---------------------- Page: 9 ----------------------
INTERNATIONAL STANDARD ISO 11731-2:2004(E)
Water quality — Detection and enumeration of Legionella —
Part 2:
Direct membrane filtration method for waters with low bacterial
counts
WARNING — Persons using this part of ISO 11731 should be familiar with normal laboratory practice.
This part of ISO 11731 does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
1 Scope
This part of ISO 11731 describes a monitoring method for the isolation and enumeration of Legionella
organisms in water intended for human use (e.g. hot and cold water, water used for washing), for human
consumption and for treated bathing waters (e.g. swimming pools). It is especially suitable for waters expected
to contain low numbers of Legionella. As the growth of Legionella may be inhibited by overgrowth of other
bacterial colonies on the membrane, the method is only suitable for waters containing low bacterial counts.
2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods
1)
ISO 8199:— , Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 11731:1998, Water quality — Detection and enumeration of Legionella
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Legionella
genus of Gram-negative bacteria normally capable of growth in no less than 2 days on buffered charcoal
yeast extract agar containing L-cysteine and iron(III), and forming colonies, often white, purple to blue or lime
green in colour
NOTE Some species fluoresce under long wavelength UV light. The colonies have a ground-glass appearance when
viewed with a low power stereomicroscope. Growth does not occur in the absence of L-cysteine with the exception of
L. oakridgensis and L. spiritensis. L. oakridgensis and L. spiritensis require L-cysteine and iron for primary isolation but can
grow weakly in the absence of added L-cysteine thereafter.
1) To be published. (Revision of ISO 8199:1988)
© ISO 2004 – All rights reserved 1
---------------------- Page: 10 ----------------------
ISO 11731-2:2004(E)
4 Safety
The reagents used in this part of ISO 11731 should be subject to assessment in accordance with control
substances hazardous to health.
Legionella species can be handled safely by experienced microbiologists on the open bench in a conventional
microbiology laboratory conforming to containment level 2. Infection is caused by inhalation of the organism
and it is advisable therefore to assess all techniques for their ability to produce aerosols. If in any doubt, carry
out the work in a safety cabinet.
5 Principle
5.1 General
Bacteria, including Legionella organisms, in the water sample are concentrated by membrane filtration. After
filtration, the filter is treated with acid buffer added directly into the funnel to reduce the growth of non-
Legionella organisms. The filter is subsequently transferred onto a plate of agar medium selective for
Legionella and incubated. Samples expected to contain sufficient numbers of Legionella need not be
subjected to concentration prior to culture (9.1).
5.2 Enumeration
After incubation, morphologically characteristic colonies which form on the selective medium are regarded as
presumptive Legionella.
5.3 Confirmation
Presumptive colonies are confirmed as Legionella organisms by subculture to demonstrate their growth
requirement for L-cysteine and iron. Further biochemical and serological tests are needed for species
identification. Species identification may not be considered necessary for routine monitoring but is
indispensable in outbreak situations.
NOTE L. pneumophila serogroup 1 is the causative agent of most legionellosis cases and is therefore considered the
most “critical” type of Legionella to be found in the water system. Since increasing numbers of cases of legionellosis
caused by other serogroups of L. pneumophila and other Legionella species are being described, even the presence of
other Legionella species in water is considered a potential risk.
6 Culture media and reagents
6.1 General
Use chemicals of analytical grade in the preparation of media and reagents unless otherwise stated.
Alternatively, use commercially available dehydrated media and reagents. Prepare the media according to the
manufacturer's instruction and add freshly prepared (or thaw the stored material at room temperature prior to
use) selective agents or growth supplements at the concentrations recommended. Prepare media using glass
distilled water or water of equivalent quality complying with ISO 3696:1987, Grade 3. Other grades of
chemicals may be used providing they can be shown to produce the same results.
2 © ISO 2004 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 11731-2:2004(E)
6.2 Culture media
6.2.1 Buffered charcoal yeast extract agar medium (BCYE)
6.2.1.1 Composition
Yeast extract (bacteriological grade) 10,0 g
Agar 12,0g
Activated charcoal 2,0 g
1,0 g
α-Ketoglutarate, monopotassium salt
ACES buffer
(N-2-acetamido-2-aminoethane sulfonic acid) 10,0 g
Potassium hydroxide (KOH) (pellets) 2,8 g
L-cysteine hydrochloride monohydrate 0,4 g
Iron(III) pyrophosphate [Fe (P O )] 0,25g
4 2 7 3
Distilled water 1 000 ml
NOTE Check manufacturer's recommendations for concentration of agar to be added to provide adequate gelling
strength.
6.2.1.2 Preparation
6.2.1.2.1 Cysteine and iron solutions
Prepare fresh solutions of L-cysteine hydrochloride and iron(III) pyrophosphate by adding the 0,4 g and 0,25 g
respectively to 10 ml volumes of distilled water. Decontaminate each solution by filtration through a cellulose
ester membrane filter with an average pore size of 0,2 µm. Store in clean, sterile containers at (−20 ± 5) °C for
no more than 3 months.
6.2.1.2.2 ACES buffer
Add the ACES granules to 500 ml of distilled water and dissolve by standing in a water bath at 45 °C to 50 °C.
To a separate 480 ml of distilled water, add all the potassium hydroxide pellets and dissolve with gentle
shaking. To prepare the ACES buffer mix the two solutions.
NOTE ACES buffer can cause
...
INTERNATIONAL ISO
STANDARD 11731-2
First edition
2004-05-01
Water quality — Detection and
enumeration of Legionella —
Part 2:
Direct membrane filtration method for
waters with low bacterial counts
Qualité de l'eau — Recherche et dénombrement des Legionella —
Partie 2: Méthode par filtration directe sur membrane pour les eaux à
faible teneur en bactéries
Reference number
ISO 11731-2:2004(E)
©
ISO 2004
---------------------- Page: 1 ----------------------
ISO 11731-2:2004(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2004
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2004 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 11731-2:2004(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Safety. 2
5 Principle . 2
6 Culture media and reagents. 2
7 Apparatus . 6
8 Sampling . 7
9 Procedure . 7
10 Expression of results. 9
11 Test report . 9
© ISO 2004 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 11731-2:2004(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 11731-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
ISO 11731 consists of the following parts, under the general title Water quality — Detection and enumeration
of Legionella:
Part 2: Direct membrane filtration method for waters with low bacterial counts
The general method will be the subject of a future Part 1 of ISO 11731.
iv © ISO 2004 – All rights reserved
---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 11731-2:2004(E)
Water quality — Detection and enumeration of Legionella —
Part 2:
Direct membrane filtration method for waters with low bacterial
counts
WARNING — Persons using this part of ISO 11731 should be familiar with normal laboratory practice.
This part of ISO 11731 does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
1 Scope
This part of ISO 11731 describes a monitoring method for the isolation and enumeration of Legionella
organisms in water intended for human use (e.g. hot and cold water, water used for washing), for human
consumption and for treated bathing waters (e.g. swimming pools). It is especially suitable for waters expected
to contain low numbers of Legionella. As the growth of Legionella may be inhibited by overgrowth of other
bacterial colonies on the membrane, the method is only suitable for waters containing low bacterial counts.
2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods
1)
ISO 8199:— , Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 11731:1998, Water quality — Detection and enumeration of Legionella
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Legionella
genus of Gram-negative bacteria normally capable of growth in no less than 2 days on buffered charcoal
yeast extract agar containing L-cysteine and iron(III), and forming colonies, often white, purple to blue or lime
green in colour
NOTE Some species fluoresce under long wavelength UV light. The colonies have a ground-glass appearance when
viewed with a low power stereomicroscope. Growth does not occur in the absence of L-cysteine with the exception of
L. oakridgensis and L. spiritensis. L. oakridgensis and L. spiritensis require L-cysteine and iron for primary isolation but can
grow weakly in the absence of added L-cysteine thereafter.
1) To be published. (Revision of ISO 8199:1988)
© ISO 2004 – All rights reserved 1
---------------------- Page: 5 ----------------------
ISO 11731-2:2004(E)
4 Safety
The reagents used in this part of ISO 11731 should be subject to assessment in accordance with control
substances hazardous to health.
Legionella species can be handled safely by experienced microbiologists on the open bench in a conventional
microbiology laboratory conforming to containment level 2. Infection is caused by inhalation of the organism
and it is advisable therefore to assess all techniques for their ability to produce aerosols. If in any doubt, carry
out the work in a safety cabinet.
5 Principle
5.1 General
Bacteria, including Legionella organisms, in the water sample are concentrated by membrane filtration. After
filtration, the filter is treated with acid buffer added directly into the funnel to reduce the growth of non-
Legionella organisms. The filter is subsequently transferred onto a plate of agar medium selective for
Legionella and incubated. Samples expected to contain sufficient numbers of Legionella need not be
subjected to concentration prior to culture (9.1).
5.2 Enumeration
After incubation, morphologically characteristic colonies which form on the selective medium are regarded as
presumptive Legionella.
5.3 Confirmation
Presumptive colonies are confirmed as Legionella organisms by subculture to demonstrate their growth
requirement for L-cysteine and iron. Further biochemical and serological tests are needed for species
identification. Species identification may not be considered necessary for routine monitoring but is
indispensable in outbreak situations.
NOTE L. pneumophila serogroup 1 is the causative agent of most legionellosis cases and is therefore considered the
most “critical” type of Legionella to be found in the water system. Since increasing numbers of cases of legionellosis
caused by other serogroups of L. pneumophila and other Legionella species are being described, even the presence of
other Legionella species in water is considered a potential risk.
6 Culture media and reagents
6.1 General
Use chemicals of analytical grade in the preparation of media and reagents unless otherwise stated.
Alternatively, use commercially available dehydrated media and reagents. Prepare the media according to the
manufacturer's instruction and add freshly prepared (or thaw the stored material at room temperature prior to
use) selective agents or growth supplements at the concentrations recommended. Prepare media using glass
distilled water or water of equivalent quality complying with ISO 3696:1987, Grade 3. Other grades of
chemicals may be used providing they can be shown to produce the same results.
2 © ISO 2004 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 11731-2:2004(E)
6.2 Culture media
6.2.1 Buffered charcoal yeast extract agar medium (BCYE)
6.2.1.1 Composition
Yeast extract (bacteriological grade) 10,0 g
Agar 12,0g
Activated charcoal 2,0 g
1,0 g
α-Ketoglutarate, monopotassium salt
ACES buffer
(N-2-acetamido-2-aminoethane sulfonic acid) 10,0 g
Potassium hydroxide (KOH) (pellets) 2,8 g
L-cysteine hydrochloride monohydrate 0,4 g
Iron(III) pyrophosphate [Fe (P O )] 0,25g
4 2 7 3
Distilled water 1 000 ml
NOTE Check manufacturer's recommendations for concentration of agar to be added to provide adequate gelling
strength.
6.2.1.2 Preparation
6.2.1.2.1 Cysteine and iron solutions
Prepare fresh solutions of L-cysteine hydrochloride and iron(III) pyrophosphate by adding the 0,4 g and 0,25 g
respectively to 10 ml volumes of distilled water. Decontaminate each solution by filtration through a cellulose
ester membrane filter with an average pore size of 0,2 µm. Store in clean, sterile containers at (−20 ± 5) °C for
no more than 3 months.
6.2.1.2.2 ACES buffer
Add the ACES granules to 500 ml of distilled water and dissolve by standing in a water bath at 45 °C to 50 °C.
To a separate 480 ml of distilled water, add all the potassium hydroxide pellets and dissolve with gentle
shaking. To prepare the ACES buffer mix the two solutions.
NOTE ACES buffer can cause denaturation of the yeast extract if the following sequence is not followed.
6.2.1.2.3 Final medium
Add sequentially to the 980 ml of ACES buffer, the charcoal yeast extract and α-ketoglutarate. Prepare a
0,1 mol/l solution of potassium hydroxide (KOH) by dissolving 5,6 g in 1 l of distilled water. Prepare a
0,1 mol/l solution of sulfuric acid (H SO ) by carefully adding 5,3 ml of H SO (ρ = 1,84, of 95 % to 98 %
2 4 2 4
purity) to 1 l of distilled water. Use the solutions of 0,1 mol/l potassium hydroxide or 0,1 mol sulfuric acid as
appropriate to adjust the pH to 6,8 ± 0,2. Add the agar, mix and autoclave at (121 ± 3) °C for (15 ± 1) min
(6.2.4). After autoclaving allow to cool to (50 ± 2) °C in a water bath.
Add the L-cysteine and the iron(III) pyrophosphate solutions aseptically, mix well between additions.
© ISO 2004 – All rights reserved 3
---------------------- Page: 7 ----------------------
ISO 11731-2:2004(E)
Dispense in 20 ml volumes into Petri dishes of 90 mm to 100 mm diameter. Petri dishes of 60 mm may also
be used for incubating the membranes (see 9.1 and 9.2). The pH of the final medium is 6,8 ± 0,2 at 25 °C.
Allow excess moisture on the plates to dry and store at (5 ± 3) °C in airtight containers in the dark for up to
4 weeks.
6.2.2 Buffered charcoal yeast extract medium without L-cysteine (BCYE-Cys)
Prepare this medium in an identical manner to BCYE (6.2.1) but omit the L-cysteine.
6.2.3 Selective medium: buffered charcoal yeast extract medium with selective supplements (GVPC
medium)
NOTE This medium is identical to BCYE except that three antibiotic supplements and glycine are added to the BCYE
medium.
6.2.3.1 Selective supplements
The final concentrations in the GVPC medium shall be:
Ammonium-free glycine 3 g/l
Polymyxin B sulfate 80 000 IU/l
Vancomycin hydrochloride 0,001 g/l
Cycloheximide 0,08 g/l
Natamycin may be used instead of cycloheximide.
6.2.3.2 Preparation of antibiotic supplements
Add the appropriate amount (usually 200 mg) of polymyxin B sulfate to 100 ml of distilled water to achieve a
concentration of 14 545 IU/ml. Mix and decontaminate by membrane filtration as described in 6.2.1.2.
Dispense 5,5 ml volumes into sterile containers and store at (−20 ± 5) °C. For use, thaw at room temperature.
Add 20 mg of vancomycin hydrochloride to 20 ml of distilled water, mix and decontaminate by membrane
filtration (6.2.1.2). Dispense in 1 ml volumes in sterile containers and store at (−20 ± 5) °C. For use, thaw at
room temperature.
Add 2 g of cycloheximide to 100 ml of distilled water and decontaminate by membrane filtration as described
in 6.2.1.2. Dispense in 4 ml volumes in sterile containers and store at (−20 ± 5) °C. For use, thaw at room
temperature.
Antibiotic supplements may be stored for up to 6 months when frozen.
WARNING — Cycloheximide is hepatotoxic. Wear gloves and dust mask when handling this chemical
in the powder form.
6.2.3.3 Preparation of GVPC medium
Follow the instructions for preparation of B
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