Water quality - Enumeration of Clostridium perfringens - Method using membrane filtration (ISO 14189:2013)

This International Standard specifies a method for the enumeration of vegetative cells and spores of Clostridium perfringens by the membrane filtration method in samples of water intended for human consumption. However, the method can be applied to all types of water samples provided they do not contain particulate or colloidal matter that interferes with filtration.

Wasserbeschaffenheit - Zählung von Clostridium perfringens - Verfahren mittels Membranfiltration (ISO 14189:2013)

Qualité de l'eau - Dénombrement de Clostridium perfringens - Méthode de filtration sur membrane (ISO 14189:2013)

L'ISO 14189:2013 spécifie une méthode pour le dénombrement des cellules végétatives et des spores de Clostridium perfringens par filtration sur membrane dans des échantillons d'eau destinée à la consommation humaine. Cette méthode peut toutefois s'appliquer à tous les types d'échantillons d'eau, sous réserve qu'ils ne contiennent pas de matière particulaire ou colloïdale interférant avec la filtration.

Kakovost vode - Ugotavljanje števila Clostridium perfringens - Metoda membranske filtracije (ISO 14189:2013)

Ta mednarodni standard določa metodo za ugotavljanje števila vegetativnih celic in spor Clostridium perfringens z metodo membranske filtracije vzorcev vode, namenjene za prehrano ljudi. Vendar pa je mogoče to metodo uporabiti za vse vrste vodnih vzorcev, če ne vsebujejo delcev ali koloidnih snovi, ki ovirajo filtracijo.

General Information

Status
Published
Public Enquiry End Date
05-Apr-2016
Publication Date
17-May-2017
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Feb-2017
Due Date
09-Apr-2017
Completion Date
18-May-2017

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SLOVENSKI STANDARD
SIST EN ISO 14189:2017
01-junij-2017
Kakovost vode - Ugotavljanje števila Clostridium perfringens - Metoda
membranske filtracije (ISO 14189:2013)
Water quality - Enumeration of Clostridium perfringens - Method using membrane
filtration (ISO 14189:2013)
Wasserbeschaffenheit - Zählung von Clostridium perfringens - Verfahren mittels
Membranfiltration (ISO 14189:2013)

Qualité de l'eau - Dénombrement de Clostridium perfringens - Méthode de filtration sur

membrane (ISO 14189:2013)
Ta slovenski standard je istoveten z: EN ISO 14189:2016
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 14189:2017 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 14189:2017
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SIST EN ISO 14189:2017
EN ISO 14189
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2016
EUROPÄISCHE NORM
ICS 07.100.20
English Version
Water quality - Enumeration of Clostridium perfringens -
Method using membrane filtration (ISO 14189:2013)

Qualité de l'eau - Dénombrement de Clostridium Wasserbeschaffenheit - Zählung von Clostridium

perfringens - Méthode de filtration sur membrane (ISO perfringens - Verfahren mittels Membranfiltration (ISO

14189:2013) 14189:2013)
This European Standard was approved by CEN on 15 July 2016.

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, 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

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14189:2016 E

worldwide for CEN national Members.
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SIST EN ISO 14189:2017
EN ISO 14189:2016 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 14189:2017
EN ISO 14189:2016 (E)
European foreword

The text of ISO 14189:2013 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 14189:2016

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 February 2017, and conflicting national standards

shall be withdrawn at the latest by February 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.

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, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 14189:2013 has been approved by CEN as EN ISO 14189:2016 without any modification.

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SIST EN ISO 14189:2017
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SIST EN ISO 14189:2017
INTERNATIONAL ISO
STANDARD 14189
First edition
2013-11-01
Water quality — Enumeration of
Clostridium perfringens — Method
using membrane filtration
Qualité de l’eau — Dénombrement de Clostridium perfringens —
Méthode de filtration sur membrane
Reference number
ISO 14189:2013(E)
ISO 2013
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SIST EN ISO 14189:2017
ISO 14189:2013(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2013

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
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 2013 – All rights reserved
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SIST EN ISO 14189:2017
ISO 14189:2013(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Apparatus and glassware ............................................................................................................................................................................ 2

6 Culture media and reagents ...................................................................................................................................................................... 3

6.1 Basic materials ........................................................................................................................................................................................ 3

6.2 Culture media ........................................................................................................................................................................................... 3

7 Sampling ........................................................................................................................................................................................................................ 3

8 Procedure..................................................................................................................................................................................................................... 3

8.1 Transport and storage of the sample ................................................................................................................................... 3

8.2 Heat pre-treatment to select spores ..................................................................................................................................... 3

8.3 Sample dilution ....................................................................................................................................................................................... 3

8.4 Filtration ....................................................................................................................................................................................................... 4

8.5 Incubation and examination ....................................................................................................................................................... 4

8.6 Confirmation of Clostridium perfringens ..................................................................................................................... 4

9 Expression of results ........................................................................................................................................................................................ 5

10 Test report ................................................................................................................................................................................................................... 5

11 Quality assurance ................................................................................................................................................................................................ 5

Annex A (normative) Composition and preparation of culture media and reagents ........................................7

Annex B (informative) Performance data ....................................................................................................................................................... 9

Bibliography .............................................................................................................................................................................................................................12

© ISO 2013 – All rights reserved iii
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SIST EN ISO 14189:2017
ISO 14189:2013(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 meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers

to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.
iv © ISO 2013 – All rights reserved
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SIST EN ISO 14189:2017
ISO 14189:2013(E)
Introduction

Clostridium perfringens is widely recognized as a valuable indicator for faecal pollution. Within the

intestinal tract of animals and man, these Gram-positive bacteria form spores which are resistant to

heating compared with vegetative cells. C. perfringens in the intestine exists both as spores and vegetative

cells, spores are also found in environmental samples. The spores of C. perfringens survive in water for

months, much longer than vegetative faecal indicator bacteria and consequently their presence may

indicate remote or intermittent faecal pollution. Monitoring of C. perfringens has proven useful for the

assessment of the quality of water resources and to check the stages of water treatment to evaluate the

treatment-works performance. The spores are not always inactivated by routine disinfection procedures

(e.g. chlorination).
© ISO 2013 – All rights reserved v
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SIST EN ISO 14189:2017
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SIST EN ISO 14189:2017
INTERNATIONAL STANDARD ISO 14189:2013(E)
Water quality — Enumeration of Clostridium perfringens —
Method using membrane filtration

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 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 staff.
1 Scope

This International Standard specifies a method for the enumeration of vegetative cells and spores of

Clostridium perfringens by the membrane filtration method in samples of water intended for human

consumption. However, the method can be applied to all types of water samples provided they do not

contain particulate or colloidal matter that interferes with filtration.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture

ISO/TS 11133-1, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production

of culture media — Part 1: General guidelines on quality assurance for the preparation of culture media in

the laboratory
ISO 19458, Water quality — Sampling for microbiological analysis

ISO/IEC Guide 2:2004, Standardization and related activities — General vocabulary

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and the

following apply:
3.1
presumptive Clostridium perfringens

bacteria which produce all shades of black or grey to yellow brown colonies on tryptose-sulfite-cycloserine

agar, even if the colour is faint, after anaerobic incubation at (44 ± 1) °C for (21 ± 3) h

Note 1 to entry: Unlike colonies growing directly on the agar medium, colonies on the membrane do not always

display a distinct blackening, so faint colonies are included in the count.
3.2
confirmed Clostridium perfringens

bacteria that produce characteristic colonies on tryptose-sulfite-cycloserine agar and possess the

enzyme acid phosphatase
© ISO 2013 – All rights reserved 1
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SIST EN ISO 14189:2017
ISO 14189:2013(E)
4 Principle

A measured volume of the sample, or a dilution of it, is filtered through a membrane with a pore size of

0,45 µm sufficient to retain spores of clostridia. The membrane is incubated on a selective/differential

agar (tryptose-sulfite-cycloserine agar) anaerobically at (44 ± 1) °C for (21 ± 3) h. C. perfringens usually

produce black or grey to yellow brown colonies as a result of the reduction of sulfite to sulfide which

reacts with a ferric salt in the medium. Characteristic colonies are counted and confirmatory tests are

carried out. The result is calculated as the colony count per sample volume. If a count of spores alone is

required the sample is first pre-treated at (60 ± 2) °C to inactivate vegetative bacteria.

NOTE 1 The medium contains cycloserine as a selective agent to inhibit Bacillus species.

NOTE 2 Incubation at 44 °C increases the selectivity of the test for C. perfringens.

5 Apparatus and glassware

Except for disposable glassware or plastics ware which is delivered sterile, sterilize glassware as

specified in ISO 8199.
Usual microbiological equipment and particularly:
5.1 Membrane filtration apparatus, as specified in ISO 8199.
5.2 Sterile filter funnels

Use funnels either delivered sterile or sterilized as specified in ISO 8199. Alternatively flaming of funnels

made of metal prior to their use is acceptable.
NOTE For this method it is insufficient to disinfect funnels by boiling.
5.3 Sterile membrane filters, nominal pore size 0,45 µm.

The quality of membrane filters may vary from brand to brand or even from batch to batch. It is therefore

advisable to check the quality on a regular basis.
5.4 Incubators, capable of being maintained at (36 ± 2) °C and at (44 ± 1) °C.

5.5 Water bath (optional), capable of being maintained at (60 ± 2) °C equipped with a means of

circulating the water.
5.6 Autoclave, capable of being maintained at (121 ± 3) °C.
5.7 Sterile forceps
5.8 Anaerobic jars, or similar equipment.

5.9 Anaerobic gas generating system, to generate an atmosphere of approximately 90 % hydrogen

and 10 % carbon dioxide.
2 © ISO 2013 – All rights reserved
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ISO 14189:2013(E)
6 Culture media and reagents
6.1 Basic materials

For uniformity of results, in the preparation of media, use constituents of uniform quality and chemicals

of recognized analytical grade. For preparation of the media use glass-distilled water or deionized water

[1]
of equivalent purity, as specified for water grade 3 in ISO 3696 .

Alternatively, use commercially available dehydrated complete medium and reagents prepared and

used according to
...

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