EN ISO 16266:2008

Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by membrane filtration (ISO 16266:2006)

ISO 16266:2006 specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in samples of bottled water by a membrane filtration technique. This method can also be applied to other types of water with a low background flora, for example, pool waters and waters intended for human consumption.

Wasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa - Membranfiltrationsverfahren (ISO 16266:2006)

Diese Internationale Norm legt ein Verfahren zum Nachweis und zur Zählung von Pseudomonas aeruginosa in Proben von abgefülltem Wasser nach einem Membranfiltrationsverfahren fest. Dieses Verfahren ist auch für andere Wasserarten mit geringer Begleitflora geeignet, wie z. B. Schwimm  und Badebeckenwasser sowie Wasser für den menschlichen Gebrauch

Qualité de l'eau - Détection et dénombrement de Pseudomonas aeruginosa - Méthode par filtration sur membrane (ISO 16266:2006)

L'ISO 16266:2006 spécifie une méthode permettant d'isoler et de dénombrer Pseudomonas aeruginosa dans des échantillons d'eau embouteillée, par filtration sur membrane. Cette méthode peut également être appliquée à d'autres types d'eau présentant une faible flore interférente, par exemple les eaux de piscine et les eaux destinées à la consommation humaine.

Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa - Metoda membranske filtracije (ISO 16266:2006)

General Information

• Status: Published
• Publication Date: 19-Feb-2008
• Withdrawal Date: 30-Aug-2008
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 20-Feb-2008
• Due Date: 21-Nov-2008
• Completion Date: 20-Feb-2008
• Ref Project: SIST EN ISO 16266:2008 - Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by membrane filtration (ISO 16266:2006)

Relations

• Replaces: EN 12780:2002 - Water quality - Detection and enumeration of Pseudomonas aeruginosa by membrane filtration
• Effective Date: 22-Dec-2008

Overview

EN ISO 16266:2008 (ISO 16266:2006) specifies a standardized membrane filtration method for the detection and enumeration of Pseudomonas aeruginosa in bottled water and other waters with low background flora (for example, pool waters and waters intended for human consumption). The standard describes sampling volumes, culture media, incubation, colony enumeration and confirmation tests to reliably identify P. aeruginosa for water quality and public-health monitoring.

Key Topics and Requirements

• Principle: Filter a measured volume (or dilution) through a 0.45 µm membrane filter, place the filter onto selective medium, incubate and count characteristic colonies.
• Target organism definition: Colonies producing pyocyanin, fluorescing under UV (360 ± 20 nm), oxidase-positive and able to produce ammonia from acetamide are considered P. aeruginosa.
• Selective medium: Pseudomonas agar base (CN-agar) with CN supplement (cetrimide 0.2 g/L and nalidixic acid 0.015 g/L) is specified; detailed preparation, pH and storage conditions are given.
• Confirmation steps: Subculture suspicious colonies onto nutrient agar or King’s B; perform oxidase test, fluorescence checks and acetamide utilization (acetamide broth) to confirm identity.
• Laboratory practice: Media preparation (autoclave at 121 ± 3 °C for 15 min), use of analytical-grade reagents, incubation conditions, and plate storage limits are specified.
• Sampling guidance: Typical regulatory expectations cited - bottled and spring waters should be free of P. aeruginosa in any 250 ml sample; other waters often tested in 100 ml volumes.
• Quality assurance & safety: Tests must be carried out by trained personnel; precautions for handling hazardous reagents (e.g., acetamide) and following safety and QA procedures are emphasized.

Applications and Who Uses It

• Water testing laboratories performing routine microbiological analysis for bottled water, municipal supply or recreational waters.
• Bottled water and spring water producers confirming compliance with regulatory limits (absence in 250 ml).
• Public health and regulatory authorities evaluating water safety and enforcing standards.
• Pool operators and facility managers monitoring recreational waters with low background flora.
• Manufacturers of water treatment and packaging equipment needing validated microbiological methods for product verification.

Related Standards

• ISO 3696 (water for laboratory use), ISO 5667 (sampling guidance), ISO 7704 (membrane filters), ISO 8199 (enumeration guidance), ISO 19458 (microbial sampling). EN ISO 16266:2008 supersedes EN 12780:2002.

Keywords: EN ISO 16266:2008, ISO 16266, Pseudomonas aeruginosa detection, membrane filtration method, water quality testing, bottled water microbiology.

Standards Content (Sample)

SLOVENSKI STANDARD
01-julij-2008
1DGRPHãþD
SIST EN 12780:2003
Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa -
Metoda membranske filtracije (ISO 16266:2006)
Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by
membrane filtration (ISO 16266:2006)
Wasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa -
Membranfiltrationsverfahren (ISO 16266:2006)
Qualité de l'eau - Recherche et dénombrement de Pseudomonas aeruginosa - Méthode
par filtration sur membrane (ISO 16266:2006)
Ta slovenski standard je istoveten z: EN ISO 16266:2008
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 16266
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2008
ICS 13.060.70 Supersedes EN 12780:2002
English Version
Water quality - Detection and enumeration of Pseudomonas
aeruginosa - Method by membrane filtration (ISO 16266:2006)
Qualité de l'eau - Détection et dénombrement de Wasserbeschaffenheit - Nachweis und Zählung von
Pseudomonas aeruginosa - Méthode par filtration sur Pseudomonas aeruginosa - Membranfiltrationsverfahren
membrane (ISO 16266:2006) (ISO 16266:2006)
This European Standard was approved by CEN on 14 January 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 16266:2008: E
worldwide for CEN national Members.

Foreword
The text of ISO 16266:2006 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 16266:2008 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 August 2008, and conflicting national standards shall be withdrawn at
the latest by August 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.
This document supersedes EN 12780:2002.
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 16266:2006 has been approved by CEN as a EN ISO 16266:2008 without any modification.

INTERNATIONAL ISO
STANDARD 16266
First edition
2006-04-15
Water quality — Detection and
enumeration of Pseudomonas
aeruginosa — Method by membrane
filtration
Qualité de l'eau — Recherche et dénombrement de Pseudomonas
aeruginosa — Méthode par filtration sur membrane

Reference number
ISO 16266:2006(E)
©
ISO 2006
ISO 16266:2006(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 2006
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 [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2006 – All rights reserved

ISO 16266:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle. 2
5 Diluents, culture media and reagents. 2
6 Apparatus and glassware . 5
7 Sampling. 5
8 Procedure . 6
9 Expression of results . 7
10 Test report . 8
11 Performance data. 8
12 Interferences . 9
13 Quality assurance. 9
Annex A (informative) Further information about Pseudomonas aeruginosa. 10
Annex B (informative) Alternative media . 11
Bibliography . 12

ISO 16266:2006(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 16266 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This International Standard is the equivalent of EN 12780:2002.
iv © ISO 2006 – All rights reserved

ISO 16266:2006(E)
Introduction
Pseudomonas aeruginosa is an opportunistic pathogen of man that is capable of growth in water at very low
nutrient concentrations. At source and during marketing, a natural mineral water or a spring water is to be free
[1]
from Pseudomonas aeruginosa in any 250 ml sample examined (see, e.g. Council Directive 80/777/EEC and
[2]
Council Directive 96/70/EC ). Other bottled waters offered for sale are also to be free of Pseudomonas
[3]
aeruginosa in any 250 ml sample (see, e.g. Council Directive 98/83/EC ). Other waters, including pool waters
and water for human consumption, may sometimes be tested for Pseudomonas aeruginosa for reasons of public
health. In these cases, it is typical to examine 100 ml volumes.

INTERNATIONAL STANDARD ISO 16266:2006(E)

Water quality — Detection and enumeration of Pseudomonas
aeruginosa — Method by membrane filtration
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard 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 according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in
samples of bottled water by a membrane filtration technique. This method can also be applied to other types of
water with a low background flora, for example, pool waters and waters intended for human consumption.
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, Water for analytical laboratory use — Specification and test methods
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
1)
ISO 5667-2 , Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of water samples
ISO 6887-1, Microbiology of food and feeding stuffs — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
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
2)
ISO 19458 , Water quality — Sampling for microbiological analysis

1) ISO 5667-1 and ISO 5667-2 are currently undergoing joint revision, which will be published as ISO 5667-1.
2) To be published.
ISO 16266:2006(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Pseudomonas aeruginosa
micro-organisms that grow on selective media containing cetrimide and produce pyocyanin, or
micro-organisms that grow on selective media containing cetrimide, are oxidase positive, fluoresce under UV
radiation (360 ± 20) nm, and are able to produce ammonia from acetamide
4 Principle
4.1 Filtration
A measured volume of the water sample, or a dilution of the sample, is filtered through a membrane filter of
0,45 µm. The membrane filter is placed on the selective medium and incubated under the conditions specified
for the medium.
4.2 Enumeration
The numbers of presumptive Pseudomonas aeruginosa are obtained by counting the number of characteristic
colonies on the membrane filter after incubation. Pyocyanin-producing colonies are considered as confirmed
Pseudomonas aeruginosa but other fluorescing or reddish brown colonies require confirmation.
4.3 Confirmation
Subcultures of colonies requiring confirmation are made from the membrane filter onto plates of nutrient agar
(but see Annex B). After incubation, cultures that were not initially fluorescent are tested for the oxidase
reaction, and oxidase-pos
...