EN ISO 16266-2:2021

Water quality - Detection and enumeration of Pseudomonas aeruginosa - Part 2: Most probable number method (ISO 16266-2:2018)

This document specifies a method for the enumeration of Pseudomonas aeruginosa in water. The method is based on the growth of target organisms in a liquid medium and calculation of the most probable number (MPN) of organisms by reference to MPN tables.
This document is applicable to a range of types of water. For example, hospital waters, drinking water and non‑carbonated bottled waters intended for human consumption, groundwater, swimming pool and spa pool waters including those containing high background counts of heterotrophic bacteria.
This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower waters or marine waters, for which the method has not been validated. These waters are, therefore, outside the scope of this document. Laboratories can employ the method presented in this document for these matrices by undertaking appropriate validation of performance of this method prior to use.
The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa through the hydrolysis of a 7‑amino‑4‑methylcoumarin aminopeptidase substrate present in a special reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the 7‑amido‑coumarin aminopeptidase substrate releasing a product which fluoresces under ultraviolet (UV) light. The test described in this document provides a confirmed result within 24 h with no requirement for further confirmation of positive wells.

Wasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa - Teil 2: Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO 16266-2:2018)

Dieses Dokument legt ein Verfahren zur Zählung von Pseudomonas aeruginosa in Wasser fest. Das Verfahren beruht auf der Vermehrung von Zielorganismen in einem flüssigen Medium und der Berechnung der wahrscheinlichsten Keimzahl (MPN, en: most probable number) der Organismen anhand von MPN Tabellen.
Dieses Dokument ist auf eine Reihe von Wasserarten anwendbar: zum Beispiel auf Wasser für den Gebrauch in Krankenhäusern, Trinkwasser und nicht kohlensäurehaltiges, in Flaschen abgefülltes Wasser für den menschlichen Gebrauch, Grundwasser, Wasser für Schwimm- und Warmsprudelbecken, einschließlich Wasser, das eine hohe Grundanzahl heterotropher Bakterien enthält.
Dieses Dokument ist nicht anwendbar auf kohlensäurehaltiges, in Flaschen abgefülltes Wasser, aromatisiertes in Flaschen abgefülltes Wasser, Wasser in Kühltürmen oder Meerwasser, also Wasserarten, für die das Verfahren nicht validiert wurde. Diese Wasserarten liegen daher außerhalb des Anwendungsbereiches dieses Dokuments. Laboratorien können das in diesem Dokument dargestellte Verfahren für diese Matrices anwenden, indem sie vor der Verwendung eine geeignete Validierung der Leistung dieses Verfahrens durchführen.
Die Untersuchung beruht auf einer Technik zum Nachweis bakterieller Enzyme, die das Vorhandensein von P. aeruginosa durch die Hydrolyse eines in einem speziellen Reagens enthaltenen 7 Amino 4 methylcoumarin-Aminopeptidase-Substrats anzeigt. Aufgrund der reichlichen Versorgung mit Aminosäuren, Vitaminen und anderen Nährstoffen aus dem Reagens wachsen und vermehren sich die Zellen von P. aeruginosa rasch. Aktiv wachsende Stämme von P. aeruginosa verfügen über ein Enzym, welches das 7 Amino‐coumarin-aminopeptidase-Substrat aufspaltet, wodurch ein unter ultraviolettem Licht fluoreszierendes Produkt freigesetzt wird. Die in diesem Dokument beschriebene Untersuchung liefert innerhalb von 24 h ein bestätigtes Ergebnis, ohne dass eine weitere Bestätigung der positiven Vertiefungen erforderlich ist.

Qualité de l'eau - Recherche et dénombrement de Pseudomonas aeruginosa - Partie 2: Méthode du nombre le plus probable (ISO 16266-2:2018)

Le présent document spécifie une méthode permettant de dénombrer Pseudomonas aeruginosa dans l'eau. Cette méthode se fonde sur la croissance d'organismes cibles dans un milieu liquide et le calcul du nombre le plus probable (NPP) d'organismes en se référant aux tables du NPP.
Le présent document s'applique à différents types d'eau. Par exemple, les eaux destinées aux hôpitaux, l'eau potable et les eaux en bouteille non gazéifiées destinées à la consommation humaine, les eaux souterraines, les eaux de piscine et de spa, y compris celles contenant des nombres importants de bactéries hétérotrophes.
Le présent document ne s'applique pas aux eaux en bouteille gazéifiées, aux eaux en bouteille aromatisées, aux eaux de tour de refroidissement ou à l'eau de mer, pour lesquelles cette méthode n'a pas été validée. Par conséquent, ces eaux ne relèvent pas du domaine d'application du présent document. Les laboratoires peuvent utiliser la méthode présentée dans le présent document pour ces matrices en prenant les mesures adéquates de validation des performances de cette méthode avant son utilisation.
L'essai se fonde sur une technologie de recherche des enzymes bactériennes qui signale la présence de P. aeruginosa en hydrolysant un substrat de 7‑amino‑4‑méthylcoumarine aminopeptidase présent dans un réactif spécial. Les cellules de P. aeruginosa se développent et se reproduisent rapidement à l'aide de l'apport important en acides aminés, vitamines et autres nutriments présents dans le réactif. Les souches de P. aeruginosa en développement actif présentent une enzyme qui dissocie le substrat de 7‑amido‑coumarine aminopeptidase, générant un produit qui présente une fluorescence sous une lumière ultraviolette (UV). L'essai décrit dans le présent document fournit un résultat confirmé dans un délai de 24 h sans avoir besoin de confirmer les puits positifs.

Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa - 2. del: Metoda najverjetnejšega števila (ISO 16266-2:2018)

Ta dokument določa metodo za ugotavljanje števila Pseudomonas aeruginosa v vodi. Metoda temelji na rasti ciljnih organizmov v tekočem mediju in izračunu najverjetnejšega števila (MPN) organizmov z referenco na tabele MPN.
Ta dokument se uporablja za različne vrste vod, na primer vodo v bolnišnicah, pitno vodo in ustekleničeno vodo brez dodanega ogljikovega dioksida, ki je namenjena za pitje, podtalnico, vodo v bazenih in toplicah, vključno s tistimi, ki vsebujejo visoko število heterotrofnih bakterij v ozadju.
Ta dokument se ne uporablja za ustekleničeno vodo z dodanim ogljikovim dioksidom, ustekleničeno vodo z okusom, vodo v hladilnem stolpu ali morsko vodo, za katere metoda ni bila potrjena. Slednje zato ne spadajo na področje uporabe tega dokumenta. Laboratoriji lahko metodo, predstavljeno v tem dokumentu, uporabijo za te matrike, če pred uporabo ustrezno potrdijo njeno učinkovitost. Preskus temelji na tehnologiji zaznavanja bakterijskih encimov, ki signalizira prisotnost P. aeruginosa s hidrolizo substrata 7-amino-4-metilkumarin aminopeptidaze, ki je prisoten v posebnem reagentu. Celice P. aeruginosa hitro rastejo in se razmnožujejo z uporabo bogate zaloge aminokislin, vitaminov in drugih hranilnih snovi, ki so prisotne v reagentu. Aktivno rastoči sevi P. aeruginosa imajo encim, ki cepi substrat 7-amido-kumarinske aminopeptidaze, pri čemer se sprosti produkt, ki fluorescira pod ultravijolično (UV) svetlobo. Preskus, opisan v tem dokumentu, poda potrjen rezultat v 24 urah brez potrebe po nadaljnjem potrjevanju pozitivnih izvorov.

General Information

• Status: Published
• Publication Date: 16-Nov-2021
• Withdrawal Date: 30-May-2022
• 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: 17-Nov-2021
• Due Date: 01-Jun-2023
• Completion Date: 17-Nov-2021
• Ref Project: SIST EN ISO 16266-2:2022 - Water quality - Detection and enumeration of Pseudomonas aeruginosa - Part 2: Most probable number method (ISO 16266-2:2018)

Standards Content (Sample)

SLOVENSKI STANDARD
01-januar-2022
Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa - 2.
del: Metoda najverjetnejšega števila (ISO 16266-2:2018)
Water quality - Detection and enumeration of Pseudomonas aeruginosa - Part 2: Most
probable number method (ISO 16266-2:2018)
Wasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa - Teil 2:
Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO 16266-2:2018)
Qualité de l'eau - Recherche et dénombrement de Pseudomonas aeruginosa - Partie 2:
Méthode du nombre le plus probable (ISO 16266-2:2018)
Ta slovenski standard je istoveten z: EN ISO 16266-2:2021
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.

EN ISO 16266-2
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Detection and enumeration of
Pseudomonas aeruginosa - Part 2: Most probable number
method (ISO 16266-2:2018)
Qualité de l'eau - Recherche et dénombrement de Wasserbeschaffenheit - Nachweis und Zählung von
Pseudomonas aeruginosa - Partie 2: Méthode du Pseudomonas aeruginosa - Teil 2: Verfahren zur
nombre le plus probable (ISO 16266-2:2018) Bestimmung der wahrscheinlichsten Keimzahl (ISO
16266-2:2018)
This European Standard was approved by CEN on 15 November 2021.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, 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: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16266-2:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Endorsement notice . 3

European foreword
The text of ISO 16266-2:2018 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-
2:2021 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 May 2022, and conflicting national standards shall be
withdrawn at the latest by May 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 16266-2:2018 has been approved by CEN as EN ISO 16266-2:2021 without any
modification.
INTERNATIONAL ISO
STANDARD 16266-2
First edition
2018-07
Water quality — Detection and
enumeration of Pseudomonas
aeruginosa —
Part 2:
Most probable number method
Qualité de l'eau — Recherche et dénombrement de Pseudomonas
aeruginosa —
Partie 2: Méthode du nombre le plus probable
Reference number
ISO 16266-2:2018(E)
©
ISO 2018
ISO 16266-2:2018(E)
© ISO 2018
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

ISO 16266-2:2018(E)
Contents Page
Foreword . v
Introduction . vi
1  Scope . 1
2  Normative references . 1
3  Terms and definitions . 2
4  Principle . 2
5  Apparatus and gla sswa r e . 2
6  Culture media, diluents and reagents . 3
6.1  Basic materials . 3
6.2  Diluent . 3
6.3  Antifoam B . 3
7  Sampling . 4
8  Procedure . 4
8.1  Transport and storage of the samples . 4
8.2  Preparation of the sample and inoculation of media . 4
8.2.1  Preparation of 100 ml samples . 4
8.2.2  Preparation of 250 ml samples . 4
8.3  Incubation and differentiation . 4
8.4  Examination of results . 5
9  Expression of results . 5
10  Quality assurance . 5
11  Test report . 5
Annex A (informative) Further microbiological information about Pseudomonas
aeruginosa . 7
Annex B (normative) The Quanti-Tray Sealer and calculation of results . 8
Annex C (normative) Composition of the Pseudalert medium . 120
Annex D (informative) Performance characteristics . 121
Bibliography . 122
© ISO 2018 – All rights reserved
iii
ISO 16266-2:2018(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 of 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbibiological methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2018 – All rights reserved

ISO 16266-2:2018(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 from Pseudomonas aeruginosa in any 250 ml sample examined (see, for example, Council
Directive 2009/54/EC, Reference [1]). Other bottled waters offered for sale are also to be free of
Pseudomonas aeruginosa in any 250 ml sample (see e.g. Council Directive 98/83/EC, Reference [2]).
Other waters, including swimming and spa pool waters, water for human consumption and hospital
waters, may sometimes be tested for Pseudomonas aeruginosa for reasons of public health. In these
cases, it is typical to examine 100 ml volumes.
The method described in this document can be applied to a range of types of water, for example,
hospital waters, drinking water and non‐carbonated bottled waters intended for human consumption,
groundwater, swimming pool and spa pool waters including those containing high background counts
of heterotrophic bacteria (see References [3], [4], [5], [6] and [7]).
© ISO 2018 – All rights reserved
v
INTERNATIONAL STANDARD ISO 16266‐2:2
...