EN ISO 13647:2026

• Abstract:

  This document specifies a method for the enumeration of culturable microorganisms in water by counting colonies on a low-nutrient agar culture medium by spread plate inoculation after incubation at 22 °C for 7 d.; This document is applicable to:; water from the treatment process of public drinking water supplies (e.g. process water in the waterworks);; chlorinated and non-chlorinated tap water in distribution systems and containers;; bottled water; ; well water intended for human consumption.; For detailed information on the performance characteristics, see Annex B.; This document can be applied to other water matrices if the appropriate validation of performance of this method has been undertaken by the laboratory prior to use.

• Status: Published
• Publication Date: 18-Aug-2026
• ICS: 07.100.20 - Microbiology 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: 19-Aug-2026
• Due Date: 05-Feb-2026
• Completion Date: 19-Aug-2026
• Ref Project: SIST EN ISO 13647:2026 - Water quality - Enumeration of culturable microorganisms - Colony count by spread plate inoculation on R2A medium (ISO 13647:2026)

• Consolidated By: ISO 13647:2026 - Water quality — Enumeration of culturable microorganisms — Colony count by spread plate inoculation on R2A medium
• Effective Date: 12-Feb-2026

Overview

EN ISO 13647:2026 - Water quality - Enumeration of culturable microorganisms - Colony count by spread plate inoculation on R2A medium - is an international standard developed by CEN and ISO. It outlines a laboratory method for determining the number of culturable microorganisms in water samples by counting colonies grown on a low-nutrient agar medium (R2A). The method involves spread plate inoculation and incubation at 22 °C for 7 days. This standard is widely used for monitoring and assessing water quality in various water matrices, including public drinking water supplies, distribution systems, bottled water, and well water intended for human consumption.

Key Topics

• Enumeration of Culturable Microorganisms: The standard covers procedures to recover and count bacteria, yeasts, and molds able to form colonies under specific incubation conditions on R2A agar.
• Spread Plate Technique: It specifies the use of spread plating to distribute a measured sample volume across the surface of the R2A medium.
• Incubation Conditions: Incubation at 22 °C for 7 days is recommended to favor the growth of both fast- and slow-growing microorganisms.
• Sample Preparation and Dilution: Provides guidance on preparing water samples, avoiding cross-contamination, and selecting appropriate dilution levels.
• Quality Assurance: Emphasizes the need for proper laboratory controls, including equipment, media performance, and use of control strains and blanks.
• Reporting: Details requirements for expressing results as colony forming units per milliliter (cfu/ml), and necessary information for test reports.
• Applicability: Can be used for treated and untreated drinking water, chlorinated and non-chlorinated water, and bottled water. With appropriate validation, it is adaptable to other water matrices.

Applications

EN ISO 13647:2026 is practical for organizations seeking to ensure and document the microbiological safety of water supplies:

• Drinking Water Treatment and Distribution: Used by utilities to monitor the effectiveness of water treatment processes and detect changes in microbial populations that could indicate contamination or operational failures.
• Regulatory Compliance: Supports meeting legal and regulatory requirements for safe, potable water by providing a recognized analytical methodology.
• Bottled Water Quality Control: Employed by bottled water manufacturers for batch testing and validation of production hygiene.
• Well and Groundwater Monitoring: Applied in environmental and public health studies to assess water quality at the point of use, particularly in wells intended for human consumption.
• Research and Validation: Useful in scientific studies and laboratory method comparisons, especially for waters with low nutrient content.

The standard’s focus on low-nutrient (oligotrophic) conditions using R2A medium enhances the detection of microorganisms not easily recovered by standard nutrient-rich media, thus providing more comprehensive data on water microbial quality.

Related Standards

For comprehensive water quality analysis and laboratory best practices, the following related standards provide additional guidance:

• ISO 6222: Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in a nutrient agar culture medium.
• ISO 5667-5: Water quality - Sampling - Part 5: Guidance on sampling of drinking water from treatment works and piped distribution systems.
• ISO 8199: Water quality - General requirements and guidance for microbiological examinations 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.
• ISO 29201 and ISO 13843: Guidance on measurement uncertainty and establishing performance characteristics for microbiological methods.

By adopting EN ISO 13647:2026, laboratories and water supply authorities can implement robust, standardized approaches to water quality monitoring, enhancing public health protection and regulatory compliance. This standard is a vital reference for effective microbial risk management in water supply systems.

SLOVENSKI STANDARD
01-oktober-2026
Kakovost vode - Ugotavljanje števila mikroorganizmov, sposobnih tvorbe kolonij -
Štetje kolonij z nasajanjem na gojišče R2A (ISO 13647:2026)
Water quality - Enumeration of culturable microorganisms - Colony count by spread plate
inoculation on R2A medium (ISO 13647:2026)
Wasserbeschaffenheit - Zählung der kultivierbaren Mikroorganismen - Koloniezählung
durch Ausplattieren auf R2A-Medium (ISO 13647:2026)
Qualité de l'eau - Dénombrement des micro-organismes revivifiables - Comptage des
colonies par ensemencement par étalement sur un milieu gélosé R2A (ISO 13647:2026
Ta slovenski standard je istoveten z: EN ISO 13647:2026
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 13647
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2026
EUROPÄISCHE NORM
ICS 07.100.20
English Version
Water quality - Enumeration of culturable microorganisms
- Colony count by spread plate inoculation on R2A medium
(ISO 13647:2026)
Qualité de l'eau - Dénombrement des micro- Wasserbeschaffenheit - Zählung der kultivierbaren
organismes revivifiables - Comptage des colonies par Mikroorganismen - Koloniezählung durch
ensemencement par étalement sur un milieu gélosé Ausplattieren auf R2A-Medium (ISO 13647:2026)
R2A (ISO 13647:2026)
This European Standard was approved by CEN on 10 August 2026.

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, Türkiye 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
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13647:2026 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 13647:2026) 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 February 2027, and conflicting national standards
shall be withdrawn at the latest by February 2027.
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/national committee. 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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 13647:2026 has been approved by CEN as EN ISO 13647:2026 without any modification.

International
Standard
ISO 13647
First edition
Water quality — Enumeration
2026-08
of culturable microorganisms
— Colony count by spread plate
inoculation on R2A medium
Qualité de l'eau — Dénombrement des micro-organismes
revivifiables — Comptage des colonies par ensemencement par
étalement sur un milieu gélosé R2A
Reference number
ISO 13647:2026(en) © ISO 2026
ISO 13647:2026(en)
© ISO 2026
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]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO 13647:2026(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Culture medium and diluents . 2
6 Equipment and consumables . 2
7 Sampling, transport and storage . . 3
8 Procedure . 3
8.1 Preparation of the sample .3
8.2 Inoculation .3
8.3 Incubation and counting of colonies .3
9 Expression of results . 3
10 Test report . 4
11 Quality assurance . 4
Annex A (normative) Composition, preparation and performance testing of culture medium . 5
Annex B (informative) Performance characteristics . 7
Annex C (informative) Additional information about colony counting methods for heterotrophic
plate counting . 8
Annex D (informative) Determination of acceptable time between sampling and analysis . 9
Bibliography .11

iii
ISO 13647:2026(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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,
Microbiological methods, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 230, Water analysis, in accordance with the Agreement on technical cooperation between
ISO and CEN (Vienna Agreement).
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 13647:2026(en)
Introduction
Treated drinking water contains a variety of microorganisms derived from various sources, such as raw
water resource, different treatment steps within drinking water production and drinking water distribution
networks.
Enumeration of the overall colony count of culturable microorganisms provides useful information for the
assessment and surveillance of water quality. Colony counts are useful for assessing the raw water quality
and efficiency of water treatment processes and provide an indication of the cleanliness and integrity of the
drinking water distribution systems.
However, the main value of colony counts lies in the detection of changes from the expected values, based
on representative monitoring. The change in colony counts can be caused for various reasons like biofilm
formation, increase in water temperature, presence of microbially available nutrients or decrease in chlorine
concentration. Any substantial increase in count can also be either a warning of potential treatment failure
or contamination.
Nutrient rich tryptone yeast extract agar medium has been used for long time in water microbiology.
ISO 6222 provides a monitoring method for heterotrophic plate counts in tap water quality verification.
However, ISO 6222 only applies to the detection of microorganisms in nutrient rich conditions at 22 °C in 3 d
or at 36 °C in 2 d.
Reasoner’s 2 agar (R2A) medium, which has a low-nutrient composition and long incubation time (7 d at
22 °C), enables the determination of micro-organisms forming colonies after a longer incubation time.
Furthermore, the low level of nutrients in the medium minimizes the colony size and overgrowth. Therefore,
the R2A method is especially suitable for providing more information on the enumeration of heterotrophic
colony counts in water with low colony counts.
The method is well suited for monitoring water for human consumption which is low in nutrients and is
distributed in temperatures below 20 °C (see Reference [1]), for example, to measure the operational
efficiency of the treatment process of public drinking water supplies. The low-nutrient agar specified in this
document for enumeration of culturable microorganisms usually gives higher colony counts from water
samples than typically used nutrient-rich formulations of culture media (see References [2], [3] and [4]).

v
International Standard ISO 13647:2026(en)
Water quality — Enumeration of culturable microorganisms
— Colony count by spread plate inoculation on R2A medium
WARNING — Persons using this document should be familiar with normal laboratory practice. This
document does not purport to address all of the safety concerns, if any, associated with its use. It is
the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — The tests in this document shall be carried out by suitably qualified staff.
1 Scope
This document specifies a method for the enumeration of culturable microorganisms in water by counting
colonies on a low-nutrient agar culture medium by spread plate inoculation after incubation at 22 °C for 7 d.
This document is applicable to:
— water from the treatment process of public drinking water supplies (e.g. process water in the waterworks);
— chlorinated and non-chlorinated tap water in distribution systems and containers;
— bottled water;
— well water intended for human consumption.
For detailed information on the performance characteristics, see Annex B.
This document can be applied to other water matrices if the appropriate validation of performance of this
method has been undertaken by the laboratory prior to use.
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 5667-5, Water quality — Sampling — Part 5: Guidance on sampling of drinking water from treatment works
and piped distribution systems
ISO 8199:2018, Water quality — General requirements and guidance for microbiological examinations 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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/

ISO 13647:2026(en)
3.1
culturable microorganism
aerobic bacteria, yeast or mould capable of forming colonies on a culture medium
4 Principle
A test portion of the sample or dilutions of the sample are inoculated on the surface of a specified culture
medium (see Annex A) in Petri dishes by spread plate technique.
Plates are incubated at (22 ± 2) °C for (164 ± 4) h.
All visible colonies are counted using a magnifying equipment as culturable microorganisms. The colony
forming unit (cfu) per millilitre (ml) of the sample is calculated from the number of colonies counted.
NOTE 1 The spread plate technique causes no heat shock and all colonies are on top of the agar surface where they
can be distinguished readily from particles and bubbles.
NOTE 2 The low-nutrient agar in use in this document prevents the plates from becoming overgrown by faster
growing microorganisms and allows the slower growing microorganisms to become apparent due to the longer
incubation time.
NOTE 3 For certain purposes such as for waters with very low nutrient content such as de-ionised, distilled or
reverse osmosis waters (see References [5], [6] and [7]), colony counting can be done with the pour plate technique or
membrane filtration instead of the spread plate technique. For more information, see Annex C and ISO 8199.
5 Culture medium and diluents
Follow good laboratory practices in accordance with ISO 8199. Use ingredients of uniform quality and
chemicals of analytical grade. For composition, preparation and performance testing of culture medium,
follow the instructions given in Annex A and ISO 11133. Alternatively, use commercially available medium
which conforms with the composition given in Annex A and strictly follow the manufacturer’s instructions.
NOTE This document describes the use of R2A medium (see Refer
...