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

EN ISO 13647:2026 is a European Standard published by CEN that adopts ISO 13647:2026 without modification. It specifies a method for counting culturable microorganisms in water by spread plate inoculation on R2A medium after incubation at 22 °C for 7 days. Laboratories use it for drinking water, bottled water, well water and related treatment or distribution samples, and it can also be applied to other water matrices after laboratory validation.

What does EN ISO 13647:2026 specify?

EN ISO 13647:2026 specifies a microbiological water test for enumerating culturable microorganisms on a low-nutrient agar medium. The method is based on spread plate inoculation and colony counting after the defined incubation period, so it is aimed at routine water quality monitoring and comparison of results over time.

The document is organized into clauses on scope, normative references, terms and definitions, principle, culture medium and diluents, equipment and consumables, sampling, procedure, expression of results, test report, and quality assurance.

Annex	What it covers
A	Composition, preparation and performance testing of the R2A culture medium
B	Performance characteristics
C	Additional information about colony counting methods for heterotrophic plate counting
D	Acceptable time between sampling and analysis

What are the key requirements of EN ISO 13647:2026?

EN ISO 13647:2026 ties together sampling, culture medium control, incubation, colony counting, and reporting so the result is traceable and comparable. In practice, the method only gives meaningful water-quality information if the sample is handled correctly, the medium performs as expected, and the colony count is expressed in the same way every time.

• Clause 4, Principle - A sample or sample dilution is spread on the surface of the specified medium in Petri dishes, incubated at 22 ± 2 °C for 164 ± 4 h, and all visible colonies are counted. This matters because the method is built around surface growth, which makes colonies easier to distinguish from particles and bubbles.

• Clause 5, Culture medium and diluents - The document requires good laboratory practice, suitable ingredient quality, Annex A for the medium, and ISO 11133 for preparation and performance testing. Diluents are taken from ISO 8199. For the reader, this means the medium and dilutions are part of the method control, not just background laboratory work.

• Clause 6, Equipment and consumables - The method uses a sterilization autoclave, a controlled incubator, a pH meter, magnifying equipment, and a refrigerator, with laminar flow cabinet use as an option. This is important because stable temperature control and contamination control directly affect colony recovery.

• Clause 7, Sampling, transport and storage - Sampling follows ISO 19458 and ISO 5667-5, and transport and storage are at 5 °C. The document points to analysis as soon as possible and to Annex D for the storage-time study. In practice, sample history must be controlled so the count reflects the water, not storage effects.

• Clause 8, Procedure - The sample is prepared and diluted according to ISO 8199, the plates are dried, 0.1 ml is spread on a 90 mm Petri dish, and the inoculated plates are inverted and incubated. The note on duplicate plates and serial dilutions is useful because low counts and variable samples often need more than one plate to improve precision.

• Clause 9, Expression of results - Results are reported as cfu/ml, dilutions are combined as a weighted mean, and ISO 29201 is cited for measurement uncertainty. The document also states a detection limit of 10 cfu/ml when 0.1 ml is used, which helps users judge whether a result is near the method limit.

• Clauses 10 and 11 plus Annex A - The test report must identify the sample, method, result, deviations, unusual occurrences, and test date, and the laboratory must run quality control with control strains and blanks. This supports auditability and batch-to-batch confidence in the medium and technique.

• Annex B - The performance characteristics support method selection and verification, especially when the laboratory wants to compare the method with other water microbiology approaches or confirm suitability for its own matrices.

What terms does EN ISO 13647:2026 define?

• Culturable microorganism - An aerobic bacterium, yeast, or mould capable of forming colonies on a culture medium.
• Colony forming unit (cfu) - The reporting unit used for the counted colonies in a water sample.
• Reasoner’s 2 Agar (R2A) - The low-nutrient medium specified for this method, chosen to support colony development after longer incubation.
• Spread plate technique - A measured sample volume is spread over the agar surface so colonies grow on top of the plate.
• Productivity - A medium-performance measure used in Annex A to check whether the R2A batch supports growth adequately.
• Control strain - A reference microorganism used to verify that the medium performs as intended.
• Weighted mean - The calculation used when several dilutions contribute to one final result.

Who uses EN ISO 13647:2026?

EN ISO 13647:2026 is used by water microbiology laboratories, drinking water utilities, bottled water producers, and laboratories testing well water intended for human consumption. It is also relevant to quality managers and buyers who need a defined method for routine monitoring, process control, and investigation of changes in colony counts.

In practice, users apply it when they need to assess treatment process performance, monitor distribution systems, compare routine results over time, or investigate signs of biofilm growth, nutrient ingress, temperature effects, or reduced chlorine levels.

Which standards are used with EN ISO 13647:2026?

• ISO 5667-5 - Sampling guidance for drinking water from treatment works and piped distribution systems.
• ISO 19458 - Sampling for microbiological analysis, including how to take and handle samples.
• ISO 8199:2018 - General requirements for microbiological examinations by culture, including dilution, spread plate use, result calculation, and good laboratory practice.
• ISO 11133 - Preparation, storage, and performance testing of culture media.
• ISO 29201 - Guidance for estimating measurement uncertainty in microbiological enumeration.
• ISO 13843:2017 - The basis used in Annex B for performance characteristics of the quantitative method.
• ISO 17994:2014 - Used in Annex D for comparing relative recovery between storage times.
• ISO 6222 - A related water microbiology method discussed in the introduction and Annex C for comparison with nutrient-rich heterotrophic plate counts.
• ISO 23500-3 and ISO 23500-5 - Cited in Annex C in connection with membrane filtration for certain water applications.
• ISO 15883 (all parts) - Cited in Annex C as a related series for membrane filtration principles.

What does the EN ISO 13647:2026 document contain?

The document contains the full test method plus the technical material needed to run and verify it. Clause 8 gives the working procedure for preparing samples, inoculating plates, incubating them, and counting colonies, while Clause 10 defines the minimum test report content and Clause 11 covers quality assurance.

Annex A contains the R2A medium formulation, preparation steps, storage guidance, and the performance-testing setup used for batch control. Annex B summarizes method performance from a single-laboratory study, Annex C explains how the method relates to other heterotrophic plate counting techniques, and Annex D reports the study behind the acceptable sample storage time before analysis.

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
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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
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This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
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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
...