FprEN ISO 13647

SLOVENSKI STANDARD
oSIST prEN ISO 13647:2025
01-maj-2025
[Not translated]
Water quality - Enumeration of culturable microorganisms - Colony count by spread plate
inoculation on R2A medium (ISO/DIS 13647:2025)
Wasserbeschaffenheit - Zählung der kultivierbaren Mikroorganismen - Koloniezählung
durch Ausplattieren auf R2A-Medium (ISO/DIS 13647:2025)
Qualité de l'eau - Dénombrement des micro-organismes revivifiables - Comptage des
colonies par ensemencement par étalement sur un milieu gélosé R2A (ISO/DIS
13647:2025)
Ta slovenski standard je istoveten z: prEN ISO 13647
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
oSIST prEN ISO 13647:2025 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN ISO 13647:2025
oSIST prEN ISO 13647:2025
DRAFT
International
Standard
ISO/DIS 13647
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of culturable microorganisms
Voting begins on:
— Colony count by spread plate
2025-04-03
inoculation on R2A medium
Voting terminates on:
ICS: 07.100.20
2025-06-26
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 13647:2025(en)
oSIST prEN ISO 13647:2025
DRAFT
ISO/DIS 13647:2025(en)
International
Standard
ISO/DIS 13647
ISO/TC 147/SC 4
Water quality — Enumeration
Secretariat: DIN
of culturable microorganisms
Voting begins on:
— Colony count by spread plate
inoculation on R2A medium
Voting terminates on:
ICS: 07.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2025
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BE CONSIDERED IN THE LIGHT OF THEIR
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
CH-1214 Vernier, Geneva
NOTIFICATION OF ANY RELEVANT PATENT
Phone: +41 22 749 01 11
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 13647:2025(en)
ii
oSIST prEN ISO 13647:2025
ISO/DIS 13647:2025(en)
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 diluents . 2
7 Sampling, transport and storage . . 2
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 Quality assurance . 3
11 Test report . 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
oSIST prEN ISO 13647:2025
ISO/DIS 13647:2025(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 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,
Microbiological 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
oSIST prEN ISO 13647:2025
ISO/DIS 13647:2025(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 counts of culturable microorganisms provide 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.
The main value of colony counts lies in the detection of changes from those expected, based on representative
monitoring. The change in colony counts can be caused by 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 a warning of potential treatment failure and/or contamination.
Nutrient rich Tryptone Yeast Extract Agar medium has been used for long time in water microbiology
(standard method ISO 6222:1999). It provides a compliance monitoring method for heterotrophic plate
counts in tap water quality verification. However, ISO 6222 specifies the detection of microorganisms in
nutrient rich conditions at 22 °C in 3 days or at 36 °C in 2 days.
The use of a more sensitive Reasoner’s 2 Agar (R2A) medium that has a low-nutrient composition and
long incubation time (7 days at 22 °C) provides an improvement for the enumeration of culturable
microorganisms in terms of enabling the determination of micro-organisms forming colonies only after a
longer incubation time. Further, the nutrient limited conditions minimize the colony size and overgrowth.
Due to the enhanced sensitivity, the R2A method is especially suitable for bringing more information to the
enumeration heterotrophic colony counts for water with low colony count.

v
oSIST prEN ISO 13647:2025
oSIST prEN ISO 13647:2025
DRAFT International Standard ISO/DIS 13647:2025(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 — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably qualified staff.
1 Scope
This document specifies a method for the enumeration of culturable microorganisms in water by counting the
colonies on a low-nutrient agar culture medium by spread plate inoculation after incubation at 22 °C for 7 d.
The method is intended to measure the operational efficiency of the treatment process of public drinking
water supplies, including the water in distribution systems and containers. The method is particularly
suitable to monitor water for human consumption which is low in nutrients and is distributed in temperatures
below 20 °C (Reference [1]). The method can be applied to all types of water, including pool and spa waters.
The low-nutrient agar in use in this document usually gives higher colony counts from water samples than
nutrient-rich formulations of culture media typically used for enumeration of culturable microorganisms
(References [2], [3], [4]).
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, 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 terms and definitions given in ISO/IEC Guide 2 and the following apply.
ISO and IEC maintain terminological 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/
3.1
culturable microorganisms
all aerobic bacteria, yeasts and moulds capable of forming colonies on a culture medium

oSIST prEN ISO 13647:2025
ISO/DIS 13647:2025(en)
4 Principle
Inoculation of a test portion of the sample or dilutions of the sample on the surface of a specified culture
medium (see Annex A) in Petri dishes by spread-plating method.
Incubation of plates at (22 ± 2) °C for (164 ± 4) h.
Counting of all visible colonies as culturable microorganisms and calculation of the colony forming units
(cfu) per milliliter (ml) of the sample from the number of colonies counted.
NOTE 1 The spread-plating method 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 (References [5], [6], [7]), instead of spread-plating, the plate counting technique by using
pour-plating or membrane filtration might be selected for use. For more information, see Annex C and the guidance
given in ISO 8199.
5 Apparatus and glassware
Usual microbiological laboratory equipment (as specified in ISO 8199) and, in particular, the following:
5.1 Apparatus for sterilization by steam (autoclave).
Apparatus and glassware not supplied sterile shall be sterilized according to the instructions given in
ISO 8199.
5.2 Incubator, thermostatically controlled at (22 ± 2) °C.
5.3 pH meter, with an accuracy of ±0,1 at 20 °C to 25 °C.
5.4 Magnifying equipment, for colony counting with magnitude of at least 2-3 times and light above or
under the plate against dark background.
NOTE In some laboratories, the use of magnifying equipment with magnitude of 6-8 times has been successfully
used. The use of higher magnification than 2-3 times may lower the operational variability of the colony counting.
6 Culture media and diluents
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 comply with the composition given in Annex A and
strictly follow the manufacturer’s instructions.
NOTE This document describes the use of R2A medium (Reference [8]). There are other formulations available,
e.g. R3A medium tha
...