prEN ISO 29981

SLOVENSKI STANDARD
01-januar-2026
Mlečni izdelki - Štetje bifidobakterij - Tehnika štetja kolonij (ISO 29981:2024)
Milk products - Enumeration of bifidobacteria - Colony-count technique (ISO 29981:2024)
Milcherzeugnisse - Zählung von Bifidobakterien - Koloniezählverfahren (ISO
29981:2024)
Produits laitiers - Dénombrement des bifidobacteria présumés - Technique par comptage
des colonies (ISO 29981:2024)
Ta slovenski standard je istoveten z: prEN ISO 29981
ICS:
67.100.01 Mleko in mlečni proizvodi na Milk and milk products in
splošno general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

International
Standard
ISO 29981
IDF 220
Second edition
Milk products — Enumeration of
2024-11
bifidobacteria — Colony-count
technique
Produits laitiers — Dénombrement des bifidobacteria présumés
— Technique par comptage des colonies
Reference numbers
ISO 29981:2024(en)
IDF 220:2024:2024(en) © ISO and IDF 2024

ISO 29981:2024(en)
IDF 220:2024:2024(en)
© ISO and IDF 2024
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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii
ISO 29981:2024(en)
IDF 220:2024:2024(en)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General .2
4.2 Preparation of initial suspension and decimal dilutions .2
4.3 Isolation and selection for confirmation .2
4.4 Confirmation .2
4.5 Calculation .3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure . 4
9.1 General .4
9.2 Preparation of the test portion and primary dilution .4
9.2.1 Dried milk products (e.g. infant milk formulae) including dehydrated starter
and probiotic cultures .4
9.2.2 Non-dried fermented (e.g. yoghurt) and non-fermented milk-based products
(e.g. pasteurized milks) .4
9.3 Microscopic examination of initial suspension or primary dilution (optional) .5
9.4 Preparation of decimal dilution series .5
9.5 Inoculation and incubation.5
9.6 Counting of colonies .5
9.7 Confirmation .6
10 Expression of results . 6
11 Validation of the method . 6
11.1 Validation in accordance with ISO 17468 .6
11.2 Performance characteristics .6
12 Test report . 7
13 Quality assurance . 7
Annex A (normative) Flow diagram of the procedure . 8
Annex B (normative) Culture media and reagents . 9
Annex C (informative) Performance characteristics of the method .13
Bibliography .15

iii
ISO 29981:2024(en)
IDF 220:2024:2024(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 34, Food products, Subcommittee SC 5, Milk
and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.
This second edition cancels and replaces the first edition (ISO 29981 | IDF 220:2010), which has been
technically revised.
The main changes are as follows:
— diluents which can be used have been added;
— preparation of the test portion and primary dilution in cases of dried milk products has been added;
— a new culture medium, TOS agar, has been introduced;
— storage of incubated plates has been included;
— expression of results has been changed to be in accordance with ISO 7218;
— performance testing of the culture media has been introduced;
— performance characteristics, with the results of an interlaboratory study, which are based on the method
of this second edition, have been included as Annex C.
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 29981:2024(en)
IDF 220:2024:2024(en)
IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national interest
groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/
food control authorities.
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and
sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards
using the logos and reference numbers of both organizations.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF 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, IDF 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. IDF 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.
This document was prepared by IDF Standing Committee on Methods for Dairy Microbiology and ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly
by ISO and IDF.
The work was carried out by the IDF/ISO Action Team (D09) of the Standing Committee on Methods for Dairy
Microbiology under the aegis of its project leader Masamichi Muto (JP).

v
ISO 29981:2024(en)
IDF 220:2024:2024(en)
Introduction
Bifidobacteria are non-acid-fast, non-spore-forming, Gram-positive, non-motile and catalase-negative
chemoorganotrophs bacilli, which produce acetic acid, lactic acid and formic acid. Glucose is degraded
exclusively and characteristically by the fructose-6-phosphate shunt in which fructose-6-phosphate
phosphoketolase (F6PPK, EC 4.1.2.22) cleaves fructose-6-phosphate into acetyl phosphate and erythrose-4-
phosphate.
Many reports show that bifidobacteria have various physiological functions and bifidobacteria are widely
applied to foods in milk products such as yoghurt, infant formula and milk powders, and also in non-milk
products such as starter and probiotic cultures. Many bifidobacteria-containing products describe the
bacterial cell counts on the product label which is an important indicator of the functionality. An accurate
bifidobacteria enumeration method, such as the one given in this document, is important to guarantee the
bacterial cell counts.
The main technical changes listed in the Foreword, introduced in this document compared to ISO 29981 | IDF
220:2010, are considered as major (see ISO 17468). These technical changes have a major impact on the
performance characteristics of the method.

vi
ISO 29981:2024(en)
International Standard
IDF 220:2024:2024(en)
Milk products — Enumeration of bifidobacteria — Colony-
count technique
1 Scope
This document specifies a method for the selective enumeration of bifidobacteria in milk products by using
a colony-count technique at 37 °C under anaerobic conditions.
The method is applicable to milk products, such as fermented (e.g. yoghurts) and non-fermented milks
(e.g. pasteurized milks, skim milks, whey protein concentrates), milk powders and formulae (e.g. infant
formulae, follow-up formulae for older infants, products for young children) where these microorganisms
are present and viable, in combination with other lactic acid bacteria or alone. The method is also applicable
to starter and probiotic cultures. For proposed quality criteria of dairy products, see, for example, CXS 243-
[6]
2003 .
Bifidobacteria used in milk products usually belong to the following species (e.g. References [7] and [10]):
— Bifidobacterium adolescentis;
— B. animalis subsp. animalis;
— B. animalis subsp. lactis;
— B. bifidum;
— B. breve;
— B. longum subsp. infantis;
— B. longum subsp. longum.
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 6887-1, Microbiology of the food chain — 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 6887-5, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 5: Specific rules for the preparation of milk and milk products
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 19036:2019, Microbiology of the food chain — Estimation of measurement uncertainty for quantitative
determinations
ISO 29981:2024(en)
IDF 220:2024:2024(en)
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/
3.1
bifidobacteria
anaerobic microorganisms of the family Bifidobacteriaceae, usually capable of growth in/on TOS-MUP agar
or TOS agar by forming typical colonies and displaying certain characteristics in microscopic examination
Note 1 to entry: The morphology of typical colonies of bifidobacteria in/on TOS-MUP agar and TOS agar is described in
9.6. The microscopic examination and other confirmation test are described in 9.7.
4 Principle
4.1 General
The enumeration of bifidobacteria requires three successive stages as specified in Annex A.
TOS-MUP agar contains the antibiotic mupirocin lithium salt (MUP), which inhibits the growth of most lactic
acid bacteria commonly used in products, such as fermented and non-fermented milks (e.g. pasteurized
milks, skim milk, whey protein concentrate), milk powders and infant formulae, as well as starter and
probiotic cultures (see Reference [8]).
Owing to the proven selectivity of the MUP antibiotic when added to the base medium, usually there is no
growth of typical yoghurt bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus),
mesophilic cultures (e.g. Lactococcus lactis), Lactobacillus acidophilus, Lacticaseibacillus casei and
Lacticaseibacillus rhamnosus on the medium specified. This property has been tested with a representative
number of reference strains and isolates.
For the enumeration of bifidobacteria from samples containing only bifidobacteria, TOS agar with or without
the antibiotic MUP can be used.
4.2 Preparation of initial suspension and decimal dilutions
An initial dilution and decimal dilutions are prepared from the test sample.
4.3 Isolation and selection for confirmation
TOS-MUP agar or TOS agar is inoculated with a specified quantity of the test sample if the product is liquid, or
of the initial suspension in the case of other products. Other plates are prepared under the same conditions,
using decimal dilutions of the test sample or of the initial suspension.
The dishes are incubated anaerobically at 37 °C for 72 h. Alternatively, if the colony size is large enough to
count accurately, the dishes can be examined after 48 h incubation.
4.4 Confirmation
Colonies of presumptive bifidobacteria can be confirmed by microscopic examination and/or appropriate
tests (e.g. F6PPK-assay, see References [11] and [14]).
Confirmation of presumptive bifidobacteria by microscopic examination is required, but is optional in the
case of test samples containing only bifidobacteria.

ISO 29981:2024(en)
IDF 220:2024:2024(en)
4.5 Calculation
The number of bifidobacteria per millilitre or gram of the test sample is calculated from the number of
confirmed typical colonies per dish.
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218. The composition of culture media and
reagents and their preparation are specified in Annex B. For performance testing of culture media, follow
the procedures in accordance with Clause B.5 and ISO 11133.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications. The
usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following shall be used.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in ISO 7218.
6.2 Autoclave, capable of operating at a temperature of 115 °C ± 3 °C and equipped with short heating and
cooling cycles.
6.3 Equipment for culture in an anaerobic atmosphere, as specified in ISO 7218, capable of operating
at a temperature of 37 °C ± 1 °C, providing an anaerobic atmosphere of volume fraction 5 % to 20 % of
carbon dioxide; a volume fraction of approximately 70 % to 90 % of nitrogen; with a volume fraction of
approximately 10 % of hydrogen (not obligatory). The gas mixture should not contain more than a volume
fraction of 1 % of oxygen.
6.4 Refrigerator (optional), capable of operating at 5 °C ± 3 °C.
6.5 Water baths, one capable of being maintained at 37 °C ± 1 °C and another capable of being maintained
between 44 °C and 47 °C.
6.6 Sterile test tubes or flasks, of appropriate capacity. Bottles or flasks with non-toxic metallic or
plastic screwcaps may be used.
6.7 pH meter, accuracy to within ±0,1 pH unit at 25 °C.
6.8 Sterile graduated pipettes or automatic pipettes, of nominal capacities 25 ml, 10 ml, 1 ml and 0,1 ml.
6.9 Sterile Petri dishes, vented, with a diameter of approximately 90 mm.
6.10 Peristaltic blender (stomacher), with sterile bags, possibly with a device for adjusting speed and
time, as specified in ISO 7218.
6.11 Microscope (optional), preferably with phase-contrast, and with slides and cover slips, as specified by
ISO 7218.
6.12 Colony-counting equipment with a magnifying lens (optional), e.g. 8 times to 10 times, as specified
in ISO 7218.
ISO 29981:2024(en)
IDF 220:2024:2024(en)
7 Sampling
Sampling is not part of the method specified in this document. Follow the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with the sampling
of the product concerned, it is recommended that the parties concerned come to an agreement on this
subject.
Recommended sampling techniques are given in ISO 707 | IDF 50 for milk and milk products.
It is important that the laboratory receives a sample that is representative of the product under consideration.
The sample should not have been damaged or changed during transport or storage.
8 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International Standard
dealing with the product concerned: follow the procedures specified in ISO 6887-1 and ISO 6887-5. If there
is no specific International Standard available, it is recommended that the parties concerned come to an
agreement on this subject.
9 Procedure
9.1 General
Take all necessary prec
...