ISO/TS 11059:2025

Technical
Specification
ISO/TS 11059
IDF/RM 225
Second edition
Milk and milk products —
2025-12
Method for the enumeration of
Pseudomonas spp.
Lait et produits laitiers — Méthode de dénombrement des
Pseudomonas spp.
Reference numbers
IDF/RM 225:2025(en) © ISO and IDF 2025

IDF/RM 225:2025(en)
© ISO and IDF 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii
IDF/RM 225:2025(en)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General .2
4.2 Preparation of dilutions .2
4.3 Enumeration .2
4.4 Confirmation .2
4.5 Calculation .2
5 Culture media and reagents . 2
6 Equipment and consumables . 3
7 Sampling . 3
8 P reparation of test sample . 4
9 Procedure . 4
9.1 General .4
9.2 Test portion, initial suspension and dilutions .4
9.3 Inoculation and incubation.4
9.4 Counting and selection of colonies .4
9.5 Subculturing selected colonies .5
9.6 Confirmation .5
9.6.1 Oxidase reaction .5
9.6.2 Fermentation test .5
9.6.3 Interpretation of confirmation tests .5
10 Expression of results . 5
11 Te s t r ep or t . 6
12 Quality assurance . 6
Annex A (informative) Flow diagram of the main steps of the procedure . 7
Annex B (normative) Culture media and reagents . 8
Bibliography .13

iii
IDF/RM 225:2025(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 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/TS 11059 | IDF/RM 225:2009), which has
been technically revised.
The main changes are as follows:
— biochemical confirmation tests on oxidase reaction and glucose fermentation test have been aligned
with the same confirmation tests used for presumptive Enterobacteriaceae;
— the flow diagram in Annex A giving the main steps of the procedure has been added;
— in Annex B, the criteria for the performance testing of culture media and reagents have been added.
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
IDF/RM 225:2025(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 (H27) of the Standing Committee on Methods for Dairy
Microbiology under the aegis of its project leader Julie Audy (CA).

v
IDF/RM 225:2025(en)
Introduction
Pseudomonas spp. are aerobic, Gram-negative, non-spore-forming, flagellated, rod-shaped, catalase-positive
bacteria. Pseudomonas spp. are a dominant group of psychrotrophic microorganisms associated with the
contamination of raw or processed milk limiting the shelf life of fluid milk at 4 °C. The most important
species causing spoilage in the dairy sector are from the P. fluorescens group including P. fluorescens, P. fragi
and P. gessardii subgroups.
Pseudomonas spp. enter raw or processed milk from contaminated water or via biofilms in milking systems or
process equipment. Pseudomonas spp. grow in raw or processed dairy products during refrigerated storage
and some strains produce heat-stable extracellular enzymes (mainly lipases and proteases) causing a wide
range of defects from off flavour to gelification (gelation) of ultra-high temperature (UHT) treated products.
The enumeration of Pseudomonas spp. is used by the dairy sector as microbial indicator to evaluate raw milk
quality or to determine a post-pasteurization contamination in processed milk from the milk-processing
equipment.
This document is intended to provide general guidance for the examination of products not dealt with by
existing International Standards and to be taken into account by organizations preparing microbiological
test methods for application to milk and milk products and dairy environmental samples. This guidance
will not always be appropriate in every detail for certain products, and for some other products it can be
necessary to use different methods, see Reference [7]. Nevertheless, it is hoped that in all cases, every
attempt will be made to apply the guidance provided as far as possible and that deviations from it will only
be made if absolutely necessary for technical reasons.
The main technical changes listed in the Foreword, introduced in this document compared to ISO/TS 11059 |
IDF/RM 225:2009, are considered as minor (see ISO 17468).
These technical changes have a minor impact on the performance characteristics of the method.

vi
Technical Specification
IDF/RM 225:2025(en)
Milk and milk products — Method for the enumeration of
Pseudomonas spp.
1 Scope
This document specifies the enumeration of Pseudomonas spp. by the colony-count technique.
This document is applicable to:
— milk and milk products;
— environmental samples in the area of dairy production and handling.
The method allows the isolation of all pigmented and non-pigmented psychrotrophic Pseudomonas spp.
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 18593, Microbiology of the food chain — Horizontal methods for surface sampling
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
Pseudomonas spp.
species of bacteria of the genus Pseudomonas which form colonies in penicillin and pimaricin agar (PPA) at
25 °C and which show a positive oxidase reaction and absence of glucose fermentation

IDF/RM 225:2025(en)
4 Principle
4.1 General
In PPA agar, the enzymatic digest of gelatine and casein are nutrients substrates supporting the growth
of Pseudomonas. Medium osmotic balance is maintained by magnesium chloride and potassium sulfate
and promote pyocyanin production (a blue, non-fluorescent pigment soluble in water and in chloroform)
of Pseudomonas. The penicillin G and pimaricin concentrations inhibit most accompanying bacteria.
The enumeration of Pseudomonas spp. contains a confirmatory step. The flow diagram of the main steps of
the procedure is given in Annex A.
4.2 Preparation of dilutions
For the preparation of decimal dilutions from the test portion, see ISO 6887-1 and ISO 6887-5.
4.3 Enumeration
The surface of a solid selective culture medium, penicillin and pimaricin agar (PPA), is inoculated with
a specified quantity of the test sample if the product is liquid, or with a specified quantity of the initial
suspension in the case of other products.
The inoculation is done using decimal dilutions of the test sample or of the initial suspension.
If a lower limit of 10 cfu/ml (liquid samples) or 100 cfu/g (solid samples) is required, 1 ml can be examined
using one large Petri dish (140 mm diameter) or three 90 mm diameter Petri dishes.
When the number of cfu is expected to be at or near the limit of determination, the use of duplicate plates
is preferable. If duplicate plates are used, the minimum for the sum of colonies on both plates should be
10 colonies. In this case, the level of contamination is expected to be higher than 50 cfu/ml for liquid samples
or higher than 500 cfu/g for solid samples.
The plates are incubated aerobically at 25 °C and examined after 48 h to record the number of presumptive
Pseudomonas.
4.4 Confirmation
The solid non-selective medium (e.g. nutrient agar), see Clause B.4, is inoculated by streaking five randomly
selected colonies.
The plates are incubated aerobically at 25 °C for 24 h to 48 h and isolated colonies are used to conduct
biochemical tests on oxidase reaction and glucose fermentation.
Colonies of presumptive Pseudomonas spp. are confirmed by means of tests for the presence of oxidase and
absence of glucose fermentation.
4.5 Calculation
The number of Pseudomonas spp. per millilitre or gram of the test sample is calculated from the number of
confirmed typical colonies per PPA plate.
5 Culture media and reagents
Follow good laboratory practices in accordance with ISO 7218. The composition of the culture media and
reagents and their preparation are specified in Annex B. For performance testing of the culture media and
reagents, follow the procedures in accordance with Annex B and ISO 11133.

IDF/RM 225:2025(en)
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable items if these have the 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).
6.2 Freezer, capable of operating at –20 °C ± 3 °C.
6.3 Incubator, capable of operating at 25 °C ± 1 °C.
6.4 Loops, of platinum-iridium o
...