ISO 14461-1:2005

INTERNATIONAL ISO
STANDARD 14461-1
IDF
169-1
First edition
2005-05-01
Milk and milk products — Quality control
in microbiological laboratories —
Part 1:
Analyst performance assessment for
colony counts
Lait et produits laitiers — Contrôle de qualité en laboratoires
microbiologiques —
Partie 1: Évaluation de la performance des analystes effectuant les
comptages de colonies
Reference numbers
IDF 169-1:2005(E)
©
ISO and IDF 2005
IDF 169-1:2005(E)
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ii © ISO and IDF 2005 – All rights reserved

IDF 169-1:2005(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle (see Figure 1). 2
5 Diluent, culture media and reagents . 3
6 Apparatus and glassware. 5
7 Sampling . 5
8 Preparation of test sample. 6
8.1 Milk . 6
8.2 Dried milk. 6
9 Procedure. 6
9.1 General. 6
9.2 Number of decimal dilution steps . 6
9.3 Preparation of first decimal dilution . 6
9.4 Preparation of further decimal dilutions. 7
9.5 Melting the medium . 7
9.6 Preparation of binary dilutions and inoculation of the medium . 7
9.7 Incubation . 7
9.8 Randomization and counting of colonies. 8
10 Statistical evaluation . 10
10.1 Adequacy of the data set. 10
10.2 Evaluation of the complete data set (see Figure 2) . 10
Annex A (informative) Weighted mean and homogeneity testing of colony counts. 27
Annex B (Informative) BASIC programme for calculating the likelihood ratio index G . 33
Bibliography . 34

IDF 169-1:2005(E)
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 14461-1IDF 169-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC
International. It is being published jointly by ISO and IDF and separately by AOAC International.
ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products — Quality
control in microbiological laboratories:
 Part 1: Analyst performance assessment for colony counts
 Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps
iv © ISO and IDF 2005 – All rights reserved

IDF 169-1:2005(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
This part of International Standard ISO 14461-1IDF 169 was prepared by Technical Committee ISO/TC 34,
Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in
collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC
International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team, Statistics of analytical data, of the Standing
Committee on Quality assurance, statistics of analytical data and sampling, under the aegis of its project
leaders, Dr. H. Glaeser (EU) and Prof. Dr. H. Weiss (DE).
This edition of ISO 14461-1IDF 169-1, together with ISO 14461-2IDF 169-2, cancels and replaces
IDF 169:1994, which has been technically revised.
ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products — Quality
control in microbiological laboratories:
 Part 1: Analyst performance assessment for colony counts
 Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps
IDF 169-1:2005(E)
Introduction
Every microbiological method consists of several steps that are followed in a specific sequence (sub-sampling,
diluting, plating and counting). The final result has a margin of uncertainty that is determined by the variability
of all the steps involved.
In order to obtain results with a margin of uncertainty not much larger than what can be expected from the
correct application of the method, it is necessary to follow the rules of Good Laboratory Practice (GLP).
The three most important factors in obtaining a correct plate count are
 the homogeneity of the sample material,
 the exactness with which the dilutions are performed, and
 the technique of inoculation and/or counting of the plates.
By homogenizing a sample material very well, making multiple dilution series, and inoculating several plates
from the same dilution, it is possible to assess how well a laboratory can perform the colony-count technique,
taking into account the expected variability of the method.
A too large variability indicates that at least one of the steps in the performance of the method is out of control.
The identification of those steps is done by comparison of the replicate inoculations, the different dilution
levels and the dilution series. When the steps with excessive variability have been identified, the necessary
measures should be taken to bring these steps under control.

vi © ISO and IDF 2005 – All rights reserved

INTERNATIONAL STANDARD
IDF 169-1:2005(E)
Milk and milk products — Quality control in microbiological
laboratories —
Part 1:
Analyst performance assessment for colony counts
1 Scope
This part of ISO 14461IDF 169 describes a procedure for testing the performance of the colony-count
technique within a laboratory by establishing the within-laboratory variability of its technique and identifying
those steps that are associated with excessive variability.
The procedure is also suitable for checking the proper observance of Good Laboratory Practice (GLP), which
may be a prerequisite for participation in interlaboratory tests of colony-count methods.
EXAMPLE Appropriate test samples are raw milk, pasteurized milk and dried milk.
2 Normative references
The following referenced documents are indispensable for the application 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 648:1977, Laboratory glassware — One-mark pipettes
ISO 835-4, Laboratory glassware — Graduated pipettes — Part 4: Blow-out pipettes
ISO 4788, Laboratory glassware — Graduated measuring cylinders
ISO 7218, Microbiology of food and animal feedings stuffs — General rules for microbiological examinations
ISO 8261IDF 122, Milk and milk products — Preparation of samples and dilutions for microbiological
examination
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
colony-count technique
counting of the number of microorganisms as determined by the procedure specified in this part of
ISO 14461IDF 169
NOTE The number of microorganisms is expressed per gram or per millilitre of test sample.
IDF 169-1:2005(E)
4 Principle (see Figure 1)
Figure 1 — Quality assurance in the microbiological laboratory:
Design of pilot studies for plate count
4.1 A test sample is homogenized then diluted to a suitable working density. (e.g. 500 CFU to 10 000 CFU
per millilitre). A suspension is prepared.
4.2 From this first dilution, four dilution series are prepared, each consisting of 12 binary dilution steps.
NOTE Binary (two-fold) dilution steps are used, not decimal (10-fold) dilutions as is the usual practice. With binary
dilutions it is possible to count colonies on plates originating from five to six dilutions, and this large number of counts
improves considerably the testing of the dilution steps.
2 © ISO and IDF 2005 – All rights reserved

IDF 169-1:2005(E)
4.3 Three parallel plate
...