ISO 14461-2:2005

INTERNATIONAL ISO
STANDARD 14461-2
IDF
169-2
First edition
2005-04-01
Milk and milk products — Quality control
in microbiological laboratories —
Part 2:
Determination of the reliability of colony
counts of parallel plates and subsequent
dilution steps
Lait et produits laitiers — Contrôle de qualité en laboratoire
microbiologique —
Partie 2: Détermination de la fiabilité des comptages de colonies en
boîtes parallèles et des dilutions décimales suivantes

Reference numbers
IDF 169-2:2005(E)
©
ISO and IDF 2005
IDF 169-2:2005(E)
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ii © ISO and IDF 2005 – All rights reserved

IDF 169-2:2005(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 2
5 Procedure. 2
5.1 General. 2
5.2 Counting results of two parallel plates. 2
5.3 Sum of counting results of subsequent dilution steps. 2
6 Evaluation . 3
6.1 Tables of results. 3
6.2 Examples of testing parallel plates . 7
6.3 Examples of testing sums of two subsequent dilution steps . 7
7 Calculation formulae and examples. 15
7.1 In Table 1. 15
7.2 In Table 2. 16
7.3 Examples. 16

IDF 169-2:2005(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 14461-2IDF 169-2 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 169-2:2005(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.
ISO 14461-2IDF 169-2 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 14461-2IDF 169-2, together with ISO 14461-1IDF 169-1, 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 169-2:2005(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.
Too large a 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 carried out by comparison of the replicate inoculations, the different dilution
levels and the dilution series. When the steps with excessive variability have been identified, necessary
measures should be taken to bring these steps under control.
vi © ISO and IDF 2005 – All rights reserved

INTERNATIONAL STANDARD
IDF 169-2:2005(E)
Milk and milk products — Quality control in microbiological
laboratories —
Part 2:
Determination of the reliability of colony counts of parallel
plates and subsequent dilution steps
1 Scope
This part of ISO 14461IDF 169 describes a routine procedure for the evaluation of results of the enumeration
of microorganisms using colony-count methods with subsequent 10-fold dilution steps and one plate or two
parallel plates within each dilution step.
This routine procedure is applied regularly in each laboratory performing colony counts. It provides criteria for
the acceptability of differences between the results from parallel plates and subsequent dilution steps, as
follows.
a) The results (colony counts) obtained from parallel plates are compared with tabulated limits for given
colony counts. If these limits are exceeded, a technical problem when performing the parallel
determinations may be indicated.
b) The results (sums of colony counts) of two parallel plates of two subsequent 10-fold dilution steps are
compared with tabulated limits for given sums of colony counts. If these limits are exceeded, a technical
problem when performing the dilutions may be indicated.
c) If the limits mentioned above are exceeded in more cases than expected, this indicates that the test
procedure lacks reliability.
NOTE The formulae for calculating the values in Table 1 and 2 are given and explained in Clause 7.
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 14461-1IDF 169-1, Milk and milk products — Quality control in microbiological laboratories — Part 1:
Analyst performance assessment for colony counts
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply
3.1
colony count
number of microorganisms found, as determined by the method specified in ISO 14461-1|IDF 169-1
NOTE The number of microorganisms is expressed per gram or per millilitre of test sample.
IDF 169-2:2005(E)
4 Principle
The counting results obtained are compared with tabulated limits for given colony counts. Decisions are based
on the way the limits are exceeded. The tabled values are calculated and explained.
5 Procedure
5.1 General
The procedure shall be applied routinely in laboratories carrying out colony counts. A standardized method for
performing colony counts must be applied in any case.
If the applied method is not in accordance with an International Standard or another accepted standard, a
detailed description of the method shall be available and followed precisely.
In the case that a method is followed with only one plate per dilution step, the procedure described in 5.2 shall
be carried out with a certain minimum frequency (e.g. once per hundred sample units tested).
5.2 Counting results of two parallel plates
Compare the results (colony counts) of two parallel plates with the limits tabulated in Table 1.
Compare the upper and lower colony counts of an observed pair of results with the corresponding colony
counts given in Table 1. Use the upper colony count given in Table 1 as basis for the comparison. Then
compare the lower colony count given in Table 1 with the observed lower count.
A lower observed count below the lower colony count of Table 1 indicates that the difference between the
colony counts obtained with the two parallel plates is unacceptably high. (See the results of the first dilution
step in Examples 1 and 2 in Clause 7.)
A lower observed count, which is at least equal to the lower colony count, indicates that the difference is
acceptable. (See the results of the second dilution step in
...