ISO 8607:2003

INTERNATIONAL ISO
STANDARD 8607
First edition
2003-02-01
Artificial insemination of animals —
Frozen semen of breeding bulls —
Enumeration of living aerobic
microorganisms
Insémination artificielle des animaux — Semences congelées de
taureaux reproducteurs — Dénombrement des micro-organismes
aérobies vivants
Reference number
©
ISO 2003
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ii © ISO 2003 — All rights reserved

Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 2
5 Diluent and culture medium. 2
5.1 Diluent . 2
5.2 Agar medium . 3
6 Apparatus. 3
7 Sampling . 4
8 Preparation of test sample. 4
9 Procedure. 4
9.1 Test portion, initial suspension and dilutions . 4
9.2 Inoculation and incubation . 4
9.3 Control plates . 5
9.4 Interpretation of results. 5
10 Expression of results. 5
10.1 Method of calculation . 5
10.2 Precision . 7
11 Test report. 7
Annex A (normative) Confidence limits for the estimation of small numbers of colony-forming
units of microorganisms . 8
Bibliography . 9

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
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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 8607 was prepared by Technical Committee ISO/TC 34, Food products.
This first edition of ISO 8607 cancels and replaces ISO/TR 8607:1991, which has been technically revised.
iv © ISO 2003 — All rights reserved

Introduction
The quantitative microbiological control of the hygienic collection and handling of bovine semen is of great
importance in order to predict the efficiency of artificial insemination and to fulfil the requirements of
biosecurity (see reference [1]). For the same reason, the investigation of bacterial contamination and the
possible presence of facultative pathogenic microorganisms in the preserved bovine semen is also very
important.
There is a need for an international method suitable for the determination of the microbial count in frozen
semen, which indicates the hygienic status during collection, handling and storage. The aim of the colony-
count method specified in this International Standard is to enumerate the saprophytic microorganisms that are
originally present in and/or are transmitted to the bovine semen from the environment. With this method only
the total count of bacteria is detected, mainly the aerophilic and mesophilic saprophytic ones, as well as a few
facultative pathogenic microorganisms that are not very sensitive to environmental conditions.
Since samples of frozen bovine semen contain additional antibiotics, the determination of microbiological
contamination of this type of sample is slightly different from the commonly used microbiological methods.
When examining preserved semen samples in low dilutions, the number of colonies may be lower than
expected and do not follow the usual proportions. Therefore relatively high decimal dilutions should be used to
compensate for the inhibition effect of the antibiotics. As a result of the necessary high dilutions, 15 or less
colonies can be observed in each Petri dish and this result should be accepted. This differs from the usual
microbiological examinations of food where the sample dilution can be chosen in such a way that the number
of colonies is more than 15 in a Petri dish so more precise examination is possible.
Microbial cells often occur as clumps or groups in the samples. Whereas shaking samples and dilutions may
uniformly distribute the clumps of bacteria, this may not completely disrupt the clumps themselves into single
cells. Consequently, each colony that appears on the medium can arise from a clump of cells or from a single
cell and therefore it is more precise to express the result as the number of colony-forming units (CFU) of
microorganisms than to give the number of microorganisms (see reference [2]).
This International Standard does not specify a tolerable limit value for the total CFU of bacteria, which may be
a consumer requirement in trade. This should be given in commercial contracts.
A list of publications related to this International Standard is given in the Bibliography.

INTERNATIONAL STANDARD ISO 8607:2003(E)

Artificial insemination of animals — Frozen semen of breeding
bulls — Enumeration of living aerobic microorganisms
1 Scope
This International Standard specifies a method for the enumeration of living aerobic microorganisms present
in the frozen semen of breeding bulls. The colonies growing in a solid medium after aerobic incubation at
37 °C are counted. The microbiological contamination of the sample is expressed as a number of colony-
forming units of microorganisms per millilitre of the test sample.
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 6887-1, Microbiology of food and animal feeding stuffs — 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 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 6887-1 and the following apply.
3.1
semen
product of the genital organs of a male, intended for the fertilization of a female
3.2
ejaculate
quantity of semen obtained as a result of mating the male
3.3
dose
quantity of semen which is packaged individually and carries a unique identification, intended for a single
artificial insemination
3.4
series of doses
group of doses of semen obtained from one bull and prepared from one or more ejaculates, obtained on the
same day and subjected to the same treatment
3.5
living aerobic microorganisms
bacteria, yeasts and moulds which grow aerobically at 37 °C under the conditions specified in this International
Standard
3.6
colony-forming unit
CFU
single microbial cell, or clumps or a group of cells, forming one colony on the medium under the conditions
specified in this International Standard
4 Principle
Two poured plates are prepared using a specified culture medium. These are deep inoculated with a specified
quantity of test sample, followed by aerobic incubation at 37 °C.
The number of CFU of microorganisms per millilitre of the test sample is calculated from the number of
colonies obtained.
5 Diluent and culture medium
For general guidance, see ISO 7218.
Chemical products shall be of recognized analytical quality an
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