EN ISO 8261:2001

SLOVENSKI STANDARD
SIST EN ISO 8261:2001
01-december-2001
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Milk and milk products - General guidance for the preparation of test samples, initial
suspensions and decimal dilutions for microbiological examination (ISO 8261:2001)
Milch und Milchprodukte - Allgemeiner Leitfaden für die Vorbereitung von
Untersuchungsproben und die Herstellung von Anschüttelungen und
Dezimalverdünnungen für mikrobiologische Untersuchungen (ISO 8261:2001)
Lait et produits laitiers - Lignes directrices générales pour la préparation des échantillons
pour essai, de la suspension mere et des dilutions décimales en vue de l'examen
microbiologique (ISO 8261:2001)
Ta slovenski standard je istoveten z: EN ISO 8261:2001
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.100.01 0OHNRLQPOHþQLSURL]YRGLQD Milk and milk products in
VSORãQR general
SIST EN ISO 8261:2001 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 8261:2001

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SIST EN ISO 8261:2001
EUROPEAN STANDARD
EN ISO 8261
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2001
ICS 07.100.30
English version
Milk and milk products - General guidance for the preparation of
test samples, initial suspensions and decimal dilutions for
microbiological examination (ISO 8261:2001)
Lait et produits laitiers - Lignes directrices générales pour la Milch und Milchprodukte - Allgemeiner Leitfaden für die
préparation des échantillons pour essai, de la suspension Vorbereitung von Untersuchungsproben und die
mère et des dilutions décimales en vue de l'examen Herstellung von Anschüttelungen und
microbiologique (ISO 8261:2001) Dezimalverdünnungen für mikrobiologische
Untersuchungen (ISO 8261:2001)
This European Standard was approved by CEN on 15 June 2001.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8261:2001 E
worldwide for CEN national Members.

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SIST EN ISO 8261:2001
Page 2
EN ISO 8261:2001
Foreword
The text of the International Standard ISO 8261:2001 has been prepared by Technical
Committee ISO/TC 34 "Agricultural food products" in collaboration with Technical Committee
CEN/TC 302 "Milk and milk products - Methods of sampling and analysis", the secretariat of
which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication
of an identical text or by endorsement, at the latest by December 2001, and conflicting
national standards shall be withdrawn at the latest by December 2001.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United
Kingdom.
Endorsement notice
The text of the International Standard ISO 8261:2001 was approved by CEN as a European
Standard without any modification.
NOTE: Normative references to International Standards are listed in annex ZA (normative).

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SIST EN ISO 8261:2001
Page 3
EN ISO 8261:2001
Annex ZA (normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions
of any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications,
indicated by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 6887-1 1999 Microbiology of food and animal feeding stuffs EN ISO 6887-1 1999
- 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

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SIST EN ISO 8261:2001

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SIST EN ISO 8261:2001
INTERNATIONAL ISO
STANDARD 8261
IDF
122
Second edition
2001-06-15
Milk and milk products — General guidance
for the preparation of test samples, initial
suspensions and decimal dilutions for
microbiological examination
Lait et produits laitiers — Lignes directrices générales pour la préparation
des échantillons pour essai, de la suspension mère et des dilutions
décimales en vue de l'examen microbiologique
Reference numbers
ISO 8261:2001(E)
IDF 122:2001(E)
©
ISO and IDF 2001

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(E)
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© ISO and IDF 2001
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ii © ISO and IDF 2001 – All rights reserved

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(E)
Contents Page
Foreword.iv
Introduction.vi
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Principle.2
5 Diluents.2
5.1 Basic materials.2
5.2 Diluents for general use.2
5.3 Diluents for special purposes .3
5.4 Distribution, sterilization and storage of diluent.5
6 Apparatus .6
7 Sampling.7
8 Procedure .7
8.1 General.7
8.2 Preparation of test portion and initial suspension.7
8.3 Further decimal dilutions.10
8.4 Duration of the procedure.11
Bibliography.12
© ISO and IDF 2001 – All rights reserved iii

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 8261�IDF 122 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.
This edition cancels and replaces the first edition (ISO 8261:1989), which has been technically revised.
iv © ISO and IDF 2001 – All rights reserved

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(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 National
Committees casting a vote.
International Standard ISO 8261�IDF 122 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, Preparations of samples and dilutions for
microbiological examinations, of the Standing Committee on Microbiological methods of analysis, under the aegis
of its project leader, Mr L.J.M. Maturin (US).
This edition cancels and replaces the third edition (IDF 122C:1996).
© ISO and IDF 2001 – All rights reserved v

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(E)
Introduction
This International Standard is mainly based on ISO 6887-1. The necessary adaptations to microbiological
laboratory practice in the dairy industry and instructions specific to dairy products, especially in relation to sample
preparation, have been introduced.
The question of which diluent or diluents to specify has been the subject of discussion for some time. In this
International Standard the peptone/saline solution, as well as the buffered peptone water solution as used in
ISO 6887-1, is specified. Three other diluents which are commonly used in dairy microbiological laboratories are
also specified for general use. Furthermore, six diluents are specified for special purposes in dairy microbiological
laboratories.
vi © ISO and IDF 2001 – All rights reserved

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
INTERNATIONAL STANDARD
IDF 122:2001(E)
Milk and milk products — General guidance for the preparation of
test samples, initial suspensions and decimal dilutions for
microbiological examination
1 Scope
This International Standard describes general guidelines for the preparation of test samples, initial suspensions
and decimal dilutions for the microbiological examination of milk and milk products, including milk-based infant
foods.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
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 International Standard, the following terms and definitions apply.
3.1
initial suspension
primary dilution
suspension, solution or emulsion obtained after a weighed or measured quantity of the product under examination
(or of a test sample prepared from the product) has been mixed, if necessary, using a blender and observing
appropriate precautions, with a nine-fold quantity of dilution fluid (diluent), allowing large particles, if present, to
settle
NOTE 1 In certain cases and in particular for products giving an initial 1 + 9 suspension which is too viscous or too thick, it
may be necessary to add more diluent. On the other hand, a more concentrated primary dilution than 1 + 9 may be required for
results of tests to relate to certain specification criteria. These factors should be taken into account for subsequent operations
and/or in the expression of results.
NOTE 2 The use of the first dilution is the most appropriate for fitting the requirement of less than 10 microorganisms per
gram. If it is desirable for some enumerations in some products to fall below this threshold, it is possible to use less diluent for
the suspension. However, inoculation of this suspension may result in an unbalanced inoculum/medium ratio.
© ISO and IDF 2001 – All rights reserved 1

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(E)
NOTE 3 For appropriate precautions, see 8.1.
NOTE 4 For details of diluents, see clause 5.
3.2
further decimal dilutions
suspensions, solutions or emulsions obtained by mixing a specific volume of the primary dilution (3.1) with a nine-
fold volume of diluent, and by repeating this operation with every dilution thus prepared, until a decimal dilution
series, suitable for the inoculation of culture media, is obtained
NOTE See 8.1.
4Principle
An initial suspension (3.1) is prepared and, if necessary, further decimal dilutions (3.2) are prepared to reduce the
number of microorganisms per unit volume to facilitate microbiological examination.
5 Diluents
5.1 Basic materials
In order to improve the precision of the results, it is recommended that, for the preparation of the diluent,
dehydrated basic components or a dehydrated complete preparation be used. The manufacturer's instructions shall
be rigorously followed.
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water
or water of equivalent quality (see ISO 7218).
Any adjustment necessary to the pH of the media shall be made with solutions of sodium hydroxide (NaOH) or
hydrochloric acid (HCl) of appropriate molarities to minimize the change in media volume and thus composition; i.e.
in general, the lower the volume of medium, the higher the molarity.
5.2 Diluents for general use
5.2.1 Peptone-salt solution
5.2.1.1 Composition
Peptone of enzymatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml
5.2.1.2 Preparation
Dissolve the components in the water, by heating slightly on a hot plate (6.13) if necessary. Adjust the pH with the
appropriate solution (5.1) so that, after sterilization, it is 7,0� 0,2 at 25�C.
2 © ISO and IDF 2001 – All rights reserved

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SIST EN ISO 8261:2001
ISO 8261:2001(E)
IDF 122:2001(E)
5.2.2 Quarter-strength Ringer's solution
5.2.2.1 Composition
Sodium chloride (NaCl) 2,25 g
Potassium chloride (KCl) 0,105 g
Calcium chloride, anhydrous (CaCl)0,06g
2
Sodium hydrogen carbonate (NaHCO)0,05g
3
Water 1 000 ml
5.2.2.2 Preparation
Dissolve the salts in the water. Adjust the pH with the appropriate solution (5.1) so that, after sterilization, it is
6,9� 0,2 at 25�C.
5.2.3 Peptone solution
5.2.3.1 Composition
Peptone 1,0 g
Water 1 000 ml
5.2.3.2 Preparation
Dissolve the peptone in the water. Adjust the pH with the appropriate solution (5.1) so that, after sterilization, it is
7,0� 0,2 at 25�C.
5.2.4 Phosphate buffer solution
5.2.4.1 Composition
Potassium dihydrogen phosphate (KH PO ) 42,5 g
2 4
Water 1 000 ml
5.2.4.2 Preparation
Dissolve the salt in 500 ml of water. Adjust the pH with the appropriate solution (5.1) so that, after sterilization, it is
7,2� 0,2 at 25�C. Dilute to 1 000 ml. Store the stock solution under refrigeration.
Add 1 ml of this stock solution (at 20�C) to 1 000 ml of water for use as diluent.
5.3 Diluents for special purposes
These diluents shall only be used for the prepa
...