SIST EN ISO 13969:2004
(Main)Milk and milk products - Guidelines for a standardized description of microbial inhibitor tests (ISO 13969:2003)
Milk and milk products - Guidelines for a standardized description of microbial inhibitor tests (ISO 13969:2003)
Lait et produits laitiers - Lignes directrices pour une description normalisée des méthodes microbiologiques de dépistage d'inhibiteurs microbiens (ISO/FDIS 13969:2003)
Milch und Milchprodukte - Anleitung für eine vereinheitlichte Beschreibung mikrobiologischer Hemmstofftests (ISO 13969:2003)
Diese Internationale Norm gibt eine Anleitung für die vereinheitlichte Beschreibung mikrobiologischer Hemmstofftests für Milch und Milchprodukte. Damit soll ein Rahmen und eine Grundlage für die Bewertung bzw. Validierung mikrobiologischer Hemmstofftests geliefert werden, der einen Vergleich der durch unterschiedliche mikrobiologische Hemmstofftests und experimentelle Studien bereitgestellten Daten ermöglicht.
Lait et produits laitiers - Lignes directrices pour une description normalisée des méthodes microbiologiques de dépistage d'inhibiteurs microbiens (ISO 13969:2003)
L'ISO 13969|FIL 183:2003 indique comment établir une description normalisée des méthodes microbiologiques de dépistage d'inhibiteurs microbiens pour le lait et pour les produits laitiers. Elle a pour objet de fournir un cadre et une base d'évaluation/validation des méthodes microbiologiques de dépistage d'inhibiteurs microbiens, permettant la comparaison de données obtenues à partir de différentes méthodes et études expérimentales.
Mleko in mlečni izdelki - Navodila za standardiziran opis mikrobioloških metod za ugotavljanje zaviralnih snovi (ISO 13969:2003)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 13969:2004
01-november-2004
0OHNRLQPOHþQLL]GHONL1DYRGLOD]DVWDQGDUGL]LUDQRSLVPLNURELRORãNLKPHWRG]D
XJRWDYOMDQMH]DYLUDOQLKVQRYL ,62
Milk and milk products - Guidelines for a standardized description of microbial inhibitor
tests (ISO 13969:2003)
Milch und Milchprodukte - Anleitung für eine vereinheitlichte Beschreibung
mikrobiologischer Hemmstofftests (ISO 13969:2003)
Lait et produits laitiers - Lignes directrices pour une description normalisée des
méthodes microbiologiques de dépistage d'inhibiteurs microbiens (ISO 13969:2003)
Ta slovenski standard je istoveten z: EN ISO 13969:2004
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.100.01 0OHNRLQPOHþQLSURL]YRGLQD Milk and milk products in
VSORãQR general
SIST EN ISO 13969:2004 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 13969:2004
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SIST EN ISO 13969:2004
EUROPEAN STANDARD
EN ISO 13969
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2004
ICS 07.100.30
English version
Milk and milk products - Guidelines for a standardized
description of microbial inhibitor tests (ISO 13969:2003)
Lait et produits laitiers - Lignes directrices pour une Milch und Milchprodukte - Anleitung für eine vereinheitlichte
description normalisée des méthodes microbiologiques de Beschreibung mikrobiologischer Hemmstofftests (ISO
dépistage d'inhibiteurs microbiens (ISO 13969:2003) 13969:2003)
This European Standard was approved by CEN on 1 October 2003.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, 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
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13969:2004: E
worldwide for CEN national Members.
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SIST EN ISO 13969:2004
EN ISO 13969:2004 (E)
Foreword
The text of ISO 13969:2003 has been prepared by Technical Committee ISO/TC 34 "Agricultural
food products" of the International Organization for Standardization (ISO) and has been taken
over as EN ISO 13969:2004 by 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 February 2005, and conflicting national
standards shall be withdrawn at the latest by February 2005.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 13969:2003 has been approved by CEN as EN ISO 13969:2004 without any
modifications.
2
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SIST EN ISO 13969:2004
INTERNATIONAL ISO
STANDARD 13969
IDF
183
First edition
2003-10-01
Milk and milk products — Guidelines for
a standardized description of microbial
inhibitor tests
Lait et produits laitiers — Lignes directrices pour une description
normalisée des méthodes microbiologiques de dépistage d'inhibiteurs
microbiens
Reference numbers
ISO 13969:2003(E)
IDF 183:2003(E)
©
ISO and IDF 2003
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(E)
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Published in Switzerland
ii © ISO and IDF 2003 – All rights reserved
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(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 13969IDF 183 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 and IDF 2003 – All rights reserved iii
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(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
IDF National Committees 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. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 13969IDF 183 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 Antimicrobials and other veterinary medical
residues, of the Standing Committee on Analytical methods for additives and contaminants, under the aegis of
its project leader, Mrs G. Suhren (DE).
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(E)
Introduction
The parameters outlined in this International Standard may not need to be evaluated completely for every test,
depending on
a) the field of application of the test under study (e.g. screening or reference method, type of milk, i.e. animal
species or raw/heat treated milk),
b) the information needed [e.g. the introduction of a new substance with fixed maximum residue limit (MRL)],
and
c) the detection pattern (e.g. sensitivity of the test microorganism to a broad or narrow variety of
antimicrobial compounds).
Thus “the terms of reference” between the producer and user of a certain test should be agreed upon in the
context of these guidelines omitting, for example, those aspects that are not relevant to the intended field of
application.
A general disadvantage related to the interpretation of microbial inhibitor tests is that they are usually
evaluated in a subjective way and in very few steps, i.e. negative, questionable, and positive by comparison
with positive and/or negative control samples.
In cases where the medium contains an indicator, the type of the resultant colour change can depend upon
the type of antimicrobial present. This sometimes makes it difficult to obtain a clear distinction between
positive and negative results. Test interpretation in few steps also means that small alterations or minor colour
developments, which may be of importance in a validation programme, need major experimental effort.
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SIST EN ISO 13969:2004
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
INTERNATIONAL STANDARD
IDF 183:2003(E)
Milk and milk products — Guidelines for a standardized
description of microbial inhibitor tests
1 Scope
This International Standard gives guidance for a standardized description of microbial inhibitor tests for milk
and milk products. It is intended to give a framework and basis for the evaluation/validation of microbial
inhibitor tests, allowing the comparison of data obtained from different tests and experimental studies.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
false positives
percentage of positive results when testing negative samples
2.2
false negatives
percentage of negative results at the claimed detection level(s)
2.3
limit of detection
concentration level at which a defined percentage of samples is detected
EXAMPLE 95 % together with the respective confidence level.
3 Information needed from the developer/manufacturer
3.1 Methodology
The developer or manufacturer of the test should provide information regarding methodology by mentioning
the following:
a) description of the method;
b) principle of the method;
c) technical design of the procedure (e.g. degree of automation, data processing);
d) evaluation of test results (e.g. scores, scale and definition of what to consider “positive” or “negative”);
e) capacity (e.g. sample throughput);
f) special requirements for sampling, preservation and testing;
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(E)
g) procedure for the purpose of quality assurance by the developer/manufacturer;
h) field of application concerning
1) the intended test use (e.g. screening or confirmation), and
2) the substrate or matrix (e.g. milk from cows or other animals, milk powder or other foods).
3.2 Operating requirements
The following information should be given regarding operating requirements:
a) requirements for user experience and training;
b) requirements for safety and special laboratory service (e.g. electric power, S1-lab, waste disposal and
maximum concentration level);
c) requirements for quality control by the manufacturer and/or user.
3.3 Test specifications
The following information should be given regarding test definitions:
limit of detection: see 2.3.
3.4 Documentation
The following information should be given regarding documentation:
a) user manual, including a trouble-shooting guide;
b) suppliers of instruments, reagents, standards, technical services and customer support;
c) status of official recognition/general introduction in specified countries;
d) availability of reference material;
e) availability of internationally recognized/validated reference from ISO, IDF and AOAC International, or
others;
f) availability of, for example, literature and practical experiences.
4 Evaluation of the attributes of the microbiological inhibitor test
4.1 General
The validation of a method should always be carried out under controlled conditions, i.e. based on defined test
samples. The influence of particular conditions is described in 4.3.6.
4.2 Prerequisites
4.2.1 Milk free from antimicrobials (“negative milk”)
The cows from which milk is collected in order to serve as “negative milk” shall meet the following
requirements. If, however, a test is applied for milk of an animal species other than cows, the requirements
with respect to the status of the animal should be adjusted accordingly.
a) The clinical and sub-clinical health status shall be good, with special emphasis on udder health (less than
150 000 somatic cells per millilitre).
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(E)
b) The treatment or feeding with antimicrobial substances shall be prohibited for at least 8 weeks before milk
collection. In the case of dry cow treatment, milk should not be collected earlier than 60 days after calving
provided the dry cow period has been at least 4 weeks.
c) The cows shall be mid-lactation: more than 60 days and less than 200 days after calving, producing more
than 5 kg of milk per day.
d) The milking of at least five to seven cows shall be combined to overcome individual variations in milk
composition.
4
e) The total viable count shall be less than 10 CFU (colony-forming units) per millilitre before the
preservation process (deep-freezing, lyophilization). The possible presence of β-lactamase-producing
microorganisms shall be kept in mind in the case of β-lactam antibiotic testing.
4.2.2 Test substances
The test substances that are used in the testing procedure should be obtained from a recognized
developer/manufacturer, preferably with an analytical certificate with a guaranteed specification. The
concentration required should be calculated based on the free acid or base forms of the drug, corrected for
purity. Special considerations should be given to those substances with stability/potency problems.
Unless otherwise stated, it is preferable that the evaluation of detection limits (4.3.2) should be undertaken
using those antimicrobials and/or concentrations that the developer/manufacturer claims the test will detect.
4.2.3 Solvents
If special solvents or other chemicals are required to dissolve the substances, it should be ensured that these
solvents or chemicals in the test samples have no influence on the test result. The use of solvents other than
water should be restricted.
4.2.4 Preparation of test samples
4.2.4.1 General
The preparation of test samples can cause problems and is a very laborious task for the test evaluation
laboratories. Therefore, it might be appropriate to employ a centralized test sample preparation system, which
agrees to supply interested laboratories with test samples in stable form (e.g. lyophilized).
For large-scale evaluations (e.g. to obtain the data basis for a generalized description), all dilutions required
should be prepared in one batch to avoid day-to-day variations of weighing, diluting and differences in the
status of the “negative milk” (4.2.1).
4.2.4.2 Selection of concentrations
The selection of concentrations for the determination of the detection limits is described in 4.3.2. For
estimated purposes, if not otherwise stated, the concentration found to represent the detection limit should be
tested, together with one concentration step higher and two or three concentration steps lower than the
claimed detection limit and the corresponding negative milk. As an approximate guideline, it is recommended
to divide the concentration range giving 50 % to 100 % positives into three to four equal distant levels (linear
and logarithmic scales respectively) as demonstrated in Figure 1.
4.2.4.3 Dilution
The following precautions should be met when preparing a dilution series of test substances.
a) The preparation of the dilution series should be carried out in such a way that only the final dilution is
prepared with milk in order to avoid protein binding.
b) The proportion of the added aqueous standard solution in the final milk dilution step should be the same
for all dilutions and less than 1 %.
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SIST EN ISO 13969:2004
ISO 13969:2003(E)
IDF 183:2003(E)
4.2.4.4 Preservation
The preservation of test samples should preferably be carried out by lyophilization, if this is not deprec
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