SIST EN ISO 16297:2014
(Main)Milk - Bacterial count - Protocol for the evaluation of alternative methods (ISO 16297:2013)
Milk - Bacterial count - Protocol for the evaluation of alternative methods (ISO 16297:2013)
EN ISO 16297 gives guidelines for the evaluation of instrumental alternative methods for total bacterial count in raw milk from animals of different species.
Milch - Bestimmung der Gesamtkeimzahl - Protokoll für die Bewertung alternativer Verfahren (ISO 16297:2013)
Die vorliegende Internationale Norm gibt Anleitungen für die Bewertung alternativer instrumenteller Verfahren zur Bestimmung der Gesamtkeimzahl in Rohmilch von verschiedenen Tierarten.
ANMERKUNG Das Dokument wird als Ergänzung zu ISO 16140 und ISO 8196IDF 128 (siehe Abschnitt 2 und Literaturhinweis [1]) angesehen.
Lait - Dénombrement bactérien - Protocole pour l'évaluation des méthodes alternatives (ISO 16297:2013)
L'ISO 16297|FIL 161:2013 donne des lignes directrices pour l'évaluation de méthodes instrumentales alternatives pour le dénombrement de la flore totale dans le lait cru provenant d'animaux de différentes espèces.
Mleko - Število bakterij - Protokol za vrednotenje alternativnih metod (ISO 16297:2013)
Standard EN ISO 16297 podaja smernice za ocenjevanje bistvenih alternativnih metod za štetje bakterij v surovem mleku različnih vrst živali.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 16297:2014
01-julij-2014
Mleko - Število bakterij - Protokol za vrednotenje alternativnih metod (ISO
16297:2013)
Milk - Bacterial count - Protocol for the evaluation of alternative methods (ISO
16297:2013)
Milch - Bestimmung der Gesamtkeimzahl - Protokoll für die Bewertung alternativer
Verfahren (ISO 16297:2013)
Lait - Dénombrement bactérien - Protocole pour l'évaluation des méthodes alternatives
(ISO 16297:2013)
Ta slovenski standard je istoveten z: EN ISO 16297:2014
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 16297:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 16297:2014
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SIST EN ISO 16297:2014
EUROPEAN STANDARD
EN ISO 16297
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2014
ICS 67.100.10
English Version
Milk - Bacterial count - Protocol for the evaluation of alternative
methods (ISO 16297:2013)
Lait - Dénombrement bactérien - Protocole pour l'évaluation Milch - Bestimmung der Gesamtkeimzahl - Protokoll für die
des méthodes alternatives (ISO 16297:2013) Bewertung alternativer Verfahren (ISO 16297:2013)
This European Standard was approved by CEN on 1 May 2014.
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 CEN-CENELEC 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 CEN-CENELEC Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16297:2014 E
worldwide for CEN national Members.
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SIST EN ISO 16297:2014
EN ISO 16297:2014 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 16297:2014
EN ISO 16297:2014 (E)
Foreword
The text of ISO 16297:2013 has been prepared by Technical Committee ISO/TC 34 “Food products” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 16297:2014 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 November 2014, and conflicting national standards shall be withdrawn
at the latest by November 2014.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16297:2013 has been approved by CEN as EN ISO 16297:2014 without any modification.
3
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SIST EN ISO 16297:2014
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SIST EN ISO 16297:2014
INTERNATIONAL ISO
STANDARD 16297
IDF
161
First edition
2013-06-01
Milk — Bacterial count — Protocol for
the evaluation of alternative methods
Lait — Dénombrement bactérien — Protocole pour l’évaluation des
méthodes alternatives
Reference numbers
ISO 16297:2013(E)
IDF 161:2013(E)
©
ISO and IDF 2013
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO/IDF 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or
by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO or IDF at the respective address below or ISO’s member body in the
country of the requester.
ISO copyright office International Dairy Federation
Case postale 56 • CH-1211 Geneva 20 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels
Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail copyright@iso.org E-mail info@fil-idf.org
Web www.iso.org Web www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Transformation of results . 1
5 Attributes of the alternative method . 2
5.1 Description of the method to be evaluated . 2
5.2 Measuring range . 2
5.3 Carry-over . 3
5.4 Stability . 5
5.5 Precision . 5
6 The alternative method as an estimate of the reference method .6
6.1 Evaluation of factors affecting the estimation . 6
6.2 Measurement protocol . 7
6.3 Calculations . 7
6.4 Attributes of the alternative method expressed in units of the reference method .10
7 Rating of the elaborated attributes .10
Annex A (informative) Expression of precision parameters .11
Bibliography .13
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(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 16297|IDF 161 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk
and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.
iv © ISO and IDF 2013 – All rights reserved
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
Foreword
IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector
worldwide. IDF membership comprises National Committees in every member country as well as
regional dairy associations having signed a formal agreement on cooperation with IDF. All members of
IDF have the right to be represented at the IDF Standing Committees carrying out the technical work.
IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and
milk products.
The main task of Standing Committees is to prepare International Standards. Draft International
Standards adopted by the Standing Committees are circulated to the National Committees for
endorsement prior to publication as an International Standard. 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 document may be the subject of
patent rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 16297|IDF 161 was prepared by the International Dairy Federation (IDF) and Technical
Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published
jointly by IDF and ISO.
All work was carried out by Joint ISO-IDF Project Group (S07) of the Standing Committee on Statistics
and automation under the aegis of its project leader, Mrs. I. Andersson (SE).
This first edition of ISO 16297|IDF 161 cancels and replaces IDF 161A:1995, which has been
technically revised.
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
Introduction
Any quantitative measurement in microbiology should consider that there is a requirement for the
microbiological state in a sample to be regarded as one point within the co-ordinates of a multidimensional
system, which is to be projected on to the one-dimensional scale of the method applied, i.e. plate count,
flow cytometry. Aspects such as flora (types and numbers of microorganisms and their distribution),
growth phase, sub-lethal damage, metabolic activity, and history, influence to a greater or lesser extent
any parameter that is measured. It is evident that any projection of an n-dimensional situation on to an
one-dimensional scale is bound to provide a picture of the real situation that is rather restricted. In this
respect one has to bow to the inevitable, regardless of which method of measurement is preferred.
The term reference (or official or anchor) method in this International Standard means a method
internationally recognized by experts, used in legislation or by agreement between the parties. There
are requirements for evaluation of an alternative method to refer to the reference method and to be
based on the examination of suitable samples for its intended use.
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
INTERNATIONAL STANDARD
IDF 161:2013(E)
Milk — Bacterial count — Protocol for the evaluation of
alternative methods
1 Scope
This International Standard gives guidelines for the evaluation of instrumental alternative methods for
total bacterial count in raw milk from animals of different species.
NOTE The document is considered complementary to ISO 16140 and ISO 8196|IDF 128 (see Clause 2 and
Reference [1]).
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 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1: General
principles and definitions
ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method
for the determination of repeatability and reproducibility of a standard measurement method
ISO 8196-1|IDF 128-1, Milk — Definition and evaluation of the overall accuracy of alternative methods of
milk analysis — Part 1: Analytical attributes of alternative methods
ISO 8196-2|IDF 128-2, Milk — Definition and evaluation of the overall accuracy of alternative methods of
milk analysis — Part 2: Calibration and quality control in the dairy laboratory
ISO 16140-1, Microbiology of food and animal feed — Method validation — Part 1: Vocabulary
ISO 16140-2, Microbiology of food and animal feed — Method validation — Part 2: Protocol for the validation
of alternative (proprietary) methods against a reference method
ISO 21187|IDF 196:2004, Milk — Quantitative determination of bacteriological quality — Guidance for
establishing and verifying a conversion relationship between routine method results and anchor method results
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 8196-1|IDF 128-1 and
ISO 8196-2|IDF 128-2 apply.
For the definitions of precision, repeatability and reproducibility, see ISO 5725-1, ISO 5725-2,
ISO 8196-1|IDF 128-1, and ISO 16140-1.
4 Transformation of results
A prerequisite for statistics most common in the evaluation of measuring methods is the approximation
of a normal distribution of the data. The exponential multiplication of microorganisms usually leads to
a right-tailed distribution of quantitative microbiological parameters. Thus, in general, transformation
of the raw data is necessary for approximation of normality. This is usually a common logarithmic
transformation or a square root transformation for low bacteria levels. The most appropriate
transformation can be checked by comparing histograms. All statistics are then computed from the
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
transformed data, unless specified otherwise, and only the final results are re-transformed to give a
more expressive idea of the situation to the user (see also Annex A).
5 Attributes of the alternative method
NOTE The parameters outlined in this clause do not need to be evaluated completely for every alternative
method. For example, the measuring range (see 5.2) of the plate loop method is determined by the loop(s) used.
5.1 Description of the method to be evaluated
5.1.1 Description
The description of the method under study shall be in line with the checklist in 5.1.2.
Most of the information is found in the specification of the method given by the responsible supplier or
any other source (author) of the technique specified.
5.1.2 Checklist
a) Principle of the method.
b) Parameter or unit.
c) Technical design of the measurement procedure.
d) Field of application:
1) purpose: e.g. research, screening, milk grading;
2) matrix: e.g. raw milk from cows.
e) Supplier(s) of instrument, reagents, standards.
f) Specification of the method given by the producer or the author:
1) prerequisites for sampling (often compared to the situation of fat analysis);
2) possibilities for sample preservation [reagent(s), storage condition(s)];
3) quantitative (units: method under study or reference method) and qualitative (the kind of
microorganisms covered) spectrum;
4) precision (in units of the method under study or in reference method units);
5) accuracy of the estimate (in reference method units);
6) samples per hour;
7) list of references.
5.2 Measuring range
5.2.1 Lower limit of quantification
The lower limit of quantification is defined as the average of milk without bacteria plus the n-fold of its
standard deviation; generally, n = 10. See also ISO 16140-2.
Analyse milk without bacteria or with a very low concentration of bacteria. Transform data by calculating
square root from each result. Calculate the mean, x , and the standard deviation, s, of the transformed
results. Calculate the lower limit of quantification as xn+ s .
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SIST EN ISO 16297:2014
ISO 16297:2013(E)
IDF 161:2013(E)
5.2.2 Upper limit of quantification
The upper limit of quantification is determined by the highest possible reading of the method or by
methodological limitations, e.g. coincidence effects, inaccuracy in the upper measuring range, clogging
of filters. Coincidence is when two or more elements of the measurand are detected simultaneously and
identified as only one unit. For example, with flow cytometry, if two bacterial cells pass the detector
simultaneously, they are detected as one. The coincidence effect is higher with higher concentrations
of a measurand.
Upper limit of quantification is determined as the highest concentration where the instrument is still
linear according to 5.2.3.
5.2.3 Linearity of the instrument signal
The relationship between the instrument readings and the expected values shall be linear within the
concerned range of bacterial counts. Deviations from linearity may stem from non-specific signals and
coincidence effects.
A linearity check is at first performed visually using appropriate graphs to obtain an impression of the
shape of the relationship. Whenever deviation from linearity appears evident, a quantitative parameter
is calculated to indicate whether the observed trend is acceptable or not.
To achieve this, use a high bacterial count milk diluted serially with low bacterial count milk, resulting
in a set of at least 10 samples covering the concentration range of interest.
Measure all samples at least four times and calculate the average result for each sample. This gives the
measured value per sample. Use the measured values for the high count milk and the low count milk to
calculate values for the intermediate samples from the applied mixing ratios. This results in an expected
value for each sample. Then apply linear regression with the expected values per sample, C , on the x-axis
e
and the measured values per sample, C , on the y-axis. Calculate the residuals ΔC = C , – (
...
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