SIST EN ISO 16140-7:2025

SLOVENSKI STANDARD
01-januar-2025
Mikrobiologija v prehranski verigi - Validacija metode - 7. del: Protokol za
validacijo metod za identifikacijo mikroorganizmov (ISO 16140-7:2024)
Microbiology of the food chain - Method validation - Part 7: Protocol for the validation of
identification methods of microorganisms (ISO 16140-7:2024)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 7: Arbeitsvorschrift für
die Validierung von Identifizierungsverfahren von Mikroorganismen (ISO 16140-7:2024)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 7: Protocole
pour la validation de méthodes d’identification des micro-organismes (ISO 16140-
7:2024)
Ta slovenski standard je istoveten z: EN ISO 16140-7:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 16140-7
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 7:
Protocol for the validation of identification methods of
microorganisms (ISO 16140-7:2024)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 7: Protocole pour la validation de Verfahrensvalidierung - Teil 7: Arbeitsvorschrift für die
méthodes d'identification des micro-organismes (ISO Validierung von Identifizierungsverfahren von
16140-7:2024) Mikroorganismen (ISO 16140 7:2024)
This European Standard was approved by CEN on 6 October 2024.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-7:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 16140-7:2024) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
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 May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 16140-7:2024 has been approved by CEN as EN ISO 16140-7:2024 without any
modification.
International
Standard
ISO 16140-7
First edition
Microbiology of the food chain —
2024-11
Method validation —
Part 7:
Protocol for the validation
of identification methods of
microorganisms
Microbiologie de la chaîne alimentaire — Validation des
méthodes —
Partie 7: Protocole pour la validation de méthodes
d’identification des micro-organismes
Reference number
ISO 16140-7:2024(en) © ISO 2024

ISO 16140-7:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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 at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 16140-7:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General principles for the validation of identification methods of microorganisms . 4
5 Strains . 4
6 Performance characteristics of an identification method . 5
6.1 General .5
6.2 Description of the concept and limitation(s) of the identification method .5
6.3 Identification reliability of the identification method .6
6.3.1 Number of strains to be tested .6
6.3.2 Selection of the strains .7
6.3.3 Testing of the strains .7
6.3.4 Expression and interpretation of results .8
6.4 Evaluation .9
7 Interlaboratory study .10
7.1 General .10
7.2 Data sets to be obtained .10
7.3 Protocol .11
7.4 Expression of results . .11
7.5 Interpretation and evaluation . 12
Annex A (informative) Guidelines for the validation of methods for the identification
of microorganisms in ecosystems . 14
Annex B (normative) Points to be considered when selecting strains for an identification
reliability study .21
Annex C (informative) Expression, interpretation and evaluation of results.22
Annex D (informative) Illustrations on the validation of methods for the identification
of microorganisms in ecosystems .30
Bibliography .35

iii
ISO 16140-7:2024(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical Committee
CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 16140 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

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ISO 16140-7:2024(en)
Introduction
0.1  The ISO 16140 series
The ISO 16140 series has been expanded in response to the need for various ways to validate or verify test
methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists of several parts with the
general title, Microbiology of the food chain — Method validation:
— Part 1: Vocabulary;
— Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;
— Part 3: Protocol for the verification of reference methods and validated alternative methods in a single
laboratory;
— Part 4: Protocol for method validation in a single laboratory;
— Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;
— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and
typing procedures;
— Part 7: Protocol for the validation of identification methods of microorganisms.
ISO 17468 is a closely linked International Standard, which establishes technical rules for the development
and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.
— The first stage is the validation of the method. Validation is conducted using a study in a single laboratory
followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5, ISO 16140-6 and as described in
this document). In the case when a method is validated within one laboratory (see ISO 16140-4), no
interlaboratory study is conducted.
— The second stage is method verification, where a laboratory demonstrates that it can satisfactorily
perform a validated method. This is described in ISO 16140-3. Verification is only applicable to methods
that have been validated using an interlaboratory study.
In general, two types of methods are distinguished: reference methods and alternative methods.
A reference method is defined in ISO 16140-1:2016, 2.59, as an “internationally recognized and widely
accepted method”. The note to entry clarifies that “these are ISO standards and standards jointly published
by ISO and CEN or other regional/national standards of equivalent standing”.
In the ISO 16140 series, reference methods include standardized reference (ISO and CEN) methods as
defined in ISO 17468:2023, 3.7, as a “reference method described in a standard”.
An alternative method (method submitted for validation) is defined in ISO 16140-1:2016, 2.4, as a “method
of analysis that detects or quantifies, for a given category of products, the same analyte as is detected or
quantified using the corresponding reference method”. The note to entry clarifies that: “The method can be
proprietary. The term ‘alternative’ is used to refer to the entire ‘test procedure and reaction system’. This
term includes all ingredients, whether material or otherwise, required for implementing the method”.
ISO 16140-4 addresses validation within a single laboratory. The results are therefore only valid for the
laboratory that conducted the study. In this case, verification (as described in ISO 16140-3) is not applicable.
ISO 16140-5 describes protocols for non-proprietary methods where a more rapid validation is required or
when the method to be validated is highly specialized and the number of participating laboratories required
by ISO 16140-2 cannot be reached. ISO 16140-4 and ISO 16140-5 can be used for validation against a
reference method. ISO 16140-4 (regarding qualitative and quantitative methods) and ISO 16140-5 (regarding
quantitative methods only) can also be used for validation without a reference method.

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ISO 16140-7:2024(en)
The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above. It
also guides the user in selecting the right part of the ISO 16140 series, taking into account the purpose of the
study and the remarks given above.
Figure 1 — Flow chart for application of ISO 16140-2 to ISO 16140-5
NOTE 1 In this document, the words “category”, “type” and/or “item” are sometimes combined with “(food)” to
improve readability. However, the word “(food)” is interchangeable with “(feed)” and other areas of the food chain as
mentioned in Clause 1.
ISO 16140-6 and this document (ISO 16140-7) are somewhat different from the other parts in the ISO 16140
series in that they relate to very specific situations.
ISO 16140-6 is restricted to the confirmation procedure of a method to be validated [e.g. the biochemical
confirmation of Enterobacteriaceae (see ISO 21528-2)]. The confirmation procedure advances a suspected
(presumptive) result to a confirmed positive result. The validation of alternative typing techniques
(e.g. serotyping of Salmonella) is also covered by ISO 16140-6. The validation study in ISO 16140-6 clearly
specifies the selective agar(s) from which strains can be confirmed using the alternative confirmation
method. If successfully validated, the alternative confirmation method can only be used if strains are
recovered on an agar that was used and was shown to be acceptable within the validation study. Figure 2
shows the possibilities where an alternative confirmation method validated in accordance with ISO 16140-6
can be applied (see text in the boxes).

vi
ISO 16140-7:2024(en)
Figure 2 — Use of validated alternative confirmation methods (see ISO 16140-6)
EXAMPLE 1 An example application of a validated alternative confirmation method is as follows.
An alternative confirmation method based on ELISA has been validated to replace the biochemical confirmation for
Salmonella as described in ISO 6579-1. In the validation study, XLD (mandatory agar in accordance with ISO 6579-1)
plus BGA and a specified chromogenic agar (two optional agars for second plating in accordance with ISO 6579-1) were
used as the agars to start the confirmation. The validated confirmation method can be used to replace the biochemical
confirmation under the following conditions:
— by laboratories using ISO 6579-1; or
— by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for confirmation; or
— by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation from XLD and/or
BGA agar and/or the specified chromogenic agar.
The validated confirmation method cannot be used under the following conditions:
— by laboratories using an ISO 16140-2 validated alternative method that refers only to agars other than those
included in the validation to start the confirmation (e.g. Hektoen agar and SS agar only); or
— by laboratories using an ISO 16140-2 validated alternative method that refers only to a confirmation procedure
that does not require isolation on agar.
This document (ISO 16140-7) addresses the validation of identification procedures (e.g. molecular
identification using multiplex PCR or DNA sequencing or mass spectrometry). This document differs from
the other parts in the ISO 16140 series, as it is intended for microbial identification for which there is no
reference method and, therefore, it is not possible to run a method comparison study. The validation study in
this document specifies the identification method principle, the identification database and algorithm when
appropriate, and the agar(s) from which strains can be identified. If properly characterized and successfully
validated, the identification method can only be validly used on strains recovered on the agars covered and
shown to have been acceptable within the validation study.
NOTE 2 Whole-genome sequencing (WGS) in accordance with ISO 23418 will eventually be a reference method
for all microorganisms, but the implementation of this technique is still at an early stage. Therefore, the use of WGS
cannot currently be requested as a reference method for a large panel of strains.
Figure 3 shows the possibilities where an alternative confirmation method validated in accordance with
ISO 16140-6 and an alternative identification method validated in accordance with this document can be
applied within a reference method or an ISO 16140-2 validated detection or enumeration method. The result
provided by the ISO 16140-7 validated method can be considered as additional information on the identity
of the tested colony(ies); this result cannot be taken as a confirmation result. When there is a discrepancy

vii
ISO 16140-7:2024(en)
between the results of the ISO 16140-6 validated method and the ISO 16140-7 validated method, a root
cause analysis is conducted. An ISO 16140-7 validated method can also be used to identify colonies within
methods that do not require a confirmation step.
Figure 3 — Flow chart for the application of ISO 16140-6 and this document for
the confirmation and identification of colonies within a reference method or an
ISO 16140-2 validated detection or enumeration method
If the identification method is also validated in accordance with ISO 16140-6, the same method can be used
for both confirmation and identification.
When a confirmation method is used, it is possible to apply an identification method validated in accordance
with this document for further identification.
EXAMPLE 2 An alternative confirmation method of Campylobacter genus can be validated in accordance with
ISO 16140-6 and compared to the mandatory confirmation procedure at the genus level described in ISO 10272-1.
The identification at the Campylobacter species level is optional in ISO 10272-1 and ISO 10272-2 and is therefore not
mandatory. In this instance, an identification method at the Campylobacter species level can be validated in accordance
with this document. If the method is validated by ISO 16140-6 and this document, it can be used for both confirmation
and identification purposes.
0.2  Validation and verification of identification methods of microorganisms
The procedure described in this document is intended for validation of identification methods of
microorganisms. This procedure comprises two parts: a performance characteristics study and an
interlaboratory study.
The procedure for validation of identification methods of microorganisms in a single laboratory is described
in ISO 16140-4. The procedure for verification of identification methods of microorganisms in a single
laboratory is described in ISO 16140-3.

viii
International Standard ISO 16140-7:2024(en)
Microbiology of the food chain — Method validation —
Part 7:
Protocol for the validation of identification methods of
microorganisms
1 Scope
This document specifies the general principle and the technical protocol for the validation of identification
methods of microorganisms for microbiology in the food chain. As there is no reference method, no method
comparison study can be run. Therefore, this document provides a protocol to evaluate the performance
characteristics and validate the method workflow using well-defined strains. When required, an additional
identification method can be used.
This document is applicable to the validation of identification methods of microorganisms that are used for
the analysis of isolated colonies from:
— products intended for human consumption;
— products for feeding animals;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
Identification methods only validated in accordance with this document cannot be used instead of
confirmation described in:
— the reference method;
— an alternative method validated in accordance with ISO 16140-2;
— an alternative method validated in accordance with ISO 16140-6.
In these instances, the identification method is validated in accordance with ISO 16140-6 method that is
used as a confirmation method.
This document is applicable to bacteria and fungi. Some clauses can be applicable to other (micro)organisms,
which can be determined on a case-by-case basis.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements 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 16140-1, Microbiology of the food chain — Method validation — Part 1: Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following apply.

ISO 16140-7:2024(en)
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
acceptability limit
AL
maximum acceptable proportion of deviations between the reference identities (or if not known, the
accepted reference values) of the strains or specimens and the corresponding identification results obtained
when applying the operating procedure of the candidate identification method
Note 1 to entry: In the context of this document, the reference value can be the assigned identity of the strain.
3.2
agreement
identification agreement
method under validation study provides the same identification result as the assigned, i.e. original,
identification of the tested strain
3.3
assigned identity
result of the microorganism identification displaying generally accepted molecular and/or biochemical
characteristics
[13]
EXAMPLE Bergey’s Manual of Systematics of Archaea and Bacteria .
3.4
comparison algorithm
defined calculation rules used to compare the profile of the analysed strain to the database
3.5
confirmation procedure
confirmation test
procedure or test which is carried out to verify a presumptive result
Note 1 to entry: Not all methods have a confirmation procedure
Note 2 to entry: A confirmation test can provide a positive or negative result, without yielding the identity of the
analyte.
[SOURCE: ISO 16140-1:2016, 2.17, modified — Note 2 has been included.]
3.6
database
library
collection of data categories and concept entry structure of an identification database
Note 1 to entry: An identification database usually gathers the phenotypic or molecular data information of several
strains from the same species or genus.
Note 2 to entry: Some identification methods can have a restricted scope and do not imply a database (e.g. multiplex
PCR assay).
3.7
deviation
identification deviation
method under validation study does not provide the same identification result as the assigned, i.e. original,
identification of the tested strain

ISO 16140-7:2024(en)
3.8
group
group of microbial ecosystems
specimens processed in a similar way, with similar intrinsic characteristics and a similar microbial ecology
EXAMPLE Enrichment broths.
3.9
homology
score
identity between the profile of the analysed strain and the entry(ies) in the database
Note 1 to entry: This is normally measured as % or with score value(s).
Note 2 to entry: For select identification methods (e.g. microarray), a homology score may not be obtained.
3.10
identification method
method submitted for validation
method of analysis that provides the name (identity) of the microorganism (e.g. species or higher taxonomy
ranking level)
Note 1 to entry: The method can be non-proprietary or proprietary.
Note 2 to entry: The methods can be based on various principles (e.g. phenotypic and molecular principles).
Note 3 to entry: The identification of microorganisms can help for example in determining whether it is a safety
or spoilage concern, or is a specific technological or probiotic strain, or is likely to be resistant to an inactivation
treatment.
3.11
identification procedure
identification test
procedure or test yielding the identity of the analyte (e.g. species or higher taxonomy ranking level)
3.12
level of detection
LOD
x
relative concentration of the measured analyte in proportion to the total count of the specimen, obtained by
a given measurement procedure, for which the probability of detection is x
EXAMPLE LOD is the level of detection for which 50 % of tests give a positive result.
[SOURCE: ISO 16140-1:2016, 2.35, modified — In the definition, “relative” and “in proportion to the total
count of the specimen” have been added and the Note 1 to entry has been deleted.]
3.13
profile
set of characteristics that identify or are used to identify a strain
Note 1 to entry: The profile can be phenotypic (e.g. biochemical or serological) and/or molecular (e.g. DNA fingerprint,
DNA sequence or mass spectra).
3.14
reliability
identification reliability
closeness of agreement between an identification result and the assigned identity of the tested strain
Note 1 to entry: The concept “identification reliability” is related to the identity of the analyte, i.e. genus or/and species
names(s).
Note 2 to entry: “Identification reliability” is sometimes understood as closeness of agreement with the identification
result that are being attributed to the identity of the strain given by another identification method.

ISO 16140-7:2024(en)
3.15
risk of non-identification
ratio of the probability of getting no identification result within a specified set of strains included in the
scope of validation
Note 1 to entry: This does not apply to misidentification.
3.16
scope of validation
database content and version, culture media or group of microbial ecosystems and comparison algorithm
for which a validated method for the identification of microorganisms can be used satisfactorily
[SOURCE: ISO 16140-1:2016, 2.70 modified — In the definition, “analytes, matrices, and concentrations”
has been replaced by “database content and version, culture media or group of microbial ecosystems and
comparison algorithm”.]
4 General principles for the validation of identification methods of microorganisms
The validation protocol comprises two phases:
— a performance characteristics study;
— an interlaboratory study.
NOTE 1 For proprietary methods, the validation study is conducted by an organizing laboratory (see
ISO 16140-1:2016, 2.45).
NOTE 2 It is possible, if relevant, to include inclusivity or exclusivity data obtained in an ISO 16140-2 or an
ISO 16140-6 validation study into a study related to this document.
NOTE 3 As there is no reference identification method, there is no method comparison study.
The technical rules for performing the method performance characteristics study and the interlaboratory
study are given in Clauses 6 and 7. An extended set of strains from a non-selective agar or a selective agar
when appropriate (e.g. yeasts and moulds) will be tested for both parts.
Where appropriate, the validation protocol shall also specify the selective media from which strains can be
identified using the identification method. A specified number of strains shall be tested.
NOTE 4 The term “agar” is often referred to as “solid culture medium”.
NOTE 5 Other applications of identification methods are possible (e.g. identification of microorganisms in an
ecosystem), see Annex A for further information.
NOTE 6 The identification procedure can be ensured by the collaboration of multiple laboratories. For instance, a
first laboratory can isolate the strains and run the sample preparation such as DNA extraction and sequencing; the
generated data can be analysed by comparison to the database held and managed by a second laboratory. In such a
case, during the interlaboratory study, multiple collaborators will be involved in the sample preparation and will send
the results for analysis to the laboratory managing the identification data interpretation.
5 Strains
The pure strains used for determining the identification reliability of the method shall be well-characterized
in line with the purpose of the validation study. When necessary, the identification information provided by
a second identification is used to evaluate the discrepancies between the results of the tested identification
method and the assigned identity of the strain.
NOTE 1 National, regional or international reference laboratories can be contacted during such investigations.
NOTE 2 Well-characterized strains are strains that can be obtained from national/international culture collections,
or ones that have been obtained locally and have been previously identified and given the same identity using usually
two or more identification methods that are based on dissimilar principles.

ISO 16140-7:2024(en)
NOTE 3 When appropriate, select strains with available WGS data in accordance with ISO 23418.
6 Performance characteristics of an identification method
6.1 General
The performance characteristics study is the part of the validation that is performed in one laboratory.
It consists of a description of the concept and limitation(s) of the identification method, and an identification
reliability study of the method. The results are then compared to those of the assigned identities of the
tested set of strains.
6.2 Description of the concept and limitation(s) of the identification method
The method characteristics described in Table 1 shall be carefully described in the method protocol (e.g.
standard operation procedure, instructions for use) to enable proper use of the identification method by the
end-user.
Table 1 — Essential components of the performance characteristics study
Characteristics Detailed information
Scope of validation Database description including name, version and content
Culture media
Microorganism name or type (e.g. bacteria, yeast, moulds, lactic acid bacteria,
Listeriaceae)
Comparison algorithm version
Identification method Description of the strain profile/sequence and instrument signal acquisition used for
principle identification of microorganisms
Database content List of the genera and species in the database version used in the validation study
Concept of the database Description of the types of signal profiles in the database, e.g. one unique profile select-
ed as a representative for various strains of the same species or several signal profiles
of various strains of the same species
Strains per species and Number of strains per species and genera
genera in database
A minimum of 5 strains is required to claim a species in the database content
Limitations List of the closely related species that are not differentiated by the identification
method, size of the database
Comparison algorithm Calculation of the result reporting, e.g. comparison of the instrument signal profile of
the analysed strain to each single profile in the database or comparison of the profile of
the analysed strain to unique average profiles for each species of the database.
Result reporting Description of the homology concept used to assess the identification results with high
confidence or low confidence
Strain preparation and Description of the strain preparation and method workflow, particularly when
troubleshooting obtaining homology result of low confidence or when no identification result is
generated
In some very specific cases, the minimum number of strains per species in the database can be lower than
the required 5 strains due to the lack of available strains. There should be a disclaimer in the database
content for strains that do not meet the minimum requirement.
If the identification method does not imply a database (e.g. multiplex PCR assay), the description of the
concept and limitation(s) of the identification method should be restricted to the scope of validation,
the identification method principle, the limitations, the result reporting, the strain preparation and
troubleshooting.
ISO 16140-7:2024(en)
6.3 Identification reliability of the identification method
6.3.1 Number of strains to be tested
The set of strains to be tested depends on the database content and the scope of the validation. Select, as
much as possible, one strain per species to ensure that at least 25 % of genera and 10 % of species contained
in the database are tested. However, a minimum and a maximum number of strains to be tested has been
specified at, respectively, 250 strains and 1 000 strains. For restricted database content (e.g. 25 genera and
200 species), test all the genera and species.
Figure 4 provides examples of how to determine the appropriate number of strains to be tested.
Figure 4 — Examples on how to determine the number of strains to be tested
Pure cultures from a non-selective agar of all selected strains shall be tested by the identification method.
NOTE 1 Non-selective agars (e.g. TSA) enable the recovery of a broad range of microorganisms and can favour the
recovery of some specified groups of microorganisms. For microorganisms, for which non-selective agars are not
known or are not supported by corresponding standardized reference methods for the isolation of the respective
microorganism, only selective agars can be used instead.
If the scope of the validation study also includes selective agars, refer to Table 2 for the number of relevant
genera and species to be tested for each selective agar included in the scope.
NOTE 2 Selective agars can enable the recovery of microorganisms from specific clades, (i) family, (ii) genus
or group of species (e.g. Bacillus cereus group). In this document, “group of species ” is substituted with “genus” to
improve readability. However, the word “genus” is interchangeable with “group of species”.

ISO 16140-7:2024(en)
Table 2 — Number of strains to be tested for each of the selective agars included in the scope
Taxonomy level of the strains recovered Number of strains to be tested for each of the selective agars
on the tested selective agars included in the scope
Family 100 strains, from as many different genera and species as possible,
including strains from multiple families that can grow on the selec-
tive agar
Genus 50 strains, from as many different species as possible, including
strains from multiple genera that can grow on the selective agar
Species 25 strains that can grow on the selective agar
Sub-species, types 25 strains that can grow on the selective agar
For a method with a restricted scope (e.g. multiplex PCR assay), it is recommended to test a relevant panel of
strains that should not produce identification results (negative control strains) as expected by the scope of
the method (see the example in Clause C.4).
6.3.2 Selection of the strains
A broad range of strains shall be used. Criteria used for selecting test strains shall be in accordance
with Annex B. The strains selected should take into account the measurement principle (e.g. phenotypic
or molecular based) and the database content of the identification method. Different measurement
principles can require the use of panels of different test strains, representing the diversity of the studied
microorganism(s). As much as possible, strains used in the development of the database should not be used
in the validation of the identification method unless availability of strains is limited. In this case, disclosure
shall be included in the validation study report. The rationale for the choice of the strains and their
characteristics shall be included in the validation study report (e.g. coverage of the database, closely related
species, recent changes in taxonomy).
Select strains from species able to be recovered on the test agar(s). For selective agars, select test strains
from multiple families or genera.
Each strain shall be characterized in sufficient detail for its assigned identity to be known using relevant
phenotypic or molecular tests. The method or the method principle used for the identification shall be known.
Strains should preferably have been isolated from foods, feed, the food-processing environment or from
primary production; depending on the scope of the validation. However, clinical strains can also be used.
The original source of all strains should be known, and they should be held in a local (e.g. expert laboratory),
national or international culture collection to enable them to be used in future testing if required. See
ISO 11133 for guidance on the local maintenance of stock cultures.
Results generated by a specialized reference laboratory, using an appropriate identification method, can
be used if the laboratory performing the validation study is
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