SIST EN ISO 16140-3:2021/A1:2025

Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory - Amendment 1: Protocol for verification of validated identification methods of microorganisms (ISO 16140-3:2021/Amd 1:2025)

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die Verifizierung von Referenz- und validierten alternativen Verfahren in einem Einzel-Labor - Änderung 1: Verifizierung von validierten Identifizierungsverfahren (ISO 16140-3:2021/Amd 1:2025)

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes alternatives validées - Amendement 1: Protocole pour la vérification des méthodes validées d’identification des micro-organismes (ISO 16140-3:2021/Amd 1:2025)

Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za preverjanje referenčnih in validiranih alternativnih metod, izvedenih v posameznem laboratoriju - Dopolnilo A1: Protokol za validirane metode identifikacije mikroorganizmov (ISO 16140-3:2021/Amd 1:2025)

General Information

• Status: Not Published
• Public Enquiry End Date: 25-Feb-2025
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 04-Sep-2025
• Due Date: 09-Nov-2025
• Ref Project: EN ISO 16140-3:2021/A1:2025 - Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory - Amendment 1: Protocol for verification of validated identification methods of microorganisms (ISO 16140-3:2021/Amd 1:2025)

Relations

• Amends: SIST EN ISO 16140-3:2021 - Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory (ISO 16140-3:2021)
• Effective Date: 01-Oct-2025

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 16140-3:2021/oprA1:2025
01-februar-2025
Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za
preverjanje referenčnih in validiranih alternativnih metod, izvedenih v
posameznem laboratoriju - Dopolnilo A1: Validirane metode identifikacije
mikroorganizmov - Tehnični protokol za preverjanje (ISO 16140-
3:2021/DAmd1:2024)
Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of
reference methods and validated alternative methods in a single laboratory - Amendment
1: Validated identification methods of microorganisms - Technical protocol for verification
(ISO 16140-3:2021/DAmd1:2024)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für
die Verifizierung von Referenz- und validierten alternativen Verfahren in einem Einzel-
Labor - Änderung 1: Verifizierung von validierten Identifizierungsverfahren (ISO 16140
3:2021/DAmd1:2024)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole
pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes
alternatives validées - Amendement 1: Méthodes validées d’identification des micro-
organismes - Protocole technique pour la vérification (ISO 16140-3:2021/DAmd1:2024)
Ta slovenski standard je istoveten z: EN ISO 16140-3:2021/prA1:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-3:2021/oprA1:2025 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

SIST EN ISO 16140-3:2021/oprA1:2025

SIST EN ISO 16140-3:2021/oprA1:2025
DRAFT
Amendment
ISO 16140-3:2021/
DAM 1
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Method validation —
Voting begins on:
Part 3:
2024-12-04
Protocol for the verification of
Voting terminates on:
reference methods and validated 2025-02-26
alternative methods in a single
laboratory
AMENDMENT 1: Validated
identification methods of
microorganisms — Technical protocol
for verification
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO 16140-3:2021/DAM 1:2024(en)

SIST EN ISO 16140-3:2021/oprA1:2025
DRAFT
ISO 16140-3:2021/DAM 1:2024(en)
Amendment
ISO 16140-3:2021/
DAM 1
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Method validation —
Voting begins on:
Part 3:
Protocol for the verification of
Voting terminates on:
reference methods and validated
alternative methods in a single
laboratory
AMENDMENT 1: Validated
identification methods of
microorganisms — Technical protocol
for verification
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2024
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BE CONSIDERED IN THE LIGHT OF THEIR
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
CH-1214 Vernier, Geneva
NOTIFICATION OF ANY RELEVANT PATENT
Phone: [phone]
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO 16140-3:2021/DAM 1:2024(en)

ii
SIST EN ISO 16140-3:2021/oprA1:2025
ISO 16140-3:2021/DAM 1:2024(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.

iii
SIST EN ISO 16140-3:2021/oprA1:2025

SIST EN ISO 16140-3:2021/oprA1:2025
ISO 16140-3:2021/DAM 1:2024(en)
Microbiology of the food chain — Method validation —
Part 3:
Protocol for the verification of reference methods and
validated alternative methods in a single laboratory
AMENDMENT 1: Validated identification methods of
microorganisms — Technical protocol for verification

Introduction (0.1)
Replace the third sentence with:
The ISO 16140 series consists of several parts with the general title, Microbiology of the food chain — Method
validation:
Add the following text after the sixth bullet point:
— Part 7: Protocol for the validation of identification methods of microorganisms.

Replace the reference to ISO 17468:2016, 3.5, in the sixth paragraph with:
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”.

Replace the first sentence after the NOTE 2 on Page vii with:
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)].

Replace the verb “defines” with “specifies” in the third sentence of the next paragraph:
The validation study in ISO 16140-6 clearly specifies the selective agar(s) from which strains can be
confirmed using the alternative confirmation method.

Replace Figure 2 with:
SIST EN ISO 16140-3:2021/oprA1:2025
ISO 16140-3:2021/DAM 1:2024(en)
Figure 2 — Use of validated alternative confirmation methods (see ISO 16140-6)
Replace the first sentence of the EXAMPLE below Figure 2 with:
EXAMPLE 1 An example application of a validated alternative confirmation method is as follows.

Add the following text in the end of 0.1 on Page viii:
This document (ISO 16140-7) addresses the validation of identification procedures (e.g. molecular
identification using multiplex PCR or DNA sequencing or mass spectrometry). ISO 16140-7 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 ISO 16140-7 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 3 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.
ISO 16140-7:—, 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 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.
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.
SIST EN ISO 16140-3:2021/oprA1:2025
ISO 16140-3:2021/DAM 1:2024(en)
Introduction (0.2)
Add the following text after NOTE 2 on Page ix as last bullet point:
— ISO 16140-7 for identification methods.

Clause 1
Add the following text in the last paragraph before the last sentence:
The technical protocol for the verification of validated identification methods is described in Clause 9.

Clause 3
Add the following definitions:
3.22
agreement
identification agreement
method under validation study provides the same identification result as the assigned, i.e. original,
identification of the tested strain
[SOURCE: ISO 16140-7:—, 3.2]
3.23
assigned identity
result of the microorganism identification displaying generally accepted molecular and/or biochemical
characteristics
[21]
EXAMPLE Bergey’s Manual of Systematics of Archaea and Bacteria.
[SOURCE: ISO 16140-7:—, 3.3]
3.24
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
[SOURCE: ISO 16140-7:—, 3.7]
3.25
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.
[SOURCE: ISO 16140-7:—, 3.10]
Clause 8
SIST EN ISO 16140-3:2021/oprA1:2025
ISO 16140-3:2021/DAM 1:2024(en)
Add a line to Table 16:
Identification method Panel of strains 100 % agreement

Clause 9
Add the following text:
9  Validated identification metho
...