ILNAS-EN ISO 16140-3:2021 - Preview only Copy via ILNAS e-Shop

ILNAS-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
Microbiologie de la chaîne alimentaire -
Validation des méthodes - Partie 3:
Protocole pour la vérification dans un
seul laboratoire de méthodes de
Mikrobiologie der Lebensmittelkette -
Verfahrensvalidierung - Teil 3:
Arbeitsvorschrift für die Verifizierung von
Referenz- und validierten alternativen

02/2021
 ILNAS-EN ISO 16140-3:2021

National Foreword

This European Standard EN ISO 16140-3:2021 was adopted as Luxembourgish

Standard ILNAS-EN ISO 16140-3:2021.

Every interested party, which is member of an organization based in Luxembourg, can

participate for FREE in the development of Luxembourgish (ILNAS), European (CEN,
CENELEC) and International (ISO, IEC) standards:

- Participate in the design of standards

- Foresee future developments
- Participate in technical committee meetings
ILNAS-EN ISO 16140-3:2021 - Preview only Copy via ILNAS e-Shop

https://portail-qualite.public.lu/fr/normes-normalisation/participer-normalisation.html

THIS PUBLICATION IS COPYRIGHT PROTECTED

Nothing from this publication may be reproduced or utilized in
any form or by any mean - electronic, mechanical, photocopying
or any other data carries without prior permission!
 
ILNAS-EN ISO 16140-3:2021
 EN ISO 16140-3
 
2
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Ͳ͹ǤͳͲͲǤ͵Ͳ

‰Ž‹•Š‡”•‹‘

 ‹ ”‘„‹‘Ž‘‰›‘ˆ–Š‡ˆ‘‘† Šƒ‹Ǧ‡–Š‘†˜ƒŽ‹†ƒ–‹‘Ǧƒ”–͵ǣ
”‘–‘ ‘Žˆ‘”–Š‡˜‡”‹ˆ‹ ƒ–‹‘‘ˆ”‡ˆ‡”‡ ‡‡–Š‘†•ƒ†
˜ƒŽ‹†ƒ–‡†ƒŽ–‡”ƒ–‹˜‡‡–Š‘†•‹ƒ•‹‰Ž‡Žƒ„‘”ƒ–‘”›ȋ 
ͳ͸ͳͶͲǦ͵ǣʹͲʹͳȌ
ILNAS-EN ISO 16140-3:2021 - Preview only Copy via ILNAS e-Shop

‹ ”‘„‹‘Ž‘‰‹‡†‡Žƒ ŠƒÁ‡ƒŽ‹‡–ƒ‹”‡ǦƒŽ‹†ƒ–‹‘†‡•  ‹”‘„‹‘Ž‘‰‹‡†‡”‡„‡•‹––‡Ž‡––‡Ǧ

±–Š‘†‡•Ǧƒ”–‹‡͵ǣ”‘–‘ ‘Ž‡’‘—”Žƒ˜±”‹ˆ‹ ƒ–‹‘†ƒ• ‡”ˆƒŠ”‡•˜ƒŽ‹†‹‡”—‰Ǧ‡‹Ž͵ǣ”„‡‹–•˜‘”• Š”‹ˆ–ˆò”†‹‡
—•‡—ŽŽƒ„‘”ƒ–‘‹”‡†‡±–Š‘†‡•†‡”±ˆ±”‡ ‡‡–†‡ ‡”‹ˆ‹œ‹‡”—‰˜‘‡ˆ‡”‡œǦ—†ƒŽ–‡”ƒ–‹˜‡‡”ˆƒŠ”‡
±–Š‘†‡•ƒŽ–‡”ƒ–‹˜‡•˜ƒŽ‹†±‡•ȋ ͳ͸ͳͶͲǦ͵ǣʹͲʹͳȌ ‹‡‹‡‡‹œ‡Ž‡ƒ„‘”ȋ ͳ͸ͳͶͲǦ͵ǣʹͲʹͳȌ

Š‹•—”‘’‡ƒ–ƒ†ƒ”†™ƒ•ƒ’’”‘˜‡†„›‘ʹͺ‡ ‡„‡”ʹͲʹͲǤ

‡„‡”•ƒ”‡„‘—†–‘ ‘’Ž›™‹–Š–Š‡Ȁ –‡”ƒŽ‡‰—Žƒ–‹‘•™Š‹ Š•–‹’—Žƒ–‡–Š‡ ‘†‹–‹‘•ˆ‘”‰‹˜‹‰–Š‹•
—”‘’‡ƒ–ƒ†ƒ”†–Š‡•–ƒ–—•‘ˆƒƒ–‹‘ƒŽ•–ƒ†ƒ”†™‹–Š‘—–ƒ›ƒŽ–‡”ƒ–‹‘Ǥ’Ǧ–‘Ǧ†ƒ–‡Ž‹•–•ƒ†„‹„Ž‹‘‰”ƒ’Š‹ ƒŽ”‡ˆ‡”‡ ‡•
‘ ‡”‹‰•— Šƒ–‹‘ƒŽ•–ƒ†ƒ”†•ƒ›„‡‘„–ƒ‹‡†‘ƒ’’Ž‹ ƒ–‹‘–‘–Š‡Ǧƒƒ‰‡‡–‡–”‡‘”–‘ƒ›
‡„‡”Ǥ

Š‹•—”‘’‡ƒ–ƒ†ƒ”†‡š‹•–•‹–Š”‡‡‘ˆˆ‹ ‹ƒŽ˜‡”•‹‘•ȋ‰Ž‹•Šǡ ”‡ Šǡ ‡”ƒȌǤ˜‡”•‹‘‹ƒ›‘–Š‡”Žƒ‰—ƒ‰‡ƒ†‡„›
–”ƒ•Žƒ–‹‘—†‡”–Š‡”‡•’‘•‹„‹Ž‹–›‘ˆƒ‡„‡”‹–‘‹–•‘™Žƒ‰—ƒ‰‡ƒ†‘–‹ˆ‹‡†–‘–Š‡Ǧƒƒ‰‡‡–
‡–”‡Šƒ•–Š‡•ƒ‡•–ƒ–—•ƒ•–Š‡‘ˆˆ‹ ‹ƒŽ˜‡”•‹‘•Ǥ

‡„‡”•ƒ”‡–Š‡ƒ–‹‘ƒŽ•–ƒ†ƒ”†•„‘†‹‡•‘ˆ—•–”‹ƒǡ‡Ž‰‹—ǡ—Ž‰ƒ”‹ƒǡ”‘ƒ–‹ƒǡ›’”—•ǡœ‡ Š‡’—„Ž‹ ǡ‡ƒ”ǡ•–‘‹ƒǡ
‹Žƒ†ǡ ”ƒ ‡ǡ ‡”ƒ›ǡ ”‡‡ ‡ǡ —‰ƒ”›ǡ ‡Žƒ†ǡ ”‡Žƒ†ǡ –ƒŽ›ǡƒ–˜‹ƒǡ‹–Š—ƒ‹ƒǡ—š‡„‘—”‰ǡƒŽ–ƒǡ‡–Š‡”Žƒ†•ǡ‘”™ƒ›ǡ
‘Žƒ†ǡ‘”–—‰ƒŽǡ‡’—„Ž‹ ‘ˆ‘”–Šƒ ‡†‘‹ƒǡ‘ƒ‹ƒǡ‡”„‹ƒǡŽ‘˜ƒ‹ƒǡŽ‘˜‡‹ƒǡ’ƒ‹ǡ™‡†‡ǡ™‹–œ‡”Žƒ†ǡ—”‡›ƒ†
‹–‡†‹‰†‘Ǥ





    

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CEN-CENELECManagement Centre: Rue de la Science 23, B-1040 Brussels

̹ʹͲʹͳ ŽŽ”‹‰Š–•‘ˆ‡š’Ž‘‹–ƒ–‹‘‹ƒ›ˆ‘”ƒ†„›ƒ›‡ƒ•”‡•‡”˜‡† ‡ˆǤ‘Ǥ ͳ͸ͳͶͲǦ͵ǣʹͲʹͳ

™‘”Ž†™‹†‡ˆ‘”ƒ–‹‘ƒŽ‡„‡”•Ǥ
 EN ISO 16140-3:2021 (E) ILNAS-EN ISO 16140-3:2021

Contents Page

European foreword....................................................................................................................................................... 3
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2
 ILNAS-EN ISO 16140-3:2021 EN ISO 16140-3:2021 (E)

European foreword

This document (EN ISO 16140-3:2021) 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 August 2021, and conflicting national standards shall
be withdrawn at the latest by August 2021.

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.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
ILNAS-EN ISO 16140-3:2021 - Preview only Copy via ILNAS e-Shop

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, Turkey and the
United Kingdom.

Endorsement notice

The text of ISO 16140-3:2021 has been approved by CEN as EN ISO 16140-3:2021 without any
modification.

3
 INTERNATIONAL
ILNAS-EN ISO 16140-3:2021
ISO
STANDARD 16140-3

First edition
2021-01

Microbiology of the food chain —

ILNAS-EN ISO 16140-3:2021 - Preview only Copy via ILNAS e-Shop

Method validation —
Part 3:
”‘–‘ ‘Žˆ‘”–Š‡˜‡”‹ϐ‹ ƒ–‹‘‘ˆ
reference methods and validated
alternative methods in a single
laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
ƒ”–‹‡͹ǣ”‘–‘ ‘Ž‡’‘—”Žƒ˜±”‹ϔ‹ ƒ–‹‘†ƒ•—•‡—ŽŽƒ„‘”ƒ–‘‹”‡†‡
méthodes de référence et de méthodes alternatives validées

Reference number
ISO 16140-3:2021(E)

© ISO 2021
 ISO 16140-3:2021(E) ILNAS-EN ISO 16140-3:2021
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COPYRIGHT PROTECTED DOCUMENT

© ISO 2021
ŽŽ”‹‰Š–•”‡•‡”˜‡†ǤŽ‡••‘–Š‡”™‹•‡•’‡ ‹ϐ‹‡†ǡ‘””‡“—‹”‡†‹–Š‡ ‘–‡š–‘ˆ‹–•‹’Ž‡‡–ƒ–‹‘ǡ‘’ƒ”–‘ˆ–Š‹•’—„Ž‹ ƒ–‹‘ƒ›
„‡”‡’”‘†— ‡†‘”—–‹Ž‹œ‡†‘–Š‡”™‹•‡‹ƒ›ˆ‘”‘”„›ƒ›‡ƒ•ǡ‡Ž‡ –”‘‹ ‘”‡ Šƒ‹ ƒŽǡ‹ Ž—†‹‰’Š‘–‘ ‘’›‹‰ǡ‘”’‘•–‹‰
‘–Š‡‹–‡”‡–‘”ƒ‹–”ƒ‡–ǡ™‹–Š‘—–’”‹‘”™”‹––‡’‡”‹••‹‘Ǥ‡”‹••‹‘ ƒ„‡”‡“—‡•–‡†ˆ”‘‡‹–Š‡” ƒ––Š‡ƒ††”‡••
„‡Ž‘™‘” ǯ•‡„‡”„‘†›‹–Š‡ ‘—–”›‘ˆ–Š‡”‡“—‡•–‡”Ǥ
 ‘’›”‹‰Š–‘ˆϐ‹ ‡
CP 401 • Ch. de Blandonnet 8
 ǦͳʹͳͶ‡”‹‡”ǡ ‡‡˜ƒ
Phone: +41 22 749 01 11
ƒ‹Žǣ ‘’›”‹‰Š–̷‹•‘Ǥ‘”‰
Website: www.iso.org
Published in Switzerland

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 ILNAS-EN ISO 16140-3:2021 ISO 16140-3:2021(E)

Contents Page

Foreword ..........................................................................................................................................................................................................................................v
Introduction................................................................................................................................................................................................................................ vi
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
͵ ‡”•ƒ††‡ϐ‹‹–‹‘• ..................................................................................................................................................................................... 1
Ͷ ‡‡”ƒŽ’”‹ ‹’Ž‡•‘ˆ˜‡”‹ϐ‹ ƒ–‹‘‘ˆ“—ƒŽ‹–ƒ–‹˜‡ȋ†‡–‡ –‹‘Ȍ‡–Š‘†•ƒ†
“—ƒ–‹ϐ‹ ƒ–‹‘‡–Š‘†• ................................................................................................................................................................................. 5
ͶǤͳ ‡‡”ƒŽ ........................................................................................................................................................................................................... 5
ͶǤʹ ’Ž‡‡–ƒ–‹‘˜‡”‹ϐ‹ ƒ–‹‘ ....................................................................................................................................................... 5
ͶǤ͵ ȋ ‘‘†Ȍ‹–‡˜‡”‹ϐ‹ ƒ–‹‘ .................................................................................................................................................................. 6
ͶǤͶ ‡“—‹”‡‡–•ˆ‘”‹’Ž‡‡–ƒ–‹‘˜‡”‹ϐ‹ ƒ–‹‘ƒ†ȋˆ‘‘†Ȍ‹–‡˜‡”‹ϐ‹ ƒ–‹‘................................... 6
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4.5 Performance characteristics........................................................................................................................................................ 9

ͷ —ƒŽ‹–ƒ–‹˜‡‡–Š‘†•Ȅ‡ Š‹ ƒŽ’”‘–‘ ‘Žˆ‘”˜‡”‹ϐ‹ ƒ–‹‘ .................................................................................... 9
5.1 Estimated LOD50 (eLOD50) determination ..................................................................................................................... 9
ͷǤʹ š’‡”‹‡–ƒŽ†‡•‹‰ .......................................................................................................................................................................... 9
5.3 Selection of (food) items.............................................................................................................................................................. 10
ͷǤͶ ”–‹ϐ‹ ‹ƒŽ ‘–ƒ‹ƒ–‹‘ ............................................................................................................................................................... 10
5.4.1 Selection of strains ...................................................................................................................................................... 10
5.4.2 Inoculation of the test portions........................................................................................................................ 11
5.5 Evaluation of results........................................................................................................................................................................ 13
5.5.1 Determination of eLOD50 using protocol 1 ............................................................................................ 13
5.5.2 Determination of eLOD50 using protocol 2 ............................................................................................ 16
5.5.3 Use of protocol 3 ........................................................................................................................................................... 17
ͷǤ͸  ‡’–ƒ„‹Ž‹–›Ž‹‹–•........................................................................................................................................................................... 18
ͷǤ͹ ‘‘– ƒ—•‡ƒƒŽ›•‹• .......................................................................................................................................................................... 18
͸ —ƒ–‹–ƒ–‹˜‡‡–Š‘†•Ȅ‡ Š‹ ƒŽ’”‘–‘ ‘Žˆ‘”˜‡”‹ϐ‹ ƒ–‹‘..............................................................................19
͸Ǥͳ –”ƒŽƒ„‘”ƒ–‘”›”‡’”‘†— ‹„‹Ž‹–›•–ƒ†ƒ”††‡˜‹ƒ–‹‘†‡–‡”‹ƒ–‹‘...................................................... 19
͸ǤͳǤͳ ‡‡”ƒŽ................................................................................................................................................................................... 19
͸ǤͳǤʹ š’‡”‹‡–ƒŽ†‡•‹‰ .................................................................................................................................................. 19
6.1.3 Selection of the (food) item ................................................................................................................................. 21
6.1.4 Natural contamination............................................................................................................................................. 21
͸ǤͳǤͷ ”–‹ϐ‹ ‹ƒŽ ‘–ƒ‹ƒ–‹‘.......................................................................................................................................... 21
6.1.6 Evaluation of results .................................................................................................................................................. 22
͸ǤͳǤ͹  ‡’–ƒ„‹Ž‹–›Ž‹‹–........................................................................................................................................................ 23
͸ǤͳǤͺ ‘‘– ƒ—•‡ƒƒŽ›•‹• ..................................................................................................................................................... 24
6.2 Estimated bias (eBias) determination ............................................................................................................................. 25
͸ǤʹǤͳ ‡‡”ƒŽ................................................................................................................................................................................... 25
͸ǤʹǤʹ š’‡”‹‡–ƒŽ†‡•‹‰ .................................................................................................................................................. 25
6.2.3 Selection of (food) items ........................................................................................................................................ 25
͸ǤʹǤͶ ”–‹ϐ‹ ‹ƒŽ ‘–ƒ‹ƒ–‹‘.......................................................................................................................................... 25
6.2.5 Evaluation of results .................................................................................................................................................. 27
͸ǤʹǤ͸  ‡’–ƒ„‹Ž‹–›Ž‹‹–........................................................................................................................................................ 27
͸ǤʹǤ͹ ‘‘– ƒ—•‡ƒƒŽ›•‹• ..................................................................................................................................................... 28
͹ ƒŽ‹†ƒ–‡†ƒŽ–‡”ƒ–‹˜‡ ‘ϐ‹”ƒ–‹‘ƒ†–›’‹‰‡–Š‘†•Ȅ‡ Š‹ ƒŽ’”‘–‘ ‘Žˆ‘”
˜‡”‹ϐ‹ ƒ–‹‘............................................................................................................................................................................................................... 28
͹Ǥͳ ‡‡”ƒŽ ........................................................................................................................................................................................................ 28
͹Ǥʹ ’Ž‡‡–ƒ–‹‘˜‡”‹ϐ‹ ƒ–‹‘ .................................................................................................................................................... 28
͹Ǥ͵ š’‡”‹‡–ƒŽ†‡•‹‰ ....................................................................................................................................................................... 28
͹Ǥ͵Ǥͳ ‡‡”ƒŽ................................................................................................................................................................................... 28
7.3.2 Strain selection ............................................................................................................................................................... 29
7.4 Evaluation of results........................................................................................................................................................................ 29
͹Ǥͷ  ‡’–ƒ„‹Ž‹–›Ž‹‹– ............................................................................................................................................................................. 30

© ISO 2021 – All rights reserved iii

 ISO 16140-3:2021(E) ILNAS-EN ISO 16140-3:2021

͹Ǥ͸ ‘‘– ƒ—•‡ƒƒŽ›•‹• .......................................................................................................................................................................... 30

ͺ —ƒ”›‘ˆƒ ‡’–ƒ„‹Ž‹–›Ž‹‹–•ˆ‘”–Š‡˜‡”‹ϐ‹ ƒ–‹‘‘ˆ˜ƒŽ‹†ƒ–‡†‡–Š‘†• ........................................30
Annex A (informative)Žƒ••‹ϐ‹ ƒ–‹‘‘ˆȋˆ‘‘†Ȍ ƒ–‡‰‘”‹‡•ƒ†•—‰‰‡•–‡†–ƒ”‰‡–
‘„‹ƒ–‹‘•ˆ‘”˜‡”‹ϐ‹ ƒ–‹‘•–—†‹‡• .........................................................................................................................................31
Annex B (informative) Guidance on how to choose challenging (food) item(s) for (food)
‹–‡˜‡”‹ϐ‹ ƒ–‹‘ ................................................................................................................................................................................................. 45
Annex C (informative)—ƒŽ‹–ƒ–‹˜‡‡–Š‘†˜‡”‹ϐ‹ ƒ–‹‘Ȅšƒ’Ž‡ ................................................................................47
Annex D (informative)—ƒ–‹–ƒ–‹˜‡‡–Š‘†˜‡”‹ϐ‹ ƒ–‹‘Ȅšƒ’Ž‡............................................................................55
Annex E (informative)ƒŽ‹†ƒ–‡†ƒŽ–‡”ƒ–‹˜‡ ‘ϐ‹”ƒ–‹‘‘”–›’‹‰‡–Š‘†˜‡”‹ϐ‹ ƒ–‹‘Ȅ
Examples .................................................................................................................................................................................................................... 60
Annex F (normative)”‘–‘ ‘Žˆ‘”–Š‡˜‡”‹ϐ‹ ƒ–‹‘‘ˆ‘Ǧ˜ƒŽ‹†ƒ–‡†”‡ˆ‡”‡ ‡‡–Š‘†•
in a single laboratory .................................................................................................................................................................................... 63
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Bibliography ............................................................................................................................................................................................................................ 70

iv © ISO 2021 – All rights reserved