INTERNATIONAL ISO

STANDARD 11290-1

Second edition
2017-05

Microbiology of the food chain —

Horizontal method for the detection
and enumeration of ‹•–‡”‹ƒ
‘‘ ›–‘‰‡‡•and of ‹•–‡”‹ƒ spp. —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de ‹•–‡”‹ƒ‘‘ ›–‘‰‡‡•et de
‹•–‡”‹ƒ spp. —
Partie 1: Méthode de recherche
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Reference number
ISO 11290-1:2017(E)

© ISO 2017
Impreso por: Generico
 ISO 11290-1:2017(E)
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

COPYRIGHT PROTECTED DOCUMENT

© ISO 2017, Published in Switzerland
ŽŽ”‹‰Š–•”‡•‡”˜‡†ǤŽ‡••‘–Š‡”™‹•‡•’‡ ‹ϐ‹‡†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
the requester.
 ‘’›”‹‰Š–‘ˆϐ‹ ‡
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
‘’›”‹‰Š–̷‹•‘Ǥ‘”‰
www.iso.org

ii
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Contents Page

Foreword ..........................................................................................................................................................................................................................................v
Introduction................................................................................................................................................................................................................................ vi
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
͵ ‡”•ƒ††‡ϐ‹‹–‹‘• ..................................................................................................................................................................................... 2
4 Principle ........................................................................................................................................................................................................................ 2
4.1 General ........................................................................................................................................................................................................... 2
ͶǤʹ ”‹ƒ”›‡”‹ Š‡–‹ƒ•‡Ž‡ –‹˜‡Ž‹“—‹†‡”‹ Š‡–‡†‹—™‹–Š”‡†— ‡†
concentration of selective agents (half-Fraser broth)........................................................................................... 2
ͶǤ͵ ‡ ‘†ƒ”›‡”‹ Š‡–™‹–Šƒ•‡Ž‡ –‹˜‡Ž‹“—‹†‡”‹ Š‡–‡†‹—™‹–Šˆ—ŽŽ
concentration of selective agents (Fraser broth) ...................................................................................................... 3
ͶǤͶ Žƒ–‹‰‘—–ƒ†‹†‡–‹ϐ‹ ƒ–‹‘ ..................................................................................................................................................... 3
ͶǤͷ ‘ϐ‹”ƒ–‹‘ ............................................................................................................................................................................................. 3
5 Culture media and reagents...................................................................................................................................................................... 3
6 Equipment and consumables .................................................................................................................................................................. 3
7 Sampling........................................................................................................................................................................................................................ 4
8 Preparation of test sample ......................................................................................................................................................................... 4
9 Procedure..................................................................................................................................................................................................................... 4
9.1 Test portion and initial suspension ....................................................................................................................................... 4
ͻǤʹ ”‹ƒ”›‡”‹ Š‡– ........................................................................................................................................................................... 4
ͻǤ͵ ‡ ‘†ƒ”›‡”‹ Š‡– ..................................................................................................................................................................... 5
ͻǤͶ Žƒ–‹‰‘—–ƒ†‹†‡–‹ϐ‹ ƒ–‹‘ ..................................................................................................................................................... 5
9.4.1 General .................................................................................................................................................................................... 5
9.4.2 Agar ‹•–‡”‹ƒaccording to Ottaviani and Agosti ........Ǥ..........................................................................5
9.4.3 Second selective medium ......................................................................................................................................... 6
ͻǤͷ ‘ϐ‹”ƒ–‹‘‘ˆ‹•–‡”‹ƒ‘‘ ›–‘‰‡‡•‘”‹•–‡”‹ƒspp. ........ǤǤǤǤǤǤ.....................................................................6
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

ͻǤͷǤͳ ‡Ž‡ –‹‘‘ˆ ‘Ž‘‹‡•ˆ‘” ‘ϐ‹”ƒ–‹‘ .......................................................................................................... 6

ͻǤͷǤʹ ‘ϐ‹”ƒ–‹‘‘ˆǤ‘‘ ›–‘‰‡‡•ǤǤǤ...........................................................................................................Ǥ..͸
ͻǤͷǤ͵ ‘ϐ‹”ƒ–‹‘‘ˆ‹•–‡”‹ƒ spp. .....Ǥ.......................................................................................................................10
ͻǤ͸ –‡”’”‡–ƒ–‹‘‘ˆ‘”’Š‘Ž‘‰‹ ƒŽƒ†’Š›•‹‘Ž‘‰‹ ƒŽ’”‘’‡”–‹‡•ƒ†‘ˆ–Š‡
biochemical reactions .................................................................................................................................................................... 11
9.7 Additional characterization of isolated strains (optional) ............................................................................. 11
10 Expression of results ..................................................................................................................................................................................... 11
11 Performance characteristics of the method.......................................................................................................................... 11
11.1 Method validation studies .......................................................................................................................................................... 11
ͳͳǤʹ ‡•‹–‹˜‹–› ................................................................................................................................................................................................. 11
ͳͳǤ͵ ’‡ ‹ϐ‹ ‹–› ................................................................................................................................................................................................. 11
11.4 Level of detection (LOD50)......................................................................................................................................................... 11
12 Test report ................................................................................................................................................................................................................ 12
13 Quality assurance ............................................................................................................................................................................................. 12
Annex A (normative) Diagram of procedure .............................................................................................................................................13
Annex B (normative) Composition and preparation of culture media and reagents .....................................14
Annex C (informative) Distinction of Listeria spp. from other genera ...........................................................................27
Annex D (informative)‡ƒ –‹‘•ˆ‘”–Š‡‹†‡–‹ϐ‹ ƒ–‹‘‘ˆListeria species ..............................................................28
Annex E (informative) Listeria selective agars ........................................................................................................................................30

© ISO 2017 – All rights reserved

Impreso por: Generico
iii
 ISO 11290-1:2017(E)

Annex F (informative) Results of the interlaboratory studies for detection of

Listeria monocytogenes............................................................................................................................................................................... 31
Bibliography ............................................................................................................................................................................................................................. 35
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

iv
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
„‘†‹‡• ȋ  ‡„‡” „‘†‹‡•ȌǤ Š‡ ™‘” ‘ˆ ’”‡’ƒ”‹‰ –‡”ƒ–‹‘ƒŽ –ƒ†ƒ”†• ‹• ‘”ƒŽŽ› ƒ””‹‡† ‘—–
–Š”‘—‰Š  –‡ Š‹ ƒŽ ‘‹––‡‡•Ǥ ƒ Š ‡„‡” „‘†› ‹–‡”‡•–‡† ‹ ƒ •—„Œ‡ – ˆ‘” ™Š‹ Š ƒ –‡ Š‹ ƒŽ
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.
 ‘ŽŽƒ„‘”ƒ–‡• Ž‘•‡Ž› ™‹–Š –Š‡ –‡”ƒ–‹‘ƒŽ Ž‡ –”‘–‡ Š‹ ƒŽ ‘‹••‹‘ ȋ Ȍ ‘ ƒŽŽ ƒ––‡”• ‘ˆ
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
†‹ˆˆ‡”‡––›’‡•‘ˆ †‘ —‡–••Š‘—Ž†„‡‘–‡†ǤŠ‹•†‘ —‡–™ƒ•†”ƒˆ–‡†‹ƒ ‘”†ƒ ‡™‹–Š–Š‡
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
––‡–‹‘‹•†”ƒ™–‘–Š‡’‘••‹„‹Ž‹–›–Šƒ–•‘‡‘ˆ–Š‡‡Ž‡‡–•‘ˆ–Š‹•†‘ —‡–ƒ›„‡–Š‡•—„Œ‡ –‘ˆ
’ƒ–‡–”‹‰Š–•Ǥ •ŠƒŽŽ‘–„‡Š‡Ž†”‡•’‘•‹„Ž‡ˆ‘”‹†‡–‹ˆ›‹‰ƒ›‘”ƒŽŽ•— Š’ƒ–‡–”‹‰Š–•Ǥ‡–ƒ‹Ž•‘ˆ
ƒ›’ƒ–‡–”‹‰Š–•‹†‡–‹ϐ‹‡††—”‹‰–Š‡†‡˜‡Ž‘’‡–‘ˆ–Š‡†‘ —‡–™‹ŽŽ„‡‹–Š‡ –”‘†— –‹‘ƒ†Ȁ‘”
on the ISO list of patent declarations received (see www.iso.org/patents).
›–”ƒ†‡ƒ‡—•‡†‹–Š‹•†‘ —‡–‹•‹ˆ‘”ƒ–‹‘‰‹˜‡ˆ‘”–Š‡ ‘˜‡‹‡ ‡‘ˆ—•‡”•ƒ††‘‡•‘–
constitute an endorsement.
‘” ƒ ‡š’Žƒƒ–‹‘ ‘ –Š‡ ˜‘Ž—–ƒ”› ƒ–—”‡ ‘ˆ •–ƒ†ƒ”†•ǡ –Š‡ ‡ƒ‹‰ ‘ˆ  •’‡ ‹ϐ‹  –‡”• ƒ†
‡š’”‡••‹‘• ”‡Žƒ–‡† –‘ ‘ˆ‘”‹–› ƒ••‡••‡–ǡ ƒ• ™‡ŽŽ ƒ• ‹ˆ‘”ƒ–‹‘ ƒ„‘—– ǯ• ƒ†Š‡”‡ ‡ –‘ –Š‡
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
Š‹• †‘ —‡– ™ƒ• ’”‡’ƒ”‡† „› –Š‡ —”‘’‡ƒ ‘‹––‡‡ ˆ‘” –ƒ†ƒ”†‹œƒ–‹‘ ȋȌ ‡ Š‹ ƒŽ
Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
Š‹••‡ ‘†‡†‹–‹‘ ƒ ‡Ž•ƒ†”‡’Žƒ ‡•–Š‡ϐ‹”•–‡†‹–‹‘ȋ ͳͳʹͻͲǦͳǣͳͻͻ͸Ȍǡ™Š‹ ŠŠƒ•„‡‡–‡ Š‹ ƒŽŽ›
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

revised. It also incorporates the amendment ISO 11290-1:1996/Amd.1:2004.

The main changes, compared to ISO 11290-1:1996, are the following.
— The detection of Listeria monocytogenesŠƒ•„‡‡‘†‹ϐ‹‡†ƒ•Ž‹•–‡†„‡Ž‘™Ǥ
Ȅ ”‹ƒ”›‡”‹ Š‡–‹ŠƒŽˆǦ ”ƒ•‡”„”‘–Šǣ‹ —„ƒ–‹‘ˆ‘”ʹͷŠάͳŠǤ
Ȅ ‡ ‘†ƒ”›‡”‹ Š‡–‹ ”ƒ•‡”„”‘–Šǣ‹ —„ƒ–‹‘ˆ‘”ʹͶŠάʹŠǤ[29]
Ȅ ƒŽˆǦ ”ƒ•‡”ƒ† ”ƒ•‡”„”‘–Š•ƒ›„‡”‡ˆ”‹‰‡”ƒ–‡†„‡ˆ‘”‡–”ƒ•ˆ‡”‘”‹•‘Žƒ–‹‘‘•‡Ž‡ –‹˜‡ƒ‰ƒ”ˆ‘”
a maximum of 72 h.
Ȅ –‘”ƒ‰‡‘ˆ‹•‘Žƒ–‹‘’Žƒ–‡•ǣ‹ —„ƒ–‡†’Žƒ–‡• ƒ„‡”‡ˆ”‹‰‡”ƒ–‡†ˆ‘”ƒƒš‹—‘ˆ–™‘†ƒ›•„‡ˆ‘”‡
reading.
Ȅ ‹ ”‘• ‘’‹  ƒ•’‡ – ˆ‘” ‘ϐ‹”ƒ–‹‘ ‹• ‘’–‹‘ƒŽ ‹ˆ –Š‡ ‹•‘Žƒ–‹‘ ƒ‰ƒ” ƒŽŽ‘™• †‹•–‹ –‹‘ „‡–™‡‡
pathogenic and non-pathogenic Listeria spp.
— CAMP test and catalase test are optional.
— Inclusion of new performance characteristics.
— Moreover, detection of Listeria•’’ǤŠƒ•„‡‡‹ Ž—†‡†‹–Š‡• ‘’‡ƒ†–Š‡–‹–Ž‡ Šƒ‰‡†ƒ ‘”†‹‰Ž›Ǥ
A list of parts in the ISO 11290 series can be found on the ISO website.

© ISO 2017 – All rights reserved

Impreso por: Generico
v
 ISO 11290-1:2017(E)

Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO 11290-1:1996
ƒ”‡ ‘•‹†‡”‡†ƒ•ƒŒ‘”ȋ•‡‡ ͳ͹Ͷ͸ͺ[28]). The technical changes were assessed and were considered
–‘Šƒ˜‡‘•‹‰‹ϐ‹ ƒ–‡ˆˆ‡ –‘–Š‡‡–Š‘†’‡”ˆ‘”ƒ ‡ Šƒ”ƒ –‡”‹•–‹ •‘”–‡•–”‡•—Ž–•Ǥ
‡ ƒ—•‡‘ˆ–Š‡Žƒ”‰‡˜ƒ”‹‡–›‘ˆˆ‘‘†ƒ†ˆ‡‡†’”‘†— –•ǡ–Š‹•Š‘”‹œ‘–ƒŽ‡–Š‘†ƒ›‘–„‡ƒ’’”‘’”‹ƒ–‡
‹‡˜‡”›†‡–ƒ‹Žˆ‘” ‡”–ƒ‹’”‘†— –•ˆ‘”™Š‹ Š‹–ƒ›„‡‡ ‡••ƒ”›–‘—•‡†‹ˆˆ‡”‡–‘”•’‡ ‹ϐ‹ ‡–Š‘†•Ǥ
Nevertheless, in all cases, this horizontal method is intended to be applied as far as possible and
†‡˜‹ƒ–‹‘•ˆ”‘–Š‹•‘Ž›„‡ƒ†‡ˆ‘”Œ—•–‹ϐ‹‡†–‡ Š‹ ƒŽ”‡ƒ•‘•Ǥ
When this document is next reviewed, account will be taken of all information then available regarding
the extent to which this horizontal method has been followed and the reasons for deviations from it in
the case of particular products.
The harmonization of test methods cannot be immediate, and for certain groups of products
–‡”ƒ–‹‘ƒŽ –ƒ†ƒ”†• ƒ†Ȁ‘” ƒ–‹‘ƒŽ •–ƒ†ƒ”†• ƒ› ƒŽ”‡ƒ†› ‡š‹•– –Šƒ– †‘ ‘– ‘’Ž› ™‹–Š –Š‹•
Š‘”‹œ‘–ƒŽ‡–Š‘†Ǥ –‹•Š‘’‡†–Šƒ–™Š‡•— Š•–ƒ†ƒ”†•ƒ”‡”‡˜‹‡™‡†ǡ–Š‡›™‹ŽŽ„‡ Šƒ‰‡†–‘ ‘’Ž›
™‹–Š–Š‹•†‘ —‡–•‘–Šƒ–‡˜‡–—ƒŽŽ›–Š‡‘Ž›”‡ƒ‹‹‰†‡’ƒ”–—”‡•ˆ”‘–Š‹•Š‘”‹œ‘–ƒŽ‡–Š‘†™‹ŽŽ
„‡–Š‘•‡‡ ‡••ƒ”›ˆ‘”™‡ŽŽǦ‡•–ƒ„Ž‹•Š‡†–‡ Š‹ ƒŽ”‡ƒ•‘•Ǥ
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

vi
Impreso por: Generico
© ISO 2017 – All rights reserved
 INTERNATIONAL STANDARD ISO 11290-1:2017(E)

Microbiology of the food chain — Horizontal method for

the detection and enumeration of ‹•–‡”‹ƒ‘‘ ›–‘‰‡‡•
and of ‹•–‡”‹ƒ spp. —
Part 1:
Detection method
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for detecting L. monocytogenes and Listeria spp. are only undertaken in properly equipped
laboratories, under the control of a skilled microbiologist, and that great care is taken in the
disposal of all incubated materials. Persons using this document should be familiar with
normal laboratory practice. This document does not purport to address all of the safety aspects,
if any, associated with its use. It is the responsibility of the user to establish appropriate
safety and health practices. In particular, it is strongly recommended that tests for detecting
L. monocytogenes are undertaken in laboratories providing biosafety level 2 conditions. It is
strongly recommended that female laboratory staff are made aware of the particular risk to the
developing foetus presented by infection of the mother through exposure to L. monocytogenes
and Listeria spp., and that pregnant personnel and persons with recognized underlying
conditions or diseases that impair cell-mediated immunity do not manipulate cultures of
L.monocytogenes and Listeria spp.

1 Scope
Š‹•†‘ —‡–•’‡ ‹ϐ‹‡•ƒŠ‘”‹œ‘–ƒŽ‡–Š‘†ˆ‘”
— the detection of L. monocytogenes, and
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

— the detection of Listeria spp. (including L. monocytogenes).

This document is applicable to
— products intended for human consumption and for the feeding of animals, and
— environmental samples in the area of food production and food handling.
–‹•’‘••‹„Ž‡–Šƒ– ‡”–ƒ‹ƒ††‹–‹‘ƒŽŽ›†‡• ”‹„‡†Listeria•’‡ ‹‡•ƒ›‘–„‡†‡–‡ –‡†‘” ‘ϐ‹”‡†„›
this method.[5],[10],[12],[14],[25],[26],[27]

2 Normative references
Š‡ ˆ‘ŽŽ‘™‹‰ †‘ —‡–• ƒ”‡ ”‡ˆ‡””‡† –‘ ‹ –Š‡ –‡š– ‹ •— Š ƒ ™ƒ› –Šƒ– •‘‡ ‘” ƒŽŽ ‘ˆ –Š‡‹” ‘–‡–
‘•–‹–—–‡• ”‡“—‹”‡‡–• ‘ˆ –Š‹• †‘ —‡–Ǥ ‘” †ƒ–‡† ”‡ˆ‡”‡ ‡•ǡ ‘Ž› –Š‡ ‡†‹–‹‘ ‹–‡† ƒ’’Ž‹‡•Ǥ ‘”
—†ƒ–‡†”‡ˆ‡”‡ ‡•ǡ–Š‡Žƒ–‡•–‡†‹–‹‘‘ˆ–Š‡”‡ˆ‡”‡ ‡††‘ —‡–ȋ‹ Ž—†‹‰ƒ›ƒ‡†‡–•Ȍƒ’’Ž‹‡•Ǥ
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media

© ISO 2017 – All rights reserved

Impreso por: Generico
1
 ISO 11290-1:2017(E)

͵ ‡”•ƒ††‡ϐ‹‹–‹‘•
‘”–Š‡’—”’‘•‡•‘ˆ–Š‹•†‘ —‡–ǡ–Š‡ˆ‘ŽŽ‘™‹‰–‡”•ƒ††‡ϐ‹‹–‹‘•ƒ’’Ž›Ǥ
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
Listeria monocytogenes
‹ ”‘‘”‰ƒ‹•• ™Š‹ Š ˆ‘” –›’‹ ƒŽ ‘Ž‘‹‡• ‘ •‘Ž‹† •‡Ž‡ –‹˜‡ ‡†‹ƒ ƒ† ™Š‹ Š †‹•’Žƒ› –Š‡
‘”’Š‘Ž‘‰‹ ƒŽǡ’Š›•‹‘Ž‘‰‹ ƒŽƒ†„‹‘ Š‡‹ ƒŽ Šƒ”ƒ –‡”‹•–‹ •†‡• ”‹„‡†™Š‡–‡•–•ƒ”‡ ƒ””‹‡†‘—–‹
accordance with this document
3.2
detection of Listeria monocytogenes
determination of the detection/non detection of Listeria monocytogenes (3.1), in a given mass or volume
‘ˆ’”‘†— –‘”ƒ•’‡ ‹ϐ‹‡†•—”ˆƒ ‡ǡ™Š‡–‡•–•ƒ”‡ ƒ””‹‡†‘—–‹ƒ ‘”†ƒ ‡™‹–Š–Š‹•†‘ —‡–
3.3
Listeria spp.
‹ ”‘‘”‰ƒ‹•• ™Š‹ Š ˆ‘” –›’‹ ƒŽ ‘Ž‘‹‡• ‘ •‘Ž‹† •‡Ž‡ –‹˜‡ ‡†‹ƒ ƒ† ™Š‹ Š †‹•’Žƒ› –Š‡
‘”’Š‘Ž‘‰‹ ƒŽǡ’Š›•‹‘Ž‘‰‹ ƒŽƒ†„‹‘ Š‡‹ ƒŽ Šƒ”ƒ –‡”‹•–‹ •†‡• ”‹„‡†™Š‡–‡•–•ƒ”‡ ƒ””‹‡†‘—–‹
accordance with this document
3.4
detection of Listeria spp.
determination of the detection/non detection of Listeria spp. (3.3), in a given mass or volume of product
‘”ƒ•’‡ ‹ϐ‹‡†•—”ˆƒ ‡ǡ™Š‡–‡•–•ƒ”‡ ƒ””‹‡†‘—–‹ƒ ‘”†ƒ ‡™‹–Š–Š‹•†‘ —‡–

4 Principle
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

4.1 General
Listeria •’’Ǥ ƒ› „‡ ’”‡•‡– ‹ •ƒŽŽ —„‡”• ƒ† ƒ”‡ ‘ˆ–‡ ƒ ‘’ƒ‹‡† „› ‘•‹†‡”ƒ„Ž› Žƒ”‰‡”
—„‡”•‘ˆ‘–Š‡”‹ ”‘‘”‰ƒ‹••ǡ–Š‡”‡ˆ‘”‡•‡Ž‡ –‹˜‡‡”‹ Š‡–‹•‡ ‡••ƒ”›Ǥ –‹•ƒŽ•‘‡ ‡••ƒ”›–‘
†‡–‡ –‹Œ—”‡†ƒ†•–”‡••‡†Listeria•’’Ǥƒ†–Š‡’”‹ƒ”›•‡Ž‡ –‹˜‡‡”‹ Š‡–‡†‹—ǡ™‹–Š”‡†— ‡†
‹Š‹„‹–‘” ‘ ‡–”ƒ–‹‘ǡˆ—Žϐ‹Ž•ƒ–Ž‡ƒ•–’ƒ”–‘ˆ–Š‹•ˆ— –‹‘Ǥ
NOTE Presence of L. monocytogenes ƒ„‡ƒ•‡†„›–Š‡’”‡•‡ ‡‘ˆ‘–Š‡”Listeria species, in particular
L. innocua or L. ivanovii.

Within the limits of this document, the detection of L. monocytogenes and of Listeria spp. necessitates
ˆ‘—”•— ‡••‹˜‡•–ƒ‰‡•ǡƒ•†‡• ”‹„‡†‹–Š‡ϐŽ‘™ Šƒ”–‹Annex A.

4.2 Primary enrichment in a selective liquid enrichment medium with reduced

concentration of selective agents (half-Fraser broth)
‘ —Žƒ–‹‘ ‘ˆ ƒ •‡Ž‡ –‹˜‡ ’”‹ƒ”› ‡”‹ Š‡– ‡†‹— ‘–ƒ‹‹‰ ŠƒŽˆ –Š‡ ‘ ‡–”ƒ–‹‘• ‘ˆ
ƒ ”‹ϐŽƒ˜‹‡ƒ†ƒŽ‹†‹š‹ ƒ ‹†ȋŠƒŽˆǦ ”ƒ•‡”„”‘–Šǡ•‡‡B.1Ȍǡ™Š‹ Š‹•ƒŽ•‘—•‡†ƒ•ƒ†‹Ž—–‹‘ϐŽ—‹†ˆ‘”–Š‡
test portion (9.1).
Incubation of the initial suspension at 30 °C for 24 h to 26 h.

2
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

4.3 Secondary enrichment with a selective liquid enrichment medium with full
concentration of selective agents (Fraser broth)
‘ —Žƒ–‹‘ ‘ˆ ˆ—ŽŽǦ•–”‡‰–Š •‡ ‘†ƒ”› Ž‹“—‹† ‡”‹ Š‡– ‡†‹— ȋ ”ƒ•‡” „”‘–ŠȌ ™‹–Š ƒ —Ž–—”‡
obtained from 4.2.
Incubation of the Fraser broth at 37 °C for 24 h.[29]

ͶǤͶ Žƒ–‹‰‘—–ƒ†‹†‡–‹ϐ‹ ƒ–‹‘

From the cultures obtained in 4.2 and 4.3, plating out on the two selective solid media:
— Agar Listeria according to Ottaviani and Agosti (see References [16] and [17] and B.3);
Ȅ ƒ›‘–Š‡”•‘Ž‹†•‡Ž‡ –‹˜‡‡†‹—ƒ––Š‡ Š‘‹ ‡‘ˆ–Š‡Žƒ„‘”ƒ–‘”› ‘’Ž‡‡–ƒ”›–‘‰ƒ”Listeria
according to Ottaviani and Agosti, using a different substrate and/or principle than the one used in
Listeria agar according to Ottaviani and Agosti (see B.4). See Annex E for information about media.
Incubation of the Agar Listeria according to Ottaviani and Agosti at 37 °C for a total of 48 h. If colonies of
presumptive L. monocytogenes or Listeria•’’Ǥƒ”‡‡˜‹†‡–ƒ–ʹͶŠ–Š‡‹ —„ƒ–‹‘ƒ›„‡•–‘’’‡†ƒ––Š‹•
stage. Incubation of the second selective medium at the appropriate temperature and examination after
the appropriate time.

ͶǤͷ ‘ϐ‹”ƒ–‹‘
Subculturing of the colonies of presumptive L. monocytogenes or Listeria spp., plated out as described
in 4.4ǡƒ† ‘ϐ‹”ƒ–‹‘„›‡ƒ•‘ˆƒ’’”‘’”‹ƒ–‡‘”’Š‘Ž‘‰‹ ƒŽƒ†Ȁ‘”„‹‘ Š‡‹ ƒŽ–‡•–•Ǥ

5 Culture media and reagents

‘” —””‡–Žƒ„‘”ƒ–‘”›’”ƒ –‹ ‡•ǡ”‡ˆ‡”–‘ ͳͳͳ͵͵Ǥ
Composition and performance testing of culture media and reagents and their preparation are
described in Annex B.
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

6 Equipment and consumables

•—ƒŽ‹ ”‘„‹‘Ž‘‰‹ ƒŽ‡“—‹’‡–ȋƒ••’‡ ‹ϐ‹‡†‹ ͹ʹͳͺȌƒ†ǡ‹’ƒ”–‹ —Žƒ”ǡ–Š‡ˆ‘ŽŽ‘™‹‰Ǥ

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).

••’‡ ‹ϐ‹‡†‹ ͹ʹͳͺǤ

6.2 Drying cabinet or incubator, capable of being maintained between 25 °C and 50 °C.

6.3 Incubatorsǡ ƒ’ƒ„Ž‡‘ˆ‘’‡”ƒ–‹‰ƒ–͵Ͳιάͳιǡ͵͹ιάͳ郐†ƒ–ʹͷιάͳιȋ‘’–‹‘ƒŽȌǤ

6.4 Water bath, capable of operating at 47 °C to 50 °C.

6.5 Sterile loopsƒ’’”‘š‹ƒ–‡Ž›͵‹†‹ƒ‡–‡”‘”ͳͲρŽǡƒ†‹‘ —Žƒ–‹‰needle or wire.

6.6 pH meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be
ƒ†‡™Š‹ Šƒ”‡ƒ —”ƒ–‡–‘άͲǡͳ’ —‹–Ǥ

6.7 Graduated pipettes or automatic pipettes, of nominal capacities 1 ml and 10 ml.

© ISO 2017 – All rights reserved

Impreso por: Generico
3
 ISO 11290-1:2017(E)

6.8 Petri dishes, for example of diameter 90 mm.

6.9 Microscopeǡ’”‡ˆ‡”ƒ„Ž›™‹–Š’Šƒ•‡Ǧ ‘–”ƒ•–ǡƒ†™‹–Š•Ž‹†‡•ƒ† ‘˜‡”•Ž‹’•Ǥ

6.10 Refrigeratorǡ ƒ’ƒ„Ž‡‘ˆ‘’‡”ƒ–‹‰ƒ–ͷιά͵ιǤ

7 Sampling
ƒ’Ž‹‰ ‹• ‘– ’ƒ”– ‘ˆ –Š‡ ‡–Š‘† •’‡ ‹ϐ‹‡† ‹ –Š‹• †‘ —‡–Ǥ ˆ –Š‡”‡ ‹• ‘ •’‡ ‹ϐ‹  –‡”ƒ–‹‘ƒŽ
Standard dealing with sampling of the product concerned, it is recommended that the parties
‘ ‡”‡† ‘‡–‘ƒƒ‰”‡‡‡–‘–Š‹••—„Œ‡ –Ǥ ‘”ˆ‘‘†ƒ†ˆ‡‡†•ƒ’Ž‡•ǡ”‡ˆ‡”–‘ Ȁͳ͹͹ʹͺ[3].
For environmental samples, use ISO 18593[2] and see Reference [24].
– ‹• ‹’‘”–ƒ– –Šƒ– –Š‡ Žƒ„‘”ƒ–‘”› ”‡ ‡‹˜‡• ƒ •ƒ’Ž‡ ™Š‹ Š ‹• –”—Ž› ”‡’”‡•‡–ƒ–‹˜‡ ƒ† Šƒ• ‘– „‡‡
damaged or changed during transport or storage (see ISO 7218).

8 Preparation of test sample

”‡’ƒ”‡–Š‡–‡•–•ƒ’Ž‡‹ƒ ‘”†ƒ ‡™‹–Š–Š‡•’‡ ‹ϐ‹  –‡”ƒ–‹‘ƒŽ–ƒ†ƒ”††‡ƒŽ‹‰™‹–Š–Š‡’”‘†— –
concerned [see ISO 6887 (all parts) and ISO 18593[2]ȐǤ ˆ–Š‡”‡‹•‘•’‡ ‹ϐ‹  –‡”ƒ–‹‘ƒŽ–ƒ†ƒ”†ǡ‹–‹•
”‡ ‘‡†‡†–Šƒ––Š‡’ƒ”–‹‡• ‘ ‡”‡† ‘‡–‘ƒƒ‰”‡‡‡–‘–Š‹••—„Œ‡ –Ǥ

9 Procedure

9.1 Test portion and initial suspension

‡ˆ‡” –‘  ͸ͺͺ͹ ȋƒŽŽ ’ƒ”–•Ȍ ƒ† ƒ› •’‡ ‹ϐ‹  –‡”ƒ–‹‘ƒŽ –ƒ†ƒ”† ƒ’’”‘’”‹ƒ–‡ –‘ –Š‡ ’”‘†— –
concerned.
‘”’”‡’ƒ”ƒ–‹‘‘ˆ–Š‡‹‹–‹ƒŽ•—•’‡•‹‘ǡ—•‡ƒ•†‹Ž—–‹‘ϐŽ—‹†–Š‡•‡Ž‡ –‹˜‡’”‹ƒ”›‡”‹ Š‡–‡†‹—
•’‡ ‹ϐ‹‡†‹B.1 (half-Fraser broth).
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

In general, to prepare the initial suspension, add a test portion of 25 g or 25 ml to 225 g or 225 ml of the
•‡Ž‡ –‹˜‡’”‹ƒ”›‡”‹ Š‡–‡†‹—ȋB.1), to obtain a tenfold dilution, and homogenize. Pre-warm
–Š‡•‡Ž‡ –‹˜‡’”‹ƒ”›‡”‹ Š‡–‡†‹—–‘”‘‘–‡’‡”ƒ–—”‡„‡ˆ‘”‡—•‡Ǥ
Š‹•†‘ —‡–Šƒ•„‡‡˜ƒŽ‹†ƒ–‡†ˆ‘”–‡•–’‘”–‹‘•‘ˆʹͷ‰‘”ŽǤ•ƒŽŽ‡”–‡•–’‘”–‹‘ƒ›„‡—•‡†ǡ
™‹–Š‘—––Š‡‡‡†ˆ‘”ƒ††‹–‹‘ƒŽ˜ƒŽ‹†ƒ–‹‘Ȁ˜‡”‹ϐ‹ ƒ–‹‘ǡ’”‘˜‹†‹‰–Šƒ––Š‡•ƒ‡”ƒ–‹‘„‡–™‡‡’”‹ƒ”›
‡”‹ Š‡–„”‘–Šƒ†–‡•–’‘”–‹‘‹•ƒ‹–ƒ‹‡†ǤŽƒ”‰‡”–‡•–’‘”–‹‘–Šƒ–Šƒ–‹‹–‹ƒŽŽ›˜ƒŽ‹†ƒ–‡†ƒ›
„‡—•‡†ǡ‹ˆƒ˜ƒŽ‹†ƒ–‹‘Ȁ˜‡”‹ϐ‹ ƒ–‹‘•–—†›Šƒ••Š‘™–Šƒ––Š‡”‡ƒ”‡‘ƒ†˜‡”•‡‡ˆˆ‡ –•‘–Š‡†‡–‡ –‹‘
of L. monocytogenes or Listeria spp.
NOTE 1 Validation can be conducted in accordance with the appropriate document of ISO 16140 (all parts)[1].
‡”‹ϐ‹ ƒ–‹‘ˆ‘”’‘‘Ž‹‰•ƒ’Ž‡• ƒ„‡ ‘†— –‡†‹ƒ ‘”†ƒ ‡™‹–Š–Š‡’”‘–‘ ‘Ž†‡• ”‹„‡†‹ ͸ͺͺ͹ǦͳǣʹͲͳ͹ǡ
‡š  ȋ˜‡”‹ϐ‹ ƒ–‹‘ ’”‘–‘ ‘Ž ˆ‘” ’‘‘Ž‹‰ •ƒ’Ž‡• ˆ‘” “—ƒŽ‹–ƒ–‹˜‡ –‡•–•ȌǤ ‡‡ ‡ˆ‡”‡ ‡• ȏ21] and [22] ƒ• –Š‡›
provide information on the particular case of Listeria pooling samples.

ʹ ‘” Žƒ”‰‡ “—ƒ–‹–‹‡•ǡ ‹– ‹• ”‡ ‘‡†‡† –‘ ’”‡Ǧ™ƒ” –Š‡ •‡Ž‡ –‹˜‡ ’”‹ƒ”› ‡”‹ Š‡– ‡†‹— –‘
͵Ͳιάͳ鄇ˆ‘”‡‹š‹‰‹–™‹–Š–Š‡–‡•–’‘”–‹‘Ǥ

9.2 Primary enrichment

 —„ƒ–‡–Š‡’”‹ƒ”›‡”‹ Š‡–‡†‹—ȋŠƒŽˆǦ ”ƒ•‡”„”‘–Šǡ•‡‡B.1), prepared in accordance with 9.1,
at 30 °C (6.3Ȍˆ‘”ʹͷŠάͳŠǤ
NOTE 1 A black coloration can develop during the incubation.

4
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

NOTE 2 It is possible to store at 5 °C (6.10) the pre-enriched sample after incubation before transfer to Fraser
broth for a maximum of 72 h.

(See Reference [20].)

9.3 Secondary enrichment

9.3.1 ˆ–‡”‹ —„ƒ–‹‘‘ˆ–Š‡‹‹–‹ƒŽ•—•’‡•‹‘ȋ’”‹ƒ”›‡”‹ Š‡–Ȍˆ‘”ʹͷŠάͳŠȋ9.2), transfer 0,1 ml

of the culture obtained in 9.2ȋ”‡‰ƒ”†Ž‡••‘ˆ‹–• ‘Ž‘—”Ȍ–‘ƒ–—„‡‘”„‘––Ž‡ ‘–ƒ‹‹‰ͳͲŽ‘ˆ•‡ ‘†ƒ”›
enrichment medium (Fraser broth) (B.2).

9.3.2 Incubate the inoculated medium (9.3.1Ȍˆ‘”ʹͶŠάʹŠƒ–͵͹ιȋ6.3).

NOTE In the case of Listeria spp. other than Listeria monocytogenes detection, additional 24 h incubation can
ƒŽŽ‘™ˆ‘””‡ ‘˜‡”›‘ˆ‘”‡•’‡ ‹‡•Ǥ

ͻǤͶ Žƒ–‹‰‘—–ƒ†‹†‡–‹ϐ‹ ƒ–‹‘

9.4.1 General

9.4.1.1 ”‘ –Š‡ ’”‹ƒ”› ‡”‹ Š‡– —Ž–—”‡ ȋ9.2Ȍ ‹ —„ƒ–‡† ˆ‘” ʹͷ Š ά ͳ Š ƒ– ͵Ͳ ι ȋ6.3), inoculate,
„› ‡ƒ• ‘ˆ ƒ Ž‘‘’ ȋ6.5Ȍǡ –Š‡ •—”ˆƒ ‡ ‘ˆ –Š‡ ϐ‹”•– •‡Ž‡ –‹˜‡ ’Žƒ–‹‰ ‡†‹—ǡ ‰ƒ” Listeria according to
Ottaviani and Agosti (B.3), to obtain well-separated colonies.

”‘ ‡‡†‹–Š‡•ƒ‡™ƒ›™‹–Š–Š‡•‡ ‘†•‡Ž‡ –‹˜‡’Žƒ–‹‰Ǧ‘—–‡†‹—‘ˆ Š‘‹ ‡ȋB.4).

NOTE Half-Fraser broth and Fraser broth can be refrigerated at 5 °C (6.10) before isolation on selective agar
for a maximum of 72 h.[20]

9.4.1.2 ”‘–Š‡•‡ ‘†ƒ”›‡”‹ Š‡–‡†‹—‹ —„ƒ–‡†ˆ‘”ʹͶŠάʹŠƒ–͵͹ιȋ6.3) (9.3.2), repeat

the procedure described in 9.4.1.1 with the two selective plating-out media.
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

9.4.1.3 Invert the Petri dishes obtained in 9.4.1.1 and 9.4.1.2 and place them in an incubator set at
37 °C (6.3) for Agar Listeria according to Ottaviani and Agosti (B.3). For the second selective medium (B.4),
follow the manufacturer’s instructions.

9.4.1.4 For Agar Listeriaƒ ‘”†‹‰–‘––ƒ˜‹ƒ‹ƒ†‰‘•–‹‹ —„ƒ–‡ˆ‘”ƒ–‘–ƒŽ‘ˆͶͺŠάʹŠǤ ˆ ‘Ž‘‹‡•‘ˆ

presumptive L. monocytogenes or Listeria•’’Ǥƒ”‡‡˜‹†‡–ƒ–ʹͶŠάʹŠ–Š‡‹ —„ƒ–‹‘ƒ›„‡•–‘’’‡†ƒ–
this stage. For second selective agar incubate for the appropriate time. Examine the dishes (9.4.1.3) for
the presence of presumptive colonies of L. monocytogenes or Listeria spp.
NOTE After incubation plates can be refrigerated at 5 °C (6.10) for a maximum of 48 h before reading.

9.4.2 Agar Listeria according to Ottaviani and Agosti

Consider as presumptive L. monocytogenes –Š‡ „Ž—‡Ǧ‰”‡‡ ‘Ž‘‹‡• •—””‘—†‡† „› ƒ ‘’ƒ“—‡ ŠƒŽ‘
ȋ–›’‹ ƒŽ ‘Ž‘‹‡•ȌǤ‘Ž‘‹‡•‘ˆL. ivanoviiƒ”‡ƒŽ•‘„Ž—‡Ǧ‰”‡‡ƒ†•—””‘—†‡†„›ƒ‘’ƒ“—‡ŠƒŽ‘Ǥ
Consider as presumptive Listeria spp. the blue-green colonies with or without opaque halo.
NOTE 1 Some strains of L. monocytogenes‡š’‘•‡†–‘•–”‡•• ‘†‹–‹‘•ǡ’ƒ”–‹ —Žƒ”Ž›ƒ ‹†•–”‡••ǡ ƒ•Š‘™ƒ
˜‡”›™‡ƒŠƒŽ‘ȋ‘”‡˜‡‘ŠƒŽ‘ȌǤ

NOTE 2 Some rare L. monocytogenesƒ”‡ Šƒ”ƒ –‡”‹œ‡†„›ƒ•Ž‘™ ȋ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž’Š‘•’Š‘Ž‹’ƒ•‡Ȍ

ƒ –‹˜‹–›Ǥ— Š„ƒ –‡”‹ƒƒ”‡†‡–‡ –‡†™Š‡–Š‡–‘–ƒŽ†—”ƒ–‹‘‘ˆ‹ —„ƒ–‹‘‹•‘”‡–Šƒǡˆ‘”‡šƒ’Ž‡ǡˆ‘—”†ƒ›•Ǥ
Some of these strains could be pathogenic.[13] No L. monocytogenes strains have been described as PIPLC
negative.

© ISO 2017 – All rights reserved

Impreso por: Generico
5
 ISO 11290-1:2017(E)

NOTE 3 Some organisms other than Listeria spp. can produce blue colonies on this medium. See Annex C and
Reference [23].

9.4.3 Second selective medium

After the appropriate time, examine the plates for the presence of colonies which are considered to be
presumptive Listeria spp. or L. monocytogenesǡ„ƒ•‡†‘–Š‡‹” Šƒ”ƒ –‡”‹•–‹ •ˆ‘”–Š‡–›’‡‘ˆ‡†‹—
used (B.4). See Annex E for more information.

ͻǤͷ ‘ϐ‹”ƒ–‹‘‘ˆListeria monocytogenes or Listeria spp.

ͻǤͷǤͳ ‡Ž‡ –‹‘‘ˆ ‘Ž‘‹‡•ˆ‘” ‘ϐ‹”ƒ–‹‘

9.5.1.1 ‘” ‘ϐ‹”ƒ–‹‘ ‘ˆ ’”‡•—’–‹˜‡ L. monocytogenesǡ –ƒ‡ ƒ– Ž‡ƒ•– ‘‡ ‘Ž‘› ’”‡•—‡† –‘ „‡
L. monocytogenes (see 9.4.2 and 9.4.3ȌǤ‡ ‘ϐ‹”‡†‹•‘Žƒ–‡’‡”•ƒ’Ž‡‹••—ˆϐ‹ ‹‡–Ǥ ˆ–Š‡ϐ‹”•– ‘Ž‘›
is negative take further colonies presumed to be L. monocytogenes from selective medium (up to a
ƒš‹—‘ˆϐ‹˜‡ ‘Ž‘‹‡•ˆ”‘‡ƒ Š’Žƒ–‡‘ˆ‡ƒ Š•‡Ž‡ –‹˜‡‡†‹—ȌǤ

Streak the selected colonies onto the surface of pre-dried plates of a non-selective agar, for example
„Ž‘‘†ƒ‰ƒ”ǡ—–”‹‡–ƒ‰ƒ”ǡ–”›’–‘‡•‘›ƒ›‡ƒ•–‡š–”ƒ –ƒ‰ƒ”ȋȌȋB.14), in a manner which will allow
isolated colonies to develop.
•‡ ‘ˆ „Ž‘‘† ƒ‰ƒ” ˆ‘” ’—”‡ —Ž–—”‡ ‡ƒ„Ž‡• ‹–‡”’”‡–ƒ–‹‘ ‘ˆ Šƒ‡‘Ž›•‹•ǡ ™Š‡ ’‘•‹–‹˜‡ǡ ƒŽ”‡ƒ†› ƒ–
that stage (see 9.5.2.5.2 and Annex DȌǤ ˆ•–”‡ƒ‹‰‘„Ž‘‘†ƒ‰ƒ”†‘‡•‘–•Š‘™Šƒ‡‘Ž›•‹•ǡ–Š‡–Š‡
Šƒ‡‘Ž›•‹•–‡•–•ŠƒŽŽ„‡†‘‡„›•–ƒ„„‹‰ȋ9.5.2.5.2) or in liquid medium (9.5.2.5.3).
Place the plates in the incubator set at 37 °C (6.3Ȍˆ‘”ͳͺŠ–‘ʹͶŠ‘”—–‹Ž‰”‘™–Š‹••ƒ–‹•ˆƒ –‘”›Ǥ
ˆ–Š‡ ‘Ž‘‹‡•ƒ”‡‘–‹•‘Žƒ–‡†ǡ’‹ ƒ–›’‹ ƒŽL. monocytogenes ‘Ž‘› onto another non-selective agar
’Žƒ–‡Ǥƒ””›‘—––Š‡ˆ‘ŽŽ‘™‹‰–‡•–•ȋ9.5.2) from colonies of a pure culture on the non-selective agar.

9.5.1.2 ‘” ‘ϐ‹”ƒ–‹‘ ‘ˆ ’”‡•—’–‹˜‡ Listeria •’’Ǥǡ –ƒ‡ ƒ– Ž‡ƒ•– ‘‡ ‘Ž‘› ’”‡•—‡† –‘ „‡
Listeria spp. (see 9.4.2 and 9.4.3ȌǤ ‡ ‘ϐ‹”‡† ‹•‘Žƒ–‡ ’‡” •ƒ’Ž‡ ‹• •—ˆϐ‹ ‹‡–Ǥ ˆ –Š‡ ϐ‹”•– ‘Ž‘› ‹•
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

negative take further colonies presumed to be Listeria spp. from selective medium (up to a maximum of
ϐ‹˜‡ ‘Ž‘‹‡•ˆ”‘‡ƒ Š’Žƒ–‡‘ˆ‡ƒ Š•‡Ž‡ –‹˜‡‡†‹—ȌǤ

‘” ‘ϐ‹”ƒ–‹‘‘ˆListeria•’’Ǥ—•‡’Žƒ–‡•‘ˆǤ
–”‡ƒ–Š‡•‡Ž‡ –‡† ‘Ž‘‹‡•‘–‘–Š‡•—”ˆƒ ‡‘ˆ’”‡Ǧ†”‹‡†’Žƒ–‡•‘ˆȋB.14), in a manner which
will allow isolated colonies to develop.
Place the plates in the incubator set at 37 °C (6.3Ȍˆ‘”ͳͺŠ–‘ʹͶŠ‘”—–‹Ž‰”‘™–Š‹••ƒ–‹•ˆƒ –‘”›Ǥ
›’‹ ƒŽ ‘Ž‘‹‡•‘ˆListeria•’’Ǥ‘ƒ”‡ͳ–‘ʹ‹†‹ƒ‡–‡”ǡ ‘˜‡šǡ ‘Ž‘—”Ž‡••ƒ†‘’ƒ“—‡
™‹–Šƒ‡–‹”‡‡†‰‡ǤŠ‡–Š‡’Žƒ–‡•ƒ”‡Š‡Ž†–‘–Š‡Ž‹‰Š–ȋƒ”–‹ϐ‹ ‹ƒŽ‘”ƒ–—”ƒŽȌƒ–ƒ„‘—–Ͷͷ†‡‰”‡‡ƒ‰Ž‡ǡ
‘Ž‘‹‡•‡šŠ‹„‹–ƒ„Ž—‡Ǧ‰”‡› ‘Ž‘—”ƒ†ƒ‰”ƒ—Žƒ”•—”ˆƒ ‡Ǥ
ˆ–Š‡ ‘Ž‘‹‡•ƒ”‡‘–‹•‘Žƒ–‡†ǡ’‹ ƒ–›’‹ ƒŽListeria•’’Ǥ ‘Ž‘›‘–‘ƒ‘–Š‡”‘Ǧ•‡Ž‡ –‹˜‡ƒ‰ƒ”’Žƒ–‡Ǥ
ƒ””›‘—––Š‡ˆ‘ŽŽ‘™‹‰–‡•–•ȋ9.5.3Ȍˆ”‘–›’‹ ƒŽ ‘Ž‘‹‡•‘ˆƒ’—”‡ —Ž–—”‡‘Ǥ

ͻǤͷǤʹ ‘ϐ‹”ƒ–‹‘‘ˆL. monocytogenes

9.5.2.1 General

ƒ””›‘—––Š‡ ‘ϐ‹”ƒ–‹‘–‡•–•ˆ‘”L. monocytogenes. Appropriate positive and negative control strains

ˆ‘”‡ƒ Š‘ˆ–Š‡ ‘ϐ‹”ƒ–‹‘–‡•–••ŠƒŽŽ„‡—•‡†Ǥ
‡”ˆ‘”ƒ–‹‹—–Š‡ƒ†ƒ–‘”›–‡•–•ƒ•Ž‹•–‡†ȋ‹„‘Ž†Ȍ‹Table 1.

6
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

ƒ„Ž‡ͳȄ‘ϐ‹”ƒ–‹‘–‡•–•ˆ‘”L. monocytogenes
Tests L. monocytogenes ‘ϐ‹”ƒ–‹‘–‡•–• Results
Mandatory Microscopic aspect a (9.5.2.4) Slim short rods or coccobacilli
Beta-haemolysis (9.5.2.5) +
L-Rhamnose (9.5.2.7) +
D-Xylose (9.5.2.7) -
Optional Catalase (9.5.2.2) +
‘–‹Ž‹–›ƒ–ʹͷιȋ9.5.2.3) +
CAMP test (9.5.2.6) +
a Microscopic aspect is optional for Agar Listeria according to Ottaviani and Agosti and for the second medium if
it allows distinction between pathogenic and non-pathogenic Listeria spp.

‡–ƒ‹Ž•‘”‡•—Ž–•ˆ‘” ‘ϐ‹”ƒ–‹‘–‡•–• ƒ„‡ˆ‘—†‹Annex D.

  ƒŽ–‡”ƒ–‹˜‡ ’”‘ ‡†—”‡ ƒ• ‡–‹‘‡† ‹  ͹ʹͳͺ ƒ „‡ —•‡† –‘ ‘ϐ‹” –Š‡ ‹•‘Žƒ–‡ ƒ• Listeria
monocytogenesǡ’”‘˜‹†‹‰–Š‡•—‹–ƒ„‹Ž‹–›‘ˆ–Š‡”‡Ž‡˜ƒ–’”‘ ‡†—”‡‹•˜‡”‹ϐ‹‡†Ǥ

ˆ•Š‘™–‘„‡”‡Ž‹ƒ„Ž‡ǡ‹‹ƒ–—”‹œ‡†‰ƒŽŽ‡”‹‡•ˆ‘”–Š‡„‹‘ Š‡‹ ƒŽ‹†‡–‹ϐ‹ ƒ–‹‘‘ˆListeria monocytogenes

ƒ›„‡—•‡†ȋ•‡‡ ͹ʹͳͺȌǤ
Rare strains of L. monocytogenes †‘ ‘– •Š‘™ „‡–ƒǦŠƒ‡‘Ž›•‹• ‘” ƒ ’‘•‹–‹˜‡ ”‡ƒ –‹‘ –‘ –Š‡ 
–‡•–—†‡”–Š‡ ‘†‹–‹‘•†‡• ”‹„‡†‹–Š‹•†‘ —‡–Ǥ ˆ–›’‹ ƒŽ ‘Ž‘‹‡•‘‰ƒ”Listeria according to
––ƒ˜‹ƒ‹ƒ†‰‘•–‹™‹–Š ƒ –‹˜‹–›‡˜‡‹ˆ‹–‹•Ž‘™ǡƒ”‡‡‰ƒ–‹˜‡ˆ‘”Šƒ‡‘Ž›•‹•ǡ‹–‹•”‡ ‘‡†‡†
–‘’‡”ˆ‘”ƒ††‹–‹‘ƒŽ–‡•–•ȋ‡Ǥ‰Ǥ ”ƒ•–ƒ‹ǡ ƒ–ƒŽƒ•‡ǡ‘–‹Ž‹–›ǡ–‡•–ǡȌǡ‹‘”†‡”–‘†‡–‡”‹‡
™Š‡–Š‡”–Š‹•‹•‘Žƒ–‡‹•ƒ‘ǦŠƒ‡‘Ž›–‹ L. monocytogenes.

9.5.2.2 Catalase reaction (optional)

ƒ‡ƒ‹•‘Žƒ–‡† ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.1ƒ†•—•’‡†‹–‹ƒ†”‘’‘ˆŠ›†”‘‰‡’‡”‘š‹†‡•‘Ž—–‹‘ȋB.6)
on a slide. The immediate formation of gas bubbles indicates a positive reaction.
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

  ƒ–ƒŽƒ•‡ ”‡ƒ –‹‘ ’‡”ˆ‘”‡† ˆ”‘ ƒ ‘Ž‘› ‘”‹‰‹ƒ–‹‰ ˆ”‘ ƒ „Ž‘‘† ƒ‰ƒ” ƒ •‘‡–‹‡• Ž‡ƒ† –‘
false-positive results.

9.5.2.3 Motility test (optional)

ƒ‡ƒ‹•‘Žƒ–‡† ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.1 and suspend it in a tube containing a non-selective nutrient

liquid medium.
Incubate in the incubator (6.3Ȍ•‡–ƒ–ʹͷιˆ‘”ͺŠ–‘ʹͶŠ—–‹Ž–Š‡‡†‹—–—”• Ž‘—†›Ǥ
Take a drop of the above culture using a loop (6.5) onto a clean glass microscope slide. Place a cover slip
on top and examine it under a microscope (6.9).
Listeria spp. (including L. monocytogenesȌƒ’’‡ƒ”ƒ••Ž‹ǡ•Š‘”–”‘†•™‹–Š–—„Ž‹‰‘–‹Ž‹–›Ǥ
—Ž–—”‡•‰”‘™ƒ––‡’‡”ƒ–—”‡•ƒ„‘˜‡ʹͷ鏃›ˆƒ‹Ž–‘‡šŠ‹„‹––Š‹•‘–‹‘ǤŽ™ƒ›• ‘’ƒ”‡–Š‡–‘
a known Listeria —Ž–—”‡Ǥ‘ ‹ǡŽƒ”‰‡”‘†•ǡ‘””‘†•™‹–Š”ƒ’‹†•™‹‹‰‘–‹Ž‹–›ƒ”‡‘–Listeria spp.
• ƒ ƒŽ–‡”ƒ–‹˜‡ –‡•– ˆ‘” ‘–‹Ž‹–›ǡ —•‹‰ ƒ ‹‘ —Žƒ–‹‰ ‡‡†Ž‡ ȋ6.5), dilute in sterile water (or other
ƒ’’”‘’”‹ƒ–‡ †‹Ž—‡–Ȍ ƒ ˆ”ƒ‰‡– ‘ˆ ‹•‘Žƒ–‡† ‘Ž‘› ‘„–ƒ‹‡† ‘ ‘Ǧ•‡Ž‡ –‹˜‡ ƒ‰ƒ”Ǥ Listeria spp.
(including L. monocytogenesȌƒ’’‡ƒ”ƒ••Ž‹ǡ•Š‘”–”‘†•™‹–Š–—„Ž‹‰‘–‹Ž‹–›Ǥ
•ƒ‘–Š‡”ƒŽ–‡”ƒ–‹˜‡–‡•–ˆ‘”‘–‹Ž‹–›ǡ—•‹‰ƒ‹‘ —Žƒ–‹‰‡‡†Ž‡ȋ6.5Ȍǡ•–ƒ„–Š‡‘–‹Ž‹–›ƒ‰ƒ”ȋB.7)
™‹–Šƒ —Ž–—”‡–ƒ‡ˆ”‘ƒ–›’‹ ƒŽ ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.1Ǥ  —„ƒ–‡ƒ–ʹͷιˆ‘”ͶͺŠάʹŠǤ

© ISO 2017 – All rights reserved

Impreso por: Generico
7
 ISO 11290-1:2017(E)

Examine for growth around the stab. Listeria •’’Ǥ ƒ”‡ ‘–‹Ž‡ǡ ‰‹˜‹‰ ƒ –›’‹ ƒŽ —„”‡ŽŽƒǦŽ‹‡ ‰”‘™–Š
’ƒ––‡”Ǥ ˆ‰”‘™–Š‹•‘–•—ˆϐ‹ ‹‡–ǡ‹ —„ƒ–‡ˆ‘”—’–‘ƒƒ††‹–‹‘ƒŽϐ‹˜‡†ƒ›•ƒ†‘„•‡”˜‡–Š‡•–ƒ„ƒ‰ƒ‹Ǥ
NOTE Some new Listeria•’‡ ‹‡•Šƒ˜‡„‡‡”‡ ‡–Ž›‹•‘Žƒ–‡†Ǥ[5],[10],[12],[14],[25],[26],[27] Most of them are not
‘„‹Ž‡‹–Š‡‘–‹Ž‹–›ƒ‰ƒ”Ǥ

ͻǤͷǤʹǤͶ ‹ ”‘• ‘’‹ ƒ•’‡ –ȋ‘’–‹‘ƒŽ‹–Š‡ ƒ•‡‘ˆ—•‡‘ˆƒ‰ƒ”•’‡ ‹ϐ‹ ˆ‘”’ƒ–Š‘‰‡‹ 

Listeria spp.)

ƒ‡ ƒ ‹ ”‘• ‘’‹  ’”‡’ƒ”ƒ–‹‘ ȋ‡Ǥ‰Ǥ –Š‡ ”ƒ •–ƒ‹ǡ ™‡– ‹ ”‘• ‘’›Ȍ ‘ ƒ ™‡ŽŽǦ•‡’ƒ”ƒ–‡† ‘Ž‘›
obtained in 9.5.1.1. Listeria spp. (including L. monocytogenes) appear as Gram positive (if this stain
‹•’‡”ˆ‘”‡†Ȍǡ•Ž‹ǡ•Š‘”–”‘†•‘” ‘ ‘„ƒ ‹ŽŽ‹ǡ™‹–Š–—„Ž‹‰‘–‹Ž‹–›™Š‡‘”‹‰‹ƒ–‹‰ˆ”‘ƒˆ”‡•Š
culture.
For Gram stain microscopic preparation see ISO 7218.

9.5.2.5 Haemolysis tests

9.5.2.5.1 General

Š‘‘•‡‘‡‘ˆ–Š‡Šƒ‡‘Ž›•‹•–‡•–•ȋ9.5.2.5.2 or 9.5.2.5.3).
NOTE There exist rare strains of L. monocytogenes™Š‹ Š†‘‘–•Š‘™„ǦŠƒ‡‘Ž›•‹•‘”ƒ’‘•‹–‹˜‡”‡ƒ –‹‘–‘
the CAMP test under the conditions described in this document.

9.5.2.5.2 Haemolysis on blood agar

ˆ –Š‡ ‘”’Š‘Ž‘‰‹ ƒŽ ƒ† ’Š›•‹‘Ž‘‰‹ ƒŽ Šƒ”ƒ –‡”‹•–‹ • ƒ”‡ ‹†‹ ƒ–‹˜‡ ‘ˆ Listeria spp., inoculate blood
agar plates (B.8Ȍ–‘†‡–‡”‹‡–Š‡Šƒ‡‘Ž›–‹ ”‡ƒ –‹‘Ǥ
”› –Š‡ ƒ‰ƒ” •—”ˆƒ ‡ ™‡ŽŽ „‡ˆ‘”‡ —•‡Ǥ ƒ‡ ƒ ‹•‘Žƒ–‡† ‘Ž‘› ‘„–ƒ‹‡† ‹ 9.5.1.1 using a wire (6.5),
then stab a section of agar. Repeat for each culture. On the same plate, if possible, stab positive
(L. monocytogenes) and negative (L. innocua) control cultures. For example, L. monocytogenes 4b WDCM
00021 or L. monocytogenes 1/2a WDCM 00109 and L. innocuaͲͲͲͳ͹ƒ›„‡—•‡†Ǥ
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

After incubation at 37 °C (6.3Ȍˆ‘”ʹͶŠάʹŠǡ‡šƒ‹‡–Š‡–‡•–•–”ƒ‹•ƒ† ‘–”‘Ž•ǤL. monocytogenes

•Š‘™ƒ””‘™ǡ Ž‡ƒ”ǡŽ‹‰Š–œ‘‡•‘ˆŠƒ‡‘Ž›•‹•ǢL. innocua show no clear zone around the stab. L. seeligeri
•Š‘™ ‘•–Ž› ƒ ™‡ƒ œ‘‡ ‘ˆ Šƒ‡‘Ž›•‹•Ǥ L. ivanovii —•—ƒŽŽ› •Š‘™ ™‹†‡ǡ Ž‡ƒ”Ž› †‡Ž‹‡ƒ–‡† œ‘‡• ‘ˆ
Šƒ‡‘Ž›•‹•Ǥšƒ‹‡–Š‡’Žƒ–‡•‹ƒ„”‹‰Š–Ž‹‰Š––‘ ‘’ƒ”‡–‡•– —Ž–—”‡•™‹–Š ‘–”‘Ž•Ǥ
ͳ Š‡Šƒ‡‘Ž›•‹•”‡ƒ –‹‘‹•‘”‡”‡ƒ†‹Ž›•‡‡„›”‡‘˜‹‰ƒ› ‘Ž‘›‰”‘™–Š‘–Š‡•—”ˆƒ ‡‘ˆ–Š‡
agar around the inoculum mark.

ʹ Š‡Šƒ‡‘Ž›•‹•–‡•– ƒ„‡’‡”ˆ‘”‡†„›•–ƒ„„‹‰–Š‡„Ž‘‘†ƒ‰ƒ”’Žƒ–‡—•‡†ˆ‘”–Š‡–‡•–Ǥ

9.5.2.5.3 Haemolysis reaction using red blood corpuscles

Š‡Šƒ‡‘Ž›–‹ ”‡ƒ –‹‘ƒ›ƒŽ•‘„‡ ƒ””‹‡†‘—–—•‹‰”‡†„Ž‘‘† ‘”’—• Ž‡•ƒ•ˆ‘ŽŽ‘™•Ǥ

‹•’‡”•‡–Š‡ ‘Ž‘›‹ͳͷͲρŽ‘ˆƒ‘Ǧ•‡Ž‡ –‹˜‡Ž‹“—‹†—–”‹‡–‡†‹—ǡ‹ —„ƒ–‡ƒ–͵͹ιȋ6.3) for 2 h.
††ͳͷͲρŽ‘ˆƒ•—•’‡•‹‘‘ˆ”‡†„Ž‘‘† ‘”’—• Ž‡•ȋB.10). Incubate at 37 °C (6.3) for 15 min to 60 min,
then refrigerate at 5 °C (6.10Ȍˆ‘”ƒ’’”‘š‹ƒ–‡Ž›ʹŠǤšƒ‹‡ˆ‘”Šƒ‡‘Ž›–‹ ƒ –‹˜‹–›Ǥ ˆ–Š‡”‡ƒ –‹‘‹•
doubtful, leave at 5 °C (6.10Ȍˆ‘”—’–‘ʹͶŠάʹŠǤ•‡†‹‡–ƒ–‹‘‘ˆ”‡†„Ž‘‘† ‘”’—• Ž‡•ȋˆ‘”ƒ–‹‘‘ˆ
a red point at the bottom of the tubes) indicates a negative reaction.
Include positive and negative controls. For control strains see 9.5.2.5.2.

8
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

9.5.2.6 CAMP test (optional)

ˆ–Š‡”‡•—Ž–‘ˆ–Š‡Šƒ‡‘Ž›•‹•–‡•–‹•†‹ˆϐ‹ —Ž––‘‹–‡”’”‡–ǡ–Š‡–‡•–‹•”‡ ‘‡†‡†–‘†‡‘•–”ƒ–‡

Ž‡ƒ”Ž›–Šƒ–Šƒ‡‘Ž›•‹•‹•†—‡–‘Ž‹•–‡”‹‘Ž›•‹ƒ –‹˜‹–›Ǥ
A ȾǦŠƒ‡‘Ž›–‹ •–”ƒ‹‘ˆStaphylococcus aureus (e.g. WDCM 00034) and a strain of Rhodococcus equi (e.g.
WDCM 0028) are required to undertake the CAMP test. Not all strains of S. aureus are suitable for the
CAMP test. Streak each of the S. aureus and R. equi cultures in single lines across the blood agar plate
(B.8.3 or B.11.4Ȍ•‘–Šƒ––Š‡–™‘ —Ž–—”‡•ƒ”‡’ƒ”ƒŽŽ‡Žƒ††‹ƒ‡–”‹ ƒŽŽ›‘’’‘•‹–‡ȋ•‡‡Figure 1). A thin,
‡˜‡‹‘ —Ž—‹•”‡“—‹”‡†ǤŠ‹• ƒ„‡‘„–ƒ‹‡†„›—•‹‰ƒ‹‘ —Žƒ–‹‘Ž‘‘’‘”ƒ™‹”‡ȋ6.5) held at
right angles to the agar.
–”‡ƒƒ™‡ŽŽ‹•‘Žƒ–‡† ‘Ž‘›‘ˆ–Š‡•–”ƒ‹—†‡”–‡•–ȋ9.5.1.1) in a similar fashion at right angles to these
cultures so that the test culture and S. aureus and R. equi cultures do not touch but at their closest are
ƒ„‘—–ͳ–‘ʹƒ’ƒ”–Ǥ‡˜‡”ƒŽ–‡•–•–”ƒ‹•ƒ›„‡•–”‡ƒ‡†‘–Š‡•ƒ‡’Žƒ–‡Ǥ

Key
1 ƒ””‘™„ƒ†‘ˆéǦŠƒ‡‘Ž›•‹•
2 ‘Šƒ‡‘Ž›•‹•
3 ™‹†‡„ƒ†‘ˆéǦŠƒ‡‘Ž›•‹•
4 L. monocytogenes
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

5 L. innocua
6 L. ivanovii

Figure 1 — Inoculation and interpretation of CAMP test plates

The vertical lines in Figure 1 represent streaks of S. aureus (S) and R. equi (R). Horizontal lines represent
•–”‡ƒ•‘ˆ–Š‡–‡•– —Ž–—”‡•Ǥ ƒ– Š‡†ƒ”‡ƒ•‹†‹ ƒ–‡–Š‡Ž‘ ƒ–‹‘•‘ˆ‡Šƒ ‡†Šƒ‡‘Ž›•‹•ǤŠ‡†‘––‡†
ƒ”‡ƒ‹†‹ ƒ–‡•–Š‡œ‘‡‘ˆ‹ϐŽ—‡ ‡‘ˆ–Š‡S. aureus culture.
‹—Ž–ƒ‡‘—•Ž›ǡ •–”‡ƒ ‘–”‘Ž —Ž–—”‡• ‘ˆ L. monocytogenes, L. innocua and L. ivanovii. For example,
L. monocytogenes 4b WDCM 00021, L. monocytogenes 1/2a WDCM 00109, L. innocua WDCM 00017 and
L. ivanoviiͲͲͲͳͺƒ›„‡—•‡†Ǥƒ‹–ƒ‹•–‘  —Ž–—”‡•ƒ••’‡ ‹ϐ‹‡†‹ ͳͳͳ͵͵Ǥ
If blood agar (B.8.3Ȍ‹•—•‡†ǡ‹ —„ƒ–‡–Š‡’Žƒ–‡•ƒ–͵͹ιˆ‘”ͳͺŠ–‘ʹͶŠǤ ˆ†‘—„Ž‡ǦŽƒ›‡”’Žƒ–‡•ȋB.11.4)
are used, incubate at 37 °C for 12 h to 18 h.
The positive reaction with R. equi‹••‡‡ƒ•ƒ™‹†‡ȋͷ–‘ͳͲȌDzƒ””‘™ǦŠ‡ƒ†dz‘ˆŠƒ‡‘Ž›•‹•ǤŠ‡
”‡ƒ –‹‘‹• ‘•‹†‡”‡†ƒ•‡‰ƒ–‹˜‡‹ˆƒ•ƒŽŽœ‘‡‘ˆ™‡ƒŠƒ‡‘Ž›•‹•‡š–‡†•‘Ž›ƒ„‘—–ͳƒ––Š‡
intersection of the test strain with the diffusion zone of the R. equi culture.
A positive reaction with S. aureusƒ’’‡ƒ”•ƒ•ƒ•ƒŽŽœ‘‡‘ˆ‡Šƒ ‡†Šƒ‡‘Ž›•‹•‡š–‡†‹‰‘Ž›ƒ„‘—–
ʹˆ”‘–Š‡–‡•–•–”ƒ‹ƒ†™‹–Š‹–Š‡™‡ƒŽ›Šƒ‡‘Ž›–‹ œ‘‡†—‡–‘‰”‘™–Š‘ˆ–Š‡S. aureus culture.
ƒ”‰‡œ‘‡•‘ˆŠƒ‡‘Ž›•‹•†‘‘–‘ —”‹–Š‡ƒ”‡ƒ‘ˆS. aureus and L. monocytogenes.

© ISO 2017 – All rights reserved

Impreso por: Generico
9
 ISO 11290-1:2017(E)

9.5.2.7 Carbohydrate utilization

Using a loop (6.5Ȍǡ ‹‘ —Žƒ–‡ ‡ƒ Š ‘ˆ –Š‡ ƒ”„‘Š›†”ƒ–‡ —–‹Ž‹œƒ–‹‘ „”‘–Š• ȋB.12) with the cultures
obtained from the non-selective agar (9.5.1.1). Incubate at 37 °C. Positive reactions (acid formation)
ƒ”‡ ‹†‹ ƒ–‡† „› ƒ ›‡ŽŽ‘™ ‘Ž‘—” ƒ† ‘ —” ‘•–Ž› ™‹–Š‹ ʹͶ Š –‘ Ͷͺ Š ˆ‘” ‹ ”‘˜‘Ž—‡• –—„‡•ǡ ƒ†
—’ –‘ ͷ †ƒ›• ˆ‘” ƒ ”‘˜‘Ž—‡• –—„‡•Ǥ ǦŠƒ‘•‡ ƒ† Ǧ›Ž‘•‡ ƒ”‡ —•‡† ˆ‘” –Š‡ ‘ϐ‹”ƒ–‹‘ ‘ˆ
L. monocytogenesǡ™Š‹ Š‹•ǦŠƒ‘•‡’‘•‹–‹˜‡ƒ†Ǧ›Ž‘•‡‡‰ƒ–‹˜‡ȋ•‡‡Annex D).
NOTE There exist rare strains of L. monocytogenes which do not ferment L-Rhamnose.[15],[18]

For microvolumes, reactions are more rapid. The level of inoculation compared to the total volume is
ƒ‹’‘”–ƒ–ˆƒ –‘”Ǥ ‘”‡ƒ Š Š‘•‡’”‘–‘ ‘Žǡ‹–‹•‹’‘”–ƒ––‘˜‡”‹ˆ›–Š‡–‹‡–ƒ‡–‘‘„–ƒ‹ƒ›‡ŽŽ‘™
coloration. It is advised to use controls. For example L. monocytogenes 4b WDCM 00021, L. innocua
WDCM 00017 and L. ivanovii  ͲͲͲͳͺ ƒ› „‡ —•‡†Ǥ ƒ‹–ƒ‹ •–‘  —Ž–—”‡• ƒ• •’‡ ‹ϐ‹‡† ‹
ISO 11133.

ͻǤͷǤ͵ ‘ϐ‹”ƒ–‹‘‘ˆListeria spp.

9.5.3.1 General

ƒ””›‘—––Š‡ ‘ϐ‹”ƒ–‹‘–‡•–•ˆ‘”Listeria•’’Ǥˆ”‘ƒ–›’‹ ƒŽ ‘Ž‘›ȋ9.5.1.2). Appropriate positive

ƒ†‡‰ƒ–‹˜‡ ‘–”‘Ž•–”ƒ‹•ˆ‘”‡ƒ Š‘ˆ–Š‡ ‘ϐ‹”ƒ–‹‘–‡•–••ŠƒŽŽ„‡—•‡†Ǥ
‡”ˆ‘”ƒ–‹‹—–Š‡ƒ†ƒ–‘”›–‡•–•ƒ•Ž‹•–‡†ȋ‹„‘Ž†Ȍ‹Table 2.
‘”ˆ—”–Š‡”–‡•–•ǡ‹ˆ‹†‡–‹ϐ‹ ƒ–‹‘‘ˆ•’‡ ‹‡•‘ˆListeria is required, see Annex D.

ƒ„Ž‡ʹȄ‘ϐ‹”ƒ–‹‘–‡•–•ˆ‘”Listeria spp.
Tests Listeria spp. Results
Mandatory Microscopic aspect (9.5.2.4) Slim short rods or coccobacilli
Catalase (9.5.2.2) +
Optional VP test (9.5.3.5) +
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

‘–‹Ž‹–›ƒ–ʹͷιȋ9.5.2.3) +

ͳ ƒŽ–‡”ƒ–‹˜‡’”‘ ‡†—”‡ƒ•‡–‹‘‡†‹ ͹ʹͳͺ ƒ„‡—•‡†–‘ ‘ϐ‹”–Š‡‹•‘Žƒ–‡ƒ•Listeria spp.,

’”‘˜‹†‹‰–Š‡•—‹–ƒ„‹Ž‹–›‘ˆ–Š‡”‡Ž‡˜ƒ–’”‘ ‡†—”‡‹•˜‡”‹ϐ‹‡†Ǥ

NOTE 2 It is possible that some new Listeria•’‡ ‹‡•”‡ ‡–Ž›‹•‘Žƒ–‡†ƒ›‘– ‘””‡•’‘†–‘–Š‹•• Š‡‡‹
’ƒ”–‹ —Žƒ”ˆ‘”‘–‹Ž‹–›ǡ–‡•–ƒ†‰”‘™–Šƒ–͵͹ιȋ•‡‡ǡˆ‘”‡šƒ’Ž‡ǡ‡ˆ‡”‡ ‡•ȏ4], [10], [25], [26] and [27]).

9.5.3.2 Catalase reaction

ƒ‡ƒ‹•‘Žƒ–‡† ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.2 and perform the test as described in 9.5.2.2.

9.5.3.3 Motility test (optional)

ƒ‡ƒ‹•‘Žƒ–‡† ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.2 and perform the test as described in 9.5.2.3.

9.5.3.4 Microscopic aspect

ƒ‡ƒ‹•‘Žƒ–‡† ‘Ž‘›‘„–ƒ‹‡†‹9.5.1.2 and perform the test as described in 9.5.2.4.

9.5.3.5 Voges – Proskauer (VP) reaction (optional)

Using a loop (6.5), inoculate a tube containing 3 ml of the VP medium (B.13.1). Incubate at 37 °C
ˆ‘” ʹͶ Š ά ʹ ŠǤ ˆ–‡” ‹ —„ƒ–‹‘ ƒ†† Ͳǡ͸ Ž ‘ˆ ͷ Ψ Ƚ-naphthol solution (B.13.2Ȍ ƒ† Ͳǡʹ Ž ‘ˆ ͶͲ Ψ
’‘–ƒ••‹— Š›†”‘š‹†‡ •‘Ž—–‹‘ ȋB.13.3). Shake well, slope the tube (to increase the area of the air-

10
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Ž‹“—‹†‹–‡”ˆƒ ‡ȌǤšƒ‹‡ƒˆ–‡”ͳͷ‹ƒ†ͳŠǤ’‘•‹–‹˜‡”‡ƒ –‹‘‹•‹†‹ ƒ–‡†„›ƒ•–”‘‰”‡† ‘Ž‘—”Ǥ

Listeria spp. are VP positive.
NOTE Some new Listeria•’‡ ‹‡•Šƒ˜‡„‡‡”‡ ‡–Ž›‹•‘Žƒ–‡†Ǥ[5],[10],[12],[14],[25],[26],[27] Most of them are VP
negative.

9.6 Interpretation of morphological and physiological properties and of the

biochemical reactions
All Listeria spp. are small, Gram-positive rods or coccobacilli that give a positive reaction in catalase test.
L. monocytogenes ƒ”‡ ‘ϐ‹”‡† ƒ ‘”†‹‰ –‘ –‡•–• Ž‹•–‡† ‹ Table 1 and Listeria •’’Ǥ ƒ”‡ ‘ϐ‹”‡†
according tests listed in Table 2.

9.7 Additional characterization of isolated strains (optional)

Isolates which are considered to be L. monocytogenes (9.6Ȍƒ›„‡•‡–ˆ‘”ˆ—”–Š‡” Šƒ”ƒ –‡”‹œƒ–‹‘–‘ƒ
recognized national or regional Listeria‡ˆ‡”‡ ‡ƒ„‘”ƒ–‘”›ǡ‘”ȋ‹ˆ‘–ƒ˜ƒ‹Žƒ„Ž‡Ȍ–‘–Š‡‘”Ž† ‡ƒŽ–Š
Organization Collaborating Centres for ListeriaǤ Š‡ †‹•’ƒ– Š •ŠƒŽŽ „‡ ƒ ‘’ƒ‹‡† „› ƒŽŽ ’‘••‹„Ž‡
information concerning the strain(s).
‘”ˆ—”–Š‡”–‡•–•ǡ‹ˆ‹†‡–‹ϐ‹ ƒ–‹‘‘ˆ•’‡ ‹‡•‘ˆListeria is required, see Annex D.

10 Expression of results
In accordance with the interpretation of the results (see 9.6), report if L. monocytogenes is detected
or not detected and/or if Listeria•’’Ǥ‹•†‡–‡ –‡†‘”‘–†‡–‡ –‡†‹–Š‡–‡•–’‘”–‹‘„›•’‡ ‹ˆ›‹‰–Š‡
mass in grams, the volume in millilitres of the sample tested, the surface in squared centimetres or per
sample device.

11 Performance characteristics of the method

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

11.1 Method validation studies

Š‡ ’‡”ˆ‘”ƒ ‡ Šƒ”ƒ –‡”‹•–‹ • ‘ˆ –Š‡ ‡–Š‘† ™‡”‡ †‡–‡”‹‡† ‹ ‹–‡”Žƒ„‘”ƒ–‘”› •–—†‹‡• –‘
†‡–‡”‹‡–Š‡•’‡ ‹ϐ‹ ‹–›ǡ•‡•‹–‹˜‹–›ƒ†ǡ™Š‡’‘••‹„Ž‡ǡ–Š‡Ž‡˜‡Ž‘ˆ†‡–‡ –‹‘ȋ50) of the method.
The data are summarized in Annex FǤŠ‡˜ƒŽ—‡•†‡”‹˜‡†ˆ”‘–Š‡‹–‡”Žƒ„‘”ƒ–‘”›•–—†‹‡•ƒ›‘–„‡
ƒ’’Ž‹ ƒ„Ž‡–‘ˆ‘‘†–›’‡•‘–Š‡”–Šƒ–Š‘•‡‰‹˜‡‹Annex F.

11.2 Sensitivity
Š‡•‡•‹–‹˜‹–›‹•†‡ϐ‹‡†ƒ•–Š‡—„‡”‘ˆ•ƒ’Ž‡•ˆ‘—†’‘•‹–‹˜‡†‹˜‹†‡†„›–Š‡—„‡”‘ˆ•ƒ’Ž‡•
tested at a given level of contamination. The results are thus dependent on the level of contamination of
the sample.

ͳͳǤ͵ ’‡ ‹ϐ‹ ‹–›

Š‡ •’‡ ‹ϐ‹ ‹–› ‹• †‡ϐ‹‡† ƒ• –Š‡ —„‡” ‘ˆ •ƒ’Ž‡• ˆ‘—† ‡‰ƒ–‹˜‡ †‹˜‹†‡† „› –Š‡ —„‡” ‘ˆ „Žƒ
samples tested.

11.4 Level of detection (LOD50)

Š‡Ž‡˜‡Ž‘ˆ†‡–‡ –‹‘ƒ–ͷͲΨȋ50Ȍ‹•–Š‡ ‘ ‡–”ƒ–‹‘ȋ ˆ—Ȁ–‡•–’‘”–‹‘Ȍˆ‘”™Š‹ Š–Š‡’”‘„ƒ„‹Ž‹–›
‘ˆ†‡–‡ –‹‘‹•ͷͲΨǤ

© ISO 2017 – All rights reserved

Impreso por: Generico
11
 ISO 11290-1:2017(E)

12 Test report
Š‡ –‡•– ”‡’‘”– •ŠƒŽŽ •’‡ ‹ˆ› –Š‡ ‡–Š‘† —•‡† ƒ† –Š‡ ”‡•—Ž–• ‘„–ƒ‹‡†Ǥ – •ŠƒŽŽ ƒŽ•‘ ‡–‹‘ ƒ›
‘’‡”ƒ–‹‰†‡–ƒ‹Ž•‘–•’‡ ‹ϐ‹‡†‹–Š‹•†‘ —‡–ǡ‘””‡‰ƒ”†‡†ƒ•‘’–‹‘ƒŽǡ–‘‰‡–Š‡”™‹–Š†‡–ƒ‹Ž•‘ˆƒ›
‹ ‹†‡–•Ž‹‡Ž›–‘Šƒ˜‡‹ϐŽ—‡ ‡†–Š‡”‡•—Ž–•ȋ•‡‡Clause 10).
Š‡–‡•–”‡’‘”–•ŠƒŽŽ‹ Ž—†‡ƒŽŽ–Š‡‹ˆ‘”ƒ–‹‘‡ ‡••ƒ”›ˆ‘”–Š‡ ‘’Ž‡–‡‹†‡–‹ϐ‹ ƒ–‹‘‘ˆ–Š‡•ƒ’Ž‡Ǥ

13 Quality assurance
Š‡ Žƒ„‘”ƒ–‘”› •Š‘—Ž† Šƒ˜‡ ƒ Ž‡ƒ”Ž› †‡ϐ‹‡† “—ƒŽ‹–› ‘–”‘Ž •›•–‡ –‘ ‡•—”‡ –Šƒ– –Š‡ ‡“—‹’‡–ǡ
reagents and techniques are suitable for the test. The use of positive controls, negative controls and
„Žƒ•ƒ”‡’ƒ”–‘ˆ–Š‡–‡•–Ǥ‡”ˆ‘”ƒ ‡–‡•–‹‰‘ˆ —Ž–—”‡‡†‹ƒ‹••’‡ ‹ϐ‹‡†‹B.5 and described in
ISO 11133.
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

12
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Annex A
(normative)

Diagram of procedure
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Figure A.1 — Diagram of procedure

© ISO 2017 – All rights reserved

Impreso por: Generico
13
 ISO 11290-1:2017(E)

Annex B
(normative)

Composition and preparation of culture media and reagents

B.1 Selective primary enrichment medium: half-Fraser broth

B.1.1 Base

B.1.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 5,0 g

œ›ƒ–‹ †‹‰‡•–‘ˆ ƒ•‡‹ 5,0 g

Meat extract 5,0 g

‡ƒ•–‡š–”ƒ – 5,0 g

Sodium chloride 20,0 g

‹•‘†‹—Š›†”‘‰‡’Š‘•’Šƒ–‡†‹Š›†”ƒ–‡ 12,0 g

‘–ƒ••‹—†‹Š›†”‘‰‡’Š‘•’Šƒ–‡ 1,35 g

Aesculin 1,0 g

Water 1 000 ml
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

B.1.1.2 Preparation

‹••‘Ž˜‡–Š‡„ƒ•‡ ‘’‘‡–•‘”–Š‡†‡Š›†”ƒ–‡† ‘’Ž‡–‡„ƒ•‡‹–Š‡™ƒ–‡”„›Š‡ƒ–‹‰‹ˆ‡ ‡••ƒ”›Ǥ

†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡʹάͲǡʹƒ–ʹͷιǤ
‹•’‡•‡–Š‡„ƒ•‡‹ϐŽƒ••‘ˆ•—‹–ƒ„Ž‡ ƒ’ƒ ‹–›–‘‘„–ƒ‹’‘”–‹‘•ƒ’’”‘’”‹ƒ–‡ˆ‘”–Š‡–‡•–ȋ•‡‡9.1).
Sterilize for 15 min in the autoclave (6.1) at 121 °C.
The lithium chloride solution (B.1.2) and nalidixic acid solution (B.1.3Ȍƒ›„‡ƒ††‡†–‘–Š‡„ƒ•‡ȋB.1.1)
before autoclaving.

B.1.2 Lithium chloride solution

B.1.2.1 Composition

Lithium chloride 3g

Water 10 ml

B.1.2.2 Preparation

Add the lithium chloride to the water.

14
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ
WARNING — Take all necessary precautions when dissolving the lithium chloride in the water
as the reaction is strongly exothermic. Also this solution irritates the mucous membranes.

B.1.3 Solution of sodium salt of nalidixic acid

B.1.3.1 Composition

Sodium salt of nalidixic acid 0,1 g

‘†‹—Š›†”‘š‹†‡ǡͲǡͲͷ‘ŽȀŽ•‘Ž—–‹‘ 10 ml

B.1.3.2 Preparation

‹••‘Ž˜‡–Š‡ƒŽ‹†‹š‹ ƒ ‹†•ƒŽ–‹–Š‡•‘†‹—Š›†”‘š‹†‡Ǥ
–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ

ǤͳǤͶ  ”‹ϐŽƒ˜‹‡Š›†”‘ ŠŽ‘”‹†‡•‘Ž—–‹‘

B.1.4.1 Composition

 ”‹ϐŽƒ˜‹‡Š›†”‘ ŠŽ‘”‹†‡ 0,25 g

Water 100 ml

B.1.4.2 Preparation

‹••‘Ž˜‡–Š‡ƒ ”‹ϐŽƒ˜‹‡Š›†”‘ ŠŽ‘”‹†‡‹–Š‡™ƒ–‡”Ǥ

–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

B.1.5 Ammonium iron(III) citrate solution

B.1.5.1 Composition

Ammonium iron(III) citrate 5,0 g

Water 100 ml

B.1.5.2 Preparation

Dissolve the ammonium iron(III) citrate in the water.

–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ

© ISO 2017 – All rights reserved

Impreso por: Generico
15
 ISO 11290-1:2017(E)

B.1.6 Complete medium

B.1.6.1 Composition

Base (B.1.1) 100 ml

Lithium chloride solution (B.1.2) 1,0 ml

Sodium salt of nalidixic acid solution (B.1.3) 0,1 ml

 ”‹ϐŽƒ˜‹‡Š›†”‘ ŠŽ‘”‹†‡•‘Ž—–‹‘ȋB.1.4) 0,5 ml

Ammonium iron(III) citrate solution (B.1.5) 1,0 ml

B.1.6.2 Preparation

‡†‹ƒ–‡Ž›„‡ˆ‘”‡—•‡ǡƒ††–Š‡ˆ‘—”•‘Ž—–‹‘•ȋB.1.2 to B.1.5) to each 100 ml portion of the base (B.1.1).

B.2 Selective secondary enrichment medium: Fraser broth

B.2.1 Base

B.2.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 5,0 g

œ›ƒ–‹ †‹‰‡•–‘ˆ ƒ•‡‹ 5,0 g

Meat extract 5,0 g

‡ƒ•–‡š–”ƒ – 5,0 g

Sodium chloride 20,0 g

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

‹•‘†‹—Š›†”‘‰‡’Š‘•’Šƒ–‡†‹Š›†”ƒ–‡ 12,0 g

‘–ƒ••‹—†‹Š›†”‘‰‡’Š‘•’Šƒ–‡ 1,35 g

Aesculin 1,0 g

Lithium chloride 3,0 g

Sodium salt of nalidixic acid 0,02 g

Water 1 000 ml

B.2.1.2 Preparation

‹••‘Ž˜‡–Š‡ ‘’‘‡–•‘”–Š‡†‡Š›†”ƒ–‡† ‘’Ž‡–‡‡†‹—‹–Š‡™ƒ–‡”ǡ„›Š‡ƒ–‹‰‹ˆ‡ ‡••ƒ”›Ǥ

†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡʹάͲǡʹƒ–ʹͷιǤ
‹•’‡•‡–Š‡‡†‹—‹–‡•––—„‡•‘ˆ•—‹–ƒ„Ž‡ ƒ’ƒ ‹–›–‘‘„–ƒ‹’‘”–‹‘•ƒ’’”‘’”‹ƒ–‡ˆ‘”–Š‡–‡•–Ǥ
Sterilize for 15 min in the autoclave at 121 °C.

ǤʹǤʹ  ”‹ϐŽƒ˜‹‡Š›†”‘ ŠŽ‘”‹†‡•‘Ž—–‹‘

See B.1.4.

16
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

B.2.3 Ammonium iron(III) citrate solution

See B.1.5.

B.2.4 Complete medium

‡†‹ƒ–‡Ž›„‡ˆ‘”‡—•‡ǡ–‘‡ƒ Š–—„‡ȋͳͲŽ˜‘Ž—‡•Ȍ‘ˆ„ƒ•‡ȋB.2.1) add 0,1 ml portions of solutions B.2.2
and B.2.3Ǥ‹š‰‡–Ž›Ǥ

B.3 Agar Listeria according to Ottaviani and Agosti[16],[17]

B.3.1 Base medium

B.3.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 18 g

œ›ƒ–‹ †‹‰‡•–‘ˆ ƒ•‡‹ 6g

‡ƒ•–‡š–”ƒ – 10 g

‘†‹—’›”—˜ƒ–‡ 2g

Glucose 2g

ƒ‰‡•‹—‰Ž› ‡”‘’Š‘•’Šƒ–‡ 1g

ƒ‰‡•‹—•—Žˆƒ–‡ȋƒŠ›†”‘—•Ȍ 0,5 g

Sodium chloride 5g

Lithium chloride 10 g

‹•‘†‹—Š›†”‘‰‡’Š‘•’Šƒ–‡ȋƒŠ›†”‘—•Ȍ 2,5 g
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

ͷǦ”‘‘ǦͶǦ ŠŽ‘”‘Ǧ͵Ǧ‹†‘Ž›ŽǦéǦĉǦ‰Ž— ‘’›”ƒ‘•‹†‡ 0,05 g

Agar 12 g to 18 g a

Water 930 ml b
a Depending on the gel strength of the agar.
b 925 ml if Amphotericin B solution is used (see B.3.5.2).

B.3.1.2 Preparation

‹••‘Ž˜‡–Š‡†‡Š›†”ƒ–‡† ‘’‘‡–•‘”†‡Š›†”ƒ–‡† ‘’Ž‡–‡„ƒ•‡‹–Š‡™ƒ–‡”„›„‘‹Ž‹‰Ǥ

Sterilize for 15 min in the autoclave at 121 °C.
†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡʹάͲǡʹǤ

B.3.2 Nalidixic acid solution

Nalidixic acid sodium salt 0,02 g

‘†‹—Š›†”‘š‹†‡ȋͲǡͲͷ‘ŽȀŽȌ 5 ml

© ISO 2017 – All rights reserved

Impreso por: Generico
17
 ISO 11290-1:2017(E)

‹••‘Ž˜‡–Š‡ƒŽ‹†‹š‹ ƒ ‹†•‘†‹—•ƒŽ–‹ͷŽ‘ˆ•‘†‹—Š›†”‘š‹†‡ƒ†•–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰Šƒ
ͲǡͶͷρ‡„”ƒ‡Ǥ

B.3.3 Ceftazidime solution

Ceftazidime 0,02 g

Water 5 ml
‹••‘Ž˜‡–Š‡ ‡ˆ–ƒœ‹†‹‡‹ͷŽ‘ˆ™ƒ–‡”ƒ†•–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ

B.3.4 Polymyxin B solution

‘Ž››š‹•—Žˆƒ–‡ 76 700 IU

Water 5 ml
‹••‘Ž˜‡–Š‡’‘Ž››š‹‹ͷŽ‘ˆ™ƒ–‡”Ǥ–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ

B.3.5 Antibiotic supplement

B.3.5.1 Cycloheximide solution

› Ž‘Š‡š‹‹†‡ 0,05 g

Ethanol 2,5 ml

Water 2,5 ml
‹••‘Ž˜‡–Š‡ › Ž‘Š‡š‹‹†‡‹ʹǡͷŽ‘ˆ‡–Šƒ‘Ž–Š‡ƒ††ʹǡͷŽ‘ˆ™ƒ–‡”Ǥ–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰Š
ƒͲǡͶͷρ‡„”ƒ‡Ǥ

B.3.5.2 Amphotericin B solution ȋƒ•ƒƒŽ–‡”ƒ–‹˜‡–‘ › Ž‘Š‡š‹‹†‡•‘Ž—–‹‘Ȍ

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Amphotericin B 0,01 g

HCl (1 mol/l) 2,5 ml

‹‡–Š›Žˆ‘”ƒ‹†‡ȋ Ȍ 7,5 ml
‹••‘Ž˜‡–Š‡ƒ’Š‘–‡”‹ ‹‹–Š‡ ŽȀ •‘Ž—–‹‘Ǥ–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ
–Š‡”–‡ Š‹“—‡•‘ˆ†‹••‘Ž—–‹‘ȋ‡Ǥ‰Ǥ‹™ƒ–‡”‘”‹Š‘–‡†‹—„ƒ•‡Ȍƒ›„‡’‡”ˆ‘”‡†ƒ ‘”†‹‰–‘
media suppliers.
WARNING — The HCl/DMF solution is toxic, handle with care.

B.3.6 Supplement
Dissolve 2 g of đ-ȽǦ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž1) in 50 ml of water.
ʹ ‰ ‘ˆ •‘› Ž‡ ‹–Š‹ ‘–ƒ‹‹‰ ƒ– Ž‡ƒ•– ͻ Ψ –‘ ͳͷ Ψ —ˆ”ƒ –‹‘ƒ–‡† ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž ƒ› „‡ —•‡†
instead of L-ȽǦ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘ŽǤ[6],[9],[19]
Stir for about 30 min until a homogeneous suspension is obtained. Autoclave at 121 °C for 15 min and
cool to 47 °C to 50 °C.

1) P 6636®•—’’Ž‹‡†„›‹‰ƒ‹•ƒ‡šƒ’Ž‡‘ˆƒ•—‹–ƒ„Ž‡’”‘†— –ƒ˜ƒ‹Žƒ„Ž‡ ‘‡” ‹ƒŽŽ›ǤŠ‹•‹ˆ‘”ƒ–‹‘‹•

‰‹˜‡ˆ‘”–Š‡ ‘˜‡‹‡ ‡‘ˆ—•‡”•‘ˆ–Š‹•†‘ —‡–ƒ††‘‡•‘– ‘•–‹–—–‡ƒ‡†‘”•‡‡–„› ‘ˆ–Š‹•’”‘†— –Ǥ

18
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

B.3.7 Complete medium

B.3.7.1 Composition

Base medium (B.3.1) 930 ml a

Nalidixic acid solution (B.3.2) 5 ml

Ceftazidime solution (B.3.3) 5 ml

‘Ž››š‹•‘Ž—–‹‘ȋB.3.4) 5 ml

› Ž‘Š‡š‹‹†‡•‘Ž—–‹‘ȋB.3.5.1) 5 ml
or Amphotericin B solution (B.3.5.2) 10 ml

Supplement (B.3.6) 50 ml
a 925 ml if Amphotericin B solution is used.

B.3.7.2 Preparation

Add the solutions to the molten base at 47 °C to 50 °C (6.4Ȍǡ‹š‹‰–Š‘”‘—‰ŠŽ›„‡–™‡‡‡ƒ Šƒ††‹–‹‘Ǥ

Š‡’ ‘ˆ–Š‡ ‘’Ž‡–‡‡†‹—•ŠƒŽŽ„‡͹ǡʹάͲǡʹƒ–ʹͷιǤ
Š‡‡†‹—•ŠƒŽŽ„‡Š‘‘‰‡‡‘—•Ž›•Ž‹‰Š–Ž›‘’ƒŽ‡• ‡–Ǥ

B.3.7.3 Preparation of agar plates

Žƒ ‡‹‡ƒ Š‡–”‹†‹•ŠͳͺŽ–‘ʹͲŽ‘ˆ–Š‡ˆ”‡•ŠŽ›’”‡’ƒ”‡† ‘’Ž‡–‡‡†‹—ǡ–Š‡ƒŽŽ‘™–‘•‘Ž‹†‹ˆ›Ǥ

B.4 Second selective solid plating-out medium

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Š‡ Š‘‹ ‡ ‘ˆ –Š‡ •‡ ‘† ‡†‹— ‹• Ž‡ˆ– –‘ –Š‡ †‹• ”‡–‹‘ ‘ˆ –Š‡ –‡•–‹‰ Žƒ„‘”ƒ–‘”›Ǥ ˆ ƒ ‘‡” ‹ƒŽ
medium is used, follow the manufacturer’s instructions regarding its preparation and use.

B.5 Performance testing for the quality assurance of the culture media
Performance testing of the culture media shall be carried out in accordance with ISO 11133, which
‹ Ž—†‡•†‡ϐ‹‹–‹‘•ˆ‘”’”‘†— –‹˜‹–›ǡ•‡Ž‡ –‹˜‹–›ƒ†•’‡ ‹ϐ‹ ‹–›ǤTable B.1 gives details of control strains
–‘„‡—•‡†ˆ‘”’‡”ˆ‘”ƒ ‡–‡•–‹‰‘ˆ —Ž–—”‡‡†‹ƒ•’‡ ‹ϐ‹‡†‹–Š‹•†‘ —‡–Ǥ
For the second isolation medium, use performance testing given in ISO 11133 or according to the
ƒ—ˆƒ –—”‡”ǯ••’‡ ‹ϐ‹ ƒ–‹‘•Ǥ
 ‘”’”‘†— –‹˜‹–›–‡•–‹‰ǡ‹–Š‡ ƒ•‡‘ˆ†‡–‡ –‹‘‘ˆListeria spp., it could be informative to add another
control strain (from another Listeria species).

© ISO 2017 – All rights reserved

Impreso por: Generico
19
 ISO 11290-1:2017(E)

Table B.1 — Performance testing of culture media for Listeria monocytogenes

Characteristic
WDCM Reference Method of reactions of
Mediaa Function Incubation Control strains Criteria
numbersc media control target micro-
organism
Half- Productiv- Listeria
Fraser ‹–› monocy- 00021b
togenes 4b
+ Escherichia 00012
colid or 00013 > 10
colonies
+ Enterococcus 00009 on
faecalisd or 00087 Blue green
Agar
— Qualitative colonies with
Listeria Listeria ac-
opaque halo
monocytogenes 00109 cording to
1/2a Ottaviani
ȋʹͷάͳȌŠȀ
and Agosti
ȋ͵ͲάͳȌι + Escherichia 00012
colid or 00013
+ Enterococcus 00009
faecalisd or 00087
‡Ž‡ –‹˜‹–› Total inhi-
Escherichia 00012
— Qualitative bition (0) —
colid or 00013
on TSA
< 100
Enterococcus 00009
— Qualitative colonies on —
faecalisd or 00087
TSA
Fraser Productiv- Listeria
‹–› monocy- 00021b
togenes 4b
+ Escheri- 00012
chia colid or 00013 > 10
+ Enterococcus 00009 colonies on
faecalisd or 00087 Agar Lis- Blue green
— Qualitative teria ac- colonies with
Listeria cording to opaque halo
monocytogenes 00109
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Ottaviani
ȋʹͶάʹȌŠȀ 1/2a and Agosti
ȋ͵͹άͳȌι + Escherichia 00012
colid or 00013
+ Enterococcus 00009
faecalisd or 00087
‡Ž‡ –‹˜‹–› Total inhi-
Escherichia 00012
— Qualitative bition (0) —
colid or 00013
on TSA
< 100
Enterococcus 00009
— Qualitative colonies on —
faecalisd or 00087
TSA
a Full names of media abbreviated terms.
b Strains to be used as a minimum.
c Refer to the reference strain catalogue available at www.wfcc .info for information on culture collection strain numbers and contact
details.
d Strain free of choice; one of the strains has to be used as a minimum.

20
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Table B.1 (continued)

Characteristic
WDCM Reference Method of reactions of
Mediaa Function Incubation Control strains Criteria
numbersc media control target micro-
organism
Agar Productiv- Listeria
Listeria ac- ‹–› monocy- 00021b
cording to togenes 4b Blue green
Good
Ottaviani — Qualitative colonies with
Listeria growth (2)
and Agosti opaque halo
monocytogenes 00109
1/2a
ȋͶͺάͶȌŠȀ
‡Ž‡ –‹˜‹–› 00012
ȋ͵͹άͳȌι Escherichia colid
or 00013 Total inhi-
— Qualitative —
Enterococcus 00009 bition (0)
faecalisd or 00087
’‡ ‹ϐ‹ ‹–› Blue green col-
Listeria innocua 00017 — Qualitative — onies without
opaque halo
a Full names of media abbreviated terms.
b Strains to be used as a minimum.
c Refer to the reference strain catalogue available at www.wfcc .info for information on culture collection strain numbers and contact
details.
d Strain free of choice; one of the strains has to be used as a minimum.

B.6 Hydrogen peroxide solution

•‡ƒƒ••ˆ”ƒ –‹‘‘ˆ͵Ψǡ‹Ǥ‡ǤͳͲ˜‘Ž—‡•‘Ž—–‹‘Ǥ

B.7 Motility agar

B.7.1 Composition
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

œ›ƒ–‹ †‹‰‡•–‘ˆ ƒ•‡‹ 20,0 g

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 6,1 g

Agar 3,5 g

Water 1 000 ml

B.7.2 Preparation
‹••‘Ž˜‡–Š‡ ‘’‘‡–•‹–Š‡™ƒ–‡”„›„‘‹Ž‹‰Ǥ
†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡ͵άͲǡʹƒ–ʹͷιǤ
Dispense the medium into tubes in quantities of about 5 ml.
Sterilize for 15 min in the autoclave at 121 °C.

© ISO 2017 – All rights reserved

Impreso por: Generico
21
 ISO 11290-1:2017(E)

B.8 Blood agar

B.8.1 Base

B.8.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 15 g

Liver digest 2,5 g

‡ƒ•–‡š–”ƒ – 5g

Sodium chloride 5g

Agar 9 g to 18 g a

Water 1 000 ml
a Depending on the gel strength of the agar.

B.8.1.2 Preparation

‹••‘Ž˜‡–Š‡ ‘’‘‡–•‹–Š‡™ƒ–‡”„›„‘‹Ž‹‰Ǥ
†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡʹάͲǡʹƒ–ʹͷιǤ
‹•’‡•‡–Š‡‡†‹—‹–‘ϐŽƒ••‘ˆ•—‹–ƒ„Ž‡ ƒ’ƒ ‹–›–‘‘„–ƒ‹’‘”–‹‘•ƒ’’”‘’”‹ƒ–‡ˆ‘”–Š‡–‡•–Ǥ
Sterilize for 15 min in the autoclave at 121 °C.

ǤͺǤʹ ‡ϐ‹„”‹ƒ–‡†„Ž‘‘†ȋ•Š‡‡’ǡ ƒŽˆ‘”„‘˜‹‡Ȍ

B.8.3 Complete medium

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

B.8.3.1 Composition

Base (B.8.1) 100 ml

‡ϐ‹„”‹ƒ–‡†„Ž‘‘†ȋB.8.2) 5 ml to 7 ml

B.8.3.2 Preparation

††–Š‡„Ž‘‘†–‘–Š‡„ƒ•‡’”‡˜‹‘—•Ž› ‘‘Ž‡†ƒ–Ͷ͹ι–‘ͷͲιȋ6.4). Mix well.

‹•’‡•‡–Š‡‡†‹—‹–‘•–‡”‹Ž‡‡–”‹†‹•Š‡•‹’‘”–‹‘•ƒ’’”‘’”‹ƒ–‡ˆ‘”–Š‡–‡•–ǤŽŽ‘™–‘•‘Ž‹†‹ˆ›Ǥ

B.9 Phosphate-buffered saline (PBS)

B.9.1 Composition

‹•‘†‹—Š›†”‘‰‡’Š‘•’Šƒ–‡†‹Š›†”ƒ–‡ 8,98 g

‘†‹—†‹Š›†”‘‰‡’Š‘•’Šƒ–‡ 2,71 g

Sodium chloride 8,5 g

Water 1 000 ml

22
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

B.9.2 Preparation
Dissolve the components in the water.
†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡʹάͲǡʹƒ–ʹͷιǤ
Sterilize in the autoclave for 15 min at 121 °C.

B.10 Red blood corpuscle suspension

ƒ‹–ƒ‹–Š‡”‡†„Ž‘‘† ‘”’—• Ž‡•ƒ–ͷιάʹ鄇ˆ‘”‡—•‡Ǥ
‡ˆ‘”‡—•‡ǡ‡šƒ‹‡ˆ‘”•‹‰•‘ˆŠƒ‡‘Ž›•‹•ȋ”‡††‡‹‰Ȍ‹–Š‡–‘’Žƒ›‡”‘ˆ–Š‡•‡”—Ǥ ˆ‘Šƒ‡‘Ž›•‹•
Šƒ•‘ —””‡†ǡ‹–”‘†— ‡ʹŽ‘ˆ–Š‡„Ž‘‘† ‘”’—• Ž‡•ˆ”‘–Š‡„‘––‘Žƒ›‡”‹–‘ͻͺŽ‘ˆ„—ˆˆ‡”ȋB.9).
ˆŠƒ‡‘Ž›•‹•Šƒ•‘ —””‡†ǡ•—•’‡†ƒ’’”‘š‹ƒ–‡Ž›ͶŽ‘ˆ–Š‡„Ž‘‘† ‘”’—• Ž‡•Žƒ›‡”‹–‘ͳͲŽ‘ˆ
„—ˆˆ‡”ƒ†‹š‰‡–Ž›ǡ–Š‡ ‡–”‹ˆ—‰‡Ǥ ˆ–Š‡•—’‡”ƒ–ƒ–Ž‹“—‹† Ž‡ƒ”Ž›„‡ ‘‡•”‡†ǡ†—‡–‘•‹‰‹ϐ‹ ƒ–
Šƒ‡‘Ž›•‹•ǡ†‘‘–—•‡–Š‡•–‘ •—•’‡•‹‘ƒ††‹• ƒ”†‹–Ǥ–Š‡”™‹•‡ǡ†‡ ƒ––Š‡•—’‡”ƒ–ƒ–Ž‹“—‹†
ƒ†ƒ††ʹŽ‘ˆ–Š‹•„Ž‘‘† ‘”’—• Ž‡•‘Ž—–‹‘–‘ͻͺŽ‘ˆ„—ˆˆ‡”Ǥ‡‡’–Š‡•—•’‡•‹‘ˆ‘”ϐ‹˜‡†ƒ›•ƒ–
ͷιάʹιǤ‹• ƒ”†‹–‹ˆŠƒ‡‘Ž›•‹•‘ —”•Ǥ

B.11 CAMP (Christie, Atkins, Munch-Petersen) medium and test strains

B.11.1 General
Blood agar plates (B.8Ȍƒ›„‡—•‡†ˆ‘”–Š‹•–‡•–ǡ„—–‹–‹•’”‡ˆ‡”ƒ„Ž‡–‘—•‡†‘—„Ž‡ǦŽƒ›‡”‡†ƒ‰ƒ”’Žƒ–‡•
™‹–Šƒ˜‡”›–Š‹„Ž‘‘†Žƒ›‡”ȋB.11.4).

B.11.2 Base
See B.8.1.

B.11.3 Blood medium

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

See B.8.3.

B.11.4 Complete medium

Dispense the base (B.11.2Ȍ‹–‘•–‡”‹Ž‡‡–”‹†‹•Š‡•‹“—ƒ–‹–‹‡•‘ˆƒ„‘—–ͳͲŽƒ†ƒŽŽ‘™–‘•‘Ž‹†‹ˆ›Ǥ
‘—”ƒ˜‡”›–Š‹Žƒ›‡”‘ˆ–Š‡„Ž‘‘†‡†‹—ȋB.11.3) using amounts not greater than 3 ml per plate.
ŽŽ‘™–‘•‘Ž‹†‹ˆ›Ǥ ˆ–Š‡„Ž‘‘†‡†‹—‹•ƒ††‡†–‘†‹•Š‡• ‘–ƒ‹‹‰–Š‡„ƒ•‡™Š‹ ŠŠƒ˜‡„‡‡’”‡’ƒ”‡†
‹ ƒ†˜ƒ ‡ǡ ‹– ƒ› „‡ ‡ ‡••ƒ”› –‘ ™ƒ” –Š‡ †‹•Š‡• ˆ‘” ʹͲ ‹ „› ’Žƒ ‹‰ –Š‡ ‹ ƒ ‹ —„ƒ–‘” •‡–
ƒ–͵͹鄇ˆ‘”‡’‘—”‹‰–Š‡„Ž‘‘†‡†‹—Žƒ›‡”Ǥ

© ISO 2017 – All rights reserved

Impreso por: Generico
23
 ISO 11290-1:2017(E)

B.12 Carbohydrate utilization broth (L-Rhamnose and D-Xylose)

B.12.1 Base

B.12.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 10 g

Meat extract 1g

Sodium chloride 5g

Bromocresol purple 0,02 g

Water 1 000 ml

B.12.1.2 Preparation

‹••‘Ž˜‡–Š‡ ‘’‘‡–•‹–Š‡™ƒ–‡”ǡ„›Š‡ƒ–‹‰‹ˆ‡ ‡••ƒ”›Ǥ

†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͸ǡͺάͲǡʹƒ–ʹͷιǤ
‹•’‡•‡–Š‡‡†‹—‹–‘–—„‡•‘ˆ•—‹–ƒ„Ž‡ ƒ’ƒ ‹–›–‘‘„–ƒ‹’‘”–‹‘•ƒ’’”‘’”‹ƒ–‡ˆ‘”–Š‡–‡•–Ǥ
Sterilize for 15 min in the autoclave at 121 °C.

B.12.2 Carbohydrate solutions

B.12.2.1 Composition

ƒ”„‘Š›†”ƒ–‡a 5g

Water 100 ml
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

aǦŠƒ‘•‡‘”Ǧ›Ž‘•‡Ǥ

B.12.2.2 Preparation

‹••‘Ž˜‡–Š‡ ‘’‘‡–•‹–Š‡™ƒ–‡”ǡƒ†•–‡”‹Ž‹œ‡„›ϐ‹Ž–”ƒ–‹‘–Š”‘—‰ŠƒͲǡͶͷρ‡„”ƒ‡Ǥ

B.12.3 Complete medium

‘”‡ƒ Š ƒ”„‘Š›†”ƒ–‡ǡƒ††ƒ•‡’–‹ ƒŽŽ›x ml of solution B.12.2 to 9x ml of the base (B.12.1).

B.13 Reagents for Voges-Proskauer (VP) Reaction

B.13.1 VP medium

B.13.1.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆƒ‹ƒŽ–‹••—‡• 7g

Sodium chloride 5g

Glucose 5g

Water 1 000 ml

24
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

B.13.1.2 Preparation

‹••‘Ž˜‡–Š‡ ‘’‘‡–•‹–Š‡™ƒ–‡”ǡ„›Š‡ƒ–‹‰‹ˆ‡ ‡••ƒ”›Ǥ

†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͸ǡͻάͲǡʹƒ–ʹͷιǤ
Dispense the medium into tubes in quantities of 3 ml.
Sterilize for 15 min in the autoclave at 121 °C.
 ‘‡” ‹ƒŽŽ›ƒ˜ƒ‹Žƒ„Ž‡’”‡Ǧ’”‡’ƒ”‡†ˆ‘”—Žƒ–‹‘•™‹–Šƒ’ ™‹–Š‹Ͳǡͷ’ —‹–‘ˆ͸ǡͻƒ”‡ƒ ‡’–ƒ„Ž‡Ǥ

B.13.2 Ƚ-Naphthol, ethanolic solution

B.13.2.1 Composition

Ƚ-Naphthol 5g

–Šƒ‘Žǡͻ͸Ψȋ˜‘Ž—‡ˆ”ƒ –‹‘Ȍ 100 ml

B.13.2.2 Preparation

Dissolve the Ƚ-naphthol in the ethanol.

WARNING — The Ƚ-naphthol is toxic, handle with care.

B.13.3 Potassium hydroxide solution

B.13.3.1 Composition

‘–ƒ••‹—Š›†”‘š‹†‡ 40 g

Water 100 ml
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

B.13.3.2 Preparation

‹••‘Ž˜‡–Š‡’‘–ƒ••‹—Š›†”‘š‹†‡‹–Š‡™ƒ–‡”

B.14 Tryptone soya yeast extract agar (TSYEA)

B.14.1 Composition

œ›ƒ–‹ †‹‰‡•–‘ˆ ƒ•‡‹ 17 g

ƒ’ƒ‹ †‹‰‡•–‘ˆ•‘›ƒ„‡ƒ‡ƒŽ 3g

‡ƒ•–‡š–”ƒ – 6g

Glucose 2,5 g

Sodium chloride 5g

‹’‘–ƒ••‹—Š›†”‘‰‡’Š‘•’Šƒ–‡ 2,5 g

© ISO 2017 – All rights reserved

Impreso por: Generico
25
 ISO 11290-1:2017(E)

Agar 12 g to 18 g a

Water 1 000 ml
a Depending on the gel strength of the agar.

B.14.2 Preparation
‹••‘Ž˜‡–Š‡†‡Š›†”ƒ–‡† ‘’‘‡–•‘”†‡Š›†”ƒ–‡† ‘’Ž‡–‡‡†‹—‹–Š‡™ƒ–‡”„›„‘‹Ž‹‰Ǥ
†Œ—•––Š‡’ ǡ‹ˆ‡ ‡••ƒ”›ǡ•‘–Šƒ–ƒˆ–‡”•–‡”‹Ž‹œƒ–‹‘‹–‹•͹ǡ͵άͲǡʹƒ–ʹͷιǤ
Sterilize for 15 min in the autoclave at 121 °C.

B.14.2.1 Preparation of agar plates

Žƒ ‡‹‡ƒ Š‡–”‹†‹•ŠͳͺŽ–‘ʹͲŽ‘ˆ–Š‡ˆ”‡•ŠŽ›’”‡’ƒ”‡† ‘’Ž‡–‡‡†‹—ǡ–Š‡ƒŽŽ‘™–‘•‘Ž‹†‹ˆ›Ǥ

Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

26
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Annex C
(informative)

Distinction of Listeria spp. from other genera

Table C.1 — Distinction of Listeria spp. from other bacterial genera or species

Gram Motility
Catalase VP test Growth at 37°C
appearance (20°C to 25°C)
Listeria spp. Gpb/Gpcb + + + +
Large Gpbb
spore bearer
Bacillus spp. a + V V +
ȋ›‘—‰
culture)
Carnobacterium spp. a Gpb - - - +
Staphylococcus spp. a Gpc + - V +
Streptococcus spp. a Gpc - - V +
- -
Lactobacillus spp. Gpb - +
(occasional +) (occasional +)
Brochothrix spp. Gpb + - + -
Kurthia spp. Gpb + + - +
Erysipelothrix spp. Gpb - - - +
-
Corynebacterium spp. Gpb + - +
(occasional +)
Enterococcus spp. a Gpc - - + +
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Cellulosimicrobium
Gpcb + + - +
funkei a
+
Kocuria kristinae a Gpc + - +
(occasional -)
Marinilactibacillus
Gpb + + + +
psychrotolerans a,c
Rothia terrae a Gpc + - + +
a Known to grow on Agar Listeria according to Ottaviani and Agosti and other chromogenic media and sometimes
forming blue or bluish colonies.
b Bacillus oleronius†‹•’Žƒ›•ƒ˜ƒ”‹ƒ„Ž‡”‡ƒ –‹‘–‘–Š‡ ”ƒ•–ƒ‹Ǣ‘•– ‡ŽŽ•ƒ’’‡ƒ”ƒ• ”ƒǦ‡‰ƒ–‹˜‡Ǥ[23]
c Distinction of M. psychrotolerans from Listeria •’’Ǥ ƒ „‡ ƒ‹†‡† „› ‹–• †‹ˆˆ‡”‡– ‘Ž‘› Šƒ”ƒ –‡”‹•–‹ • ‘ 
ȋ‘’ƒ“—‡ǡ’ƒŽ‡Ǧ›‡ŽŽ‘™‹•ŠǡŽ‡–‹ —Žƒ” ‘Ž‘‹‡•ǡʹ–‘͵‹†‹ƒ‡–‡”ƒˆ–‡”ʹͶŠ‘ˆ‹ —„ƒ–‹‘ƒ–͵͹ιȌǤ[23]
Gpb: Gram positive bacillus
Gpcb: Gram positive coccobacillus
Gpc: Gram positive coccus or coccoid
V: variable reaction
NOTE 1 Some new Listeria•’‡ ‹‡•”‡ ‡–Ž›‹•‘Žƒ–‡†ƒ›‘– ‘””‡•’‘†–‘–Š‹•• Š‡‡‹’ƒ”–‹ —Žƒ”ˆ‘”‘–‹Ž‹–›ǡ–‡•–
and growth at 37 °C (see, for example, References [5], [10], [12], [14], [25], [26] and [27]).
NOTE 2 Some rare Listeria strains are slow or not catalase positive.

© ISO 2017 – All rights reserved

Impreso por: Generico
27
 ISO 11290-1:2017(E)

Annex D
(informative)

‡ƒ –‹‘•ˆ‘”–Š‡‹†‡–‹ϐ‹ ƒ–‹‘‘ˆListeria species

Table D.1 — Main tests prescribed in this document (see 9.5)

Production of acid CAMP test
ȾǦ
Species PI-PLC L-Rham-
haemolysis D-Xylose Mannitol S. aureus R. equi
nose
L. monocytogenes + (24 h) + + - - + -
L. innocua - - V - - - -
L. ivanovii + (24 h to + - + - - +
48 h)
L. seeligeri - (+) - + - (+) -
L. welshimeri - – V + - - -
L. grayi - - V - + - -
ǤϔŽ‡‹• Šƒ‹‹ - _ + + + - -
L. marthii - - - - + - -
L. rocourtiae - - + + + - -
L. weihenstephanensis - - + + - - -
 Ǧǣ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž’Š‘•’Š‘Ž‹’ƒ•‡
V: variable reaction
(+): weak reaction
Ϊ ǣ‘”‡–ŠƒͻͲΨ‘ˆ’‘•‹–‹˜‡”‡ƒ –‹‘•
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

-: no reaction
NOTE 1 There exist rare strains of L. monocytogenes which do not show ȾǦŠƒ‡‘Ž›•‹•‘”ƒ’‘•‹–‹˜‡”‡ƒ –‹‘–‘–Š‡
test under the conditions described in this document.
NOTE 2 Some strains of L. seeligeriƒ›‰‹˜‡–Š‡•ƒ‡Šƒ‡‘Ž›•‹•”‡•—Ž–•ƒ•L. monocytogenes.
NOTE 3 Some new Listeria •’‡ ‹‡• Šƒ˜‡ „‡‡ ”‡ ‡–Ž› ‹•‘Žƒ–‡†Ǥ ††‹–‹‘ƒŽ –‡•–• ƒ› „‡ ‡ ‡••ƒ”› –‘ ‹†‡–‹ˆ› –Š‡
ƒ —”ƒ–‡Ž›.[5],[10],[12],[14],[25],[26],[27]
 Ͷ   ‘‡ ƒ–›’‹ ƒŽ L. innocua •–”ƒ‹• •Š‘™‡† ’Š‡‘–›’‹  Šƒ”ƒ –‡”‹•–‹ • ‘ˆ L. monocytogenes ƒ† ‘—Ž† „‡ ‘Ž›
†‹•–‹‰—‹•Š‡†„›ƒ‡ˆ‡”‡ ‡ƒ„‘”ƒ–‘”›Ǥ[11]
NOTE 5 There exist rare strains of L. monocytogenes which do not ferment L-Rhamnose.[15],[18]

28
Impreso por: Generico
© ISO 2017 – All rights reserved
 Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

ƒ„Ž‡ǤʹȄ††‹–‹‘ƒŽ”‡ƒ –‹‘•ˆ‘”–Š‡‹†‡–‹ϐ‹ ƒ–‹‘‘ˆListeria speciese

Esculin CAMP test
Motility ȾǦŠƒ‡‘Ž-

Impreso por: Generico

Genus Species Catalase ȋȾǦ PI-PLC a Mannitol Rhodococ- Staphylococcus DIM a MAN DAR XYL RHA MDG RIB G1P TAG
(22–30°C) ysis
glucosidase) cus equi aureus
ǤϔŽ‡‹• Šƒ‹‹ + - + - + - - - - - + + + + - - -
subsp.
coloradensis
ǤϔŽ‡‹• Šƒ‹‹ + - + - + - - - - - + + + + - - -
subsp. ϔŽ‡‹•-
chmannii

© ISO 2017 – All rights reserved

L. grayi biovar + + + - + - - - + + + - (-) Vb + - -
grayi c
L. grayi biovar + + + - + - - - + + + - + V + - -
murrayi c
L. innocua + + + - - - - - + + + - V + - - -
L. ivanovii subsp. + + + + - + + - V - + + - + + V -
ivanovii
(24 h to
48 h)
L. ivanovii subsp. + + + + - + + - V - + + - + - V -
londoniensis
(24 h to
48 h)
L. marthii + + + - - - - - - + + - - + - - -
L. monocytogenes + + + + - + - + - + + - +d + - - -
(24 h) (24 h to
72 h)
L. rocourtiae + + + - + - - - - + - + + + + - -
weak
L. seeligeri + + + - - + - + + - + + - + - - -
L. weihen- + + + - - - - - - - + + + + + - -
stephanensis
weak
L. welshimeri + + + - - - - - (+) + + + V + - - +
aǦǣ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž’Š‘•’Š‘Ž‹’ƒ•‡Ǣ ǣǦƒ”›Žƒ‹†ƒ•‡ǢǣƒŽ’ŠƒǦƒ‘•‹†ƒ•‡ǢǣǦƒ”ƒ„‹–‘ŽǢǣǦš›Ž‘•‡Ǣ ǣǦ”Šƒ‘•‡Ǣ ǣƒŽ’ŠƒǦ±–Š›ŽǦǦ‰Ž— ‘•‹†‡Ǣ ǣǦ”‹„‘•‡Ǣ
GIP: glucose-I-phosphate; TAG: D-Tagatose.
bǣ˜ƒ”‹ƒ„Ž‡ǢΪ‘”ǦǣƒŒ‘”‹–›‘ˆ•–”ƒ‹•ƒ”‡’‘•‹–‹˜‡‘”‡‰ƒ–‹˜‡ǢȋΪȌ‘”ȋǦȌǣͻͲΨ‘ˆ’‘•‹–‹˜‡‘”‡‰ƒ–‹˜‡”‡ƒ –‹‘•Ǥ
c grayi biovar is negative for Nitrate and Turanose though murrayi biovar is positive for Nitrate and Turanose.
d There exist rare rhamnose negative strains, that belong to lineage IIIB/IIIC.
e Source: National Reference Centre/WHO Collaborative Centre for Listeria, Institut Pasteur, Paris.
ISO 11290-1:2017(E)

29
 ISO 11290-1:2017(E)

Annex E
(informative)

Listeria selective agars

Detection of Listeria •’’Ǥ ”‡Ž‹‡• ‘ Ž‡ƒ˜ƒ‰‡ ‘ˆ Ǧ ȾǦǦ‰Ž— ‘•‹†‡ ‘Œ—‰ƒ–‡• „› ȾǦǦ‰Ž— ‘•‹†ƒ•‡ǡ ƒ
‡œ›‡ –Šƒ– ‹• ‘‘ –‘ ƒŽŽ Listeria spp. However, detection of L. monocytogenes and pathogenic
L. ivanovii ‹• „ƒ•‡† ‘ –Š‡ †‡–‡ –‹‘ ‘ˆ Ž‡ ‹–Š‹ƒ•‡ ȋ’Š‘•’Šƒ–‹†›Ž Š‘Ž‹‡ ’Š‘•’Š‘Ž‹’ƒ•‡  Ǧ Ȍ
ƒ†Ȁ‘”’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Ž’Š‘•’Š‘Ž‹’ƒ•‡ȋ Ȍƒ –‹˜‹–›Ǥ

Table E.1 — Non commercial Listeria selective agars (extracted from Reference [7])
Polymyxin
Lithium Indicator
(P) or  ”‹ϐŽƒ˜‹‡ Other References
chloride system a
Colistin (C)
(g/l) (mg/l) (mg/l) (mg/l)
Agars which do not differentiate between Listeria spp.
Harlequin 15 C10 2,5 ‘•ˆ‘› ‹ͷǡ CHEg + Fe Smith et al.,
listeria 2000
Cefotetan 1,
medium
› Ž‘Š‡š‹‹†‡ʹͲͲ
Lithium chlo- 5 0 0 Moxalactam 20, ‡”› Lee and
”‹†‡’Š‡›Ž‡–Š- McCain,
Ž› ‡”‹ƒŠ›†”‹†‡ͳͲǡ
anol moxa- 1986
lactam (LPM) Š‡›Ž‡–Šƒ‘ŽʹͷͲͲ
Oxford 15 C20 5 ‘•ˆ‘› ‹ͳͲǡ Ae + Fe Curtis et al.,
1989
Cefotetan 2
› Ž‘Š‡š‹‹†‡ͶͲͲͻ
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

‘†‹ϐ‹‡† 12 C10 0 Ceftazidime 20 Ae + Fe Cook, 1998

šˆ‘”†ȋȌ
PALCAM 10 P10 5 Ceftazidime 30 Ae + Fe Van Netten
et al., 1989
Mann + PR
Agars which differentiate species by haemolysis
Enhanced 10 P10 5 Ceftazidime 30 MUG Cox et al.,
Šƒ‡‘Ž›•‹• 1991 b
Sheep
agar (EHA)
blood
Listeria mono- 10 P10 0 Ceftazidime 20 Sheep Johansson,
cytogenes blood blood 1998
agar (LMBA)
‰ƒ”•™Š‹ Šƒ”‡•’‡ ‹ϐ‹ ˆ‘”’ƒ–Š‘‰‡‹ Listeria spp.
Agar Listeria 10 ‘Ž››š‹ǣ Amphotericin B 10, Chrom Ottaviani et
according to 76700 U, in al., 1997
Nalidixic acid, PCPLC
Ottaviani and column other,
Agosti add: Ceftazidime 20
a ‡ǣ ƒ‡• —Ž‹Ǣ  ‰ǣ  Ǧ‰Ž— ‘•‹†‡Ǣ Š”‘ǣ Š”‘‘‰‡‹  •—„•–”ƒ–‡Ǣ ‡ǣ ”‘ •ƒŽ–Ǣ ‡”›ǣ ‡”›ǯ• ȋͳͻͻ͵ȌǤ ‡–Š‘† ‘ˆ
‹ŽŽ—‹ƒ–‹‘Ǣƒǣƒ‹–‘ŽǢ ǣͶǦ‡–Š›Ž—„‡ŽŽ‹ˆ‡”›ŽǦéǦǦ‰Ž— ‘•‹†‡ǢǣŠ‡‘Ž”‡†

30
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Annex F
(informative)

Results of the interlaboratory studies for detection of Listeria

monocytogenes

F.1 Results of interlaboratory studies

–‡”ƒ–‹‘ƒŽ ‹–‡”Žƒ„‘”ƒ–‘”› •–—†‹‡• ˆ”‘ ƒ –‘–ƒŽ ‘ˆ ͳ͹ ‘—–”‹‡• ™‡”‡ ƒ””‹‡† ‘—–Ǥ Š‡ ˆ‘ŽŽ‘™‹‰
ˆ‘‘† –›’‡• ™‡”‡ —•‡† ‹ –Š‡ •–—†‹‡•ǣ ‘Ž†Ǧ•‘‡† •ƒŽ‘ǡ ‡˜‹”‘‡–ƒŽ •ƒ’Ž‡•ǡ ”‡ƒ†›Ǧ–‘Ǧ‡ƒ–
salads, powdered infant food formula and cheese. The food samples were each tested at two different
Ž‡˜‡Ž• ‘ˆ ‘–ƒ‹ƒ–‹‘ǡ ’Ž—• ƒ ‡‰ƒ–‹˜‡ ‘–”‘ŽǤ ƒ’Ž‡• ™‡”‡ ƒ”–‹ϐ‹ ‹ƒŽŽ› ‘–ƒ‹ƒ–‡† „‘–Š „›
L. monocytogenes and L. innocuaˆ‘”‡˜‹”‘‡–ƒŽ•ƒ’Ž‡•ƒ† Š‡‡•‡ǡƒ†„‘–Š„›L. monocytogenes
and L. welshimeriˆ‘” ‘Ž†Ǧ•‘‡†•ƒŽ‘™Š‹ Š ‘—Ž†ƒŽ–‡”•‡•‹–‹˜‹–›Ǥ ‘”‡˜‹”‘‡–ƒŽ•ƒ’Ž‡•ǡƒ
mixture of Staphylococcus epidermidis, Bacillus cereus and Pseudomonas fragi was added to each sample.
‘™†‡”‡†‹ˆƒ–ˆ‘‘†ˆ‘”—Žƒ•ƒ’Ž‡•™‡”‡ƒ”–‹ϐ‹ ‹ƒŽŽ› ‘–ƒ‹ƒ–‡†‘Ž›„›L. monocytogenes.
Š‡•–—†›™ƒ•‘”‰ƒ‹œ‡†‹ʹͲͳ͵„› ‘‘†ƒˆ‡–›ƒ„‘”ƒ–‘”›ǡƒ‹•‘•ǦŽˆ‘”–ǡ ”ƒ ‡ˆ‘” ‘Ž†Ǧ
•‘‡† •ƒŽ‘ǡ ‡˜‹”‘‡–ƒŽ •ƒ’Ž‡•ǡ ”‡ƒ†›Ǧ–‘Ǧ‡ƒ– •ƒŽƒ†•ǡ ’‘™†‡”‡† ‹ˆƒ– ˆ‘‘† ˆ‘”—Žƒǡ ƒ† „›
 Ǧ ǡ‘Ž‹‰›ǡ ”ƒ ‡ǡˆ‘” Š‡‡•‡•ƒ’Ž‡•ǡƒ•’ƒ”–‘ˆ–Š‡ƒ†ƒ–‡͵ͺͳˆ”‘–Š‡
European Commission.
Š‡˜ƒŽ—‡•‘ˆ–Š‡’‡”ˆ‘”ƒ ‡ Šƒ”ƒ –‡”‹•–‹ •†‡”‹˜‡†ˆ”‘–Š‡‹–‡”Žƒ„‘”ƒ–‘”›•–—†‹‡•ƒ”‡•Š‘™’‡”
–›’‡‘ˆ•ƒ’Ž‡‹Tables F.1 to F.5. Some storage conditions or addition of a bacteriostatic mixture in
‡”–ƒ‹ƒ–”‹ ‡•ƒ›Šƒ˜‡‹†— ‡†ƒ•–”‡••‘L. monocytogenes–Šƒ– ƒ‡š’Žƒ‹ƒŽ‘™‡”•‡•‹–‹˜‹–›
than in other matrices.
Š‡ ‹–‡”Žƒ„‘”ƒ–‘”› •–—†‹‡• ™‡”‡ ‘†— –‡† ™‹–Š ƒ ’”‹ƒ”› ‡”‹ Š‡– †—”ƒ–‹‘ ‘ˆ ʹͶ Š ά ʹ Šǡ „—–
–Š‹• †—”ƒ–‹‘ ™ƒ• ‘†‹ϐ‹‡† ƒˆ–‡”™ƒ”†• ‹ –Š‡ †‘ —‡– –‘ ʹͷ Š ά ͳ ŠǤ Š‹• Šƒ‰‡ Šƒ• ‘ ‹’ƒ – ‹
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

performance characteristics of the method, since it was adopted in order to allow growth of target
„ƒ –‡”‹ƒ–‘ƒŠ‹‰Š‡”Ž‡˜‡Ž‹’”‹ƒ”›‡”‹ Š‡–„”‘–ŠǤŠ‡—„‡”‘ˆŽƒ„‘”ƒ–‘”‹‡•–Šƒ–’‡”ˆ‘”‡†Ž‡••
–ŠƒʹͶŠ’”‹ƒ”›‡”‹ Š‡–ˆ‘”‡ƒ Š–”‹ƒŽ™ƒ•ǣ–Š”‡‡ˆ‘” ‘Ž†Ǧ•‘‡†•ƒŽ‘ǡ–™‘ˆ‘”‡˜‹”‘‡–ƒŽ
•ƒ’Ž‡•ǡϐ‹˜‡ˆ‘””‡ƒ†›Ǧ–‘Ǧ‡ƒ–•ƒŽƒ†•ǡ‘‡ˆ‘”’‘™†‡”‡†‹ˆƒ–ˆ‘‘†ˆ‘”—Žƒǡƒ†•‹šˆ‘” Š‡‡•‡Ǥ

Table F.1 — Results of data analysis obtained with cold-smoked salmon

Parameter 0 CFU/25g 8 CFU/ 25 g 74 CFU/ 25g
(blank) (low level (high level
contamination) contamination)
Number of participating collaborators 15 15 15
Number of collaborators retained after evaluation of the data 15 15 15
Number of samples 120 120 120
Number of samples retained after evaluation of the data 120 120 120
‡•‹–‹˜‹–›‹Ψ — 100 100
’‡ ‹ϐ‹ ‹–›‹Ψ 100 — —

© ISO 2017 – All rights reserved

Impreso por: Generico
31
 ISO 11290-1:2017(E)

Table F.2 — Results of data analysis obtained with environmental samples (gauze pads
immerged in diluent)
Parameter 0 CFU/ 11 CFU/ gauze 100 CFU/ gauze
gauze pad pad pad
(blank) (low level (high level
contamination) contamination)
Number of participating collaborators 15 15 15
Number of collaborators retained after evaluation of the data 14a 14a 14a
Number of samples 120 120 120
Number of samples retained after evaluation of the data 112 112 112
‡•‹–‹˜‹–›‹Ψ — 99,1b 99,1b
’‡ ‹ϐ‹ ‹–›‹Ψ 100 — —
a ‡Žƒ„‘”ƒ–‘”›‡š Ž—†‡†ˆ‘”Šƒ˜‹‰”‡ ‡‹˜‡†•ƒ’Ž‡•‘’‡‡†ȋ’”‘„Ž‡†—”‹‰–”ƒ•’‘”–ƒ–‹‘ȌǤ
b ‡ Žƒ„‘”ƒ–‘”› ȋ†‹ˆˆ‡”‡– ˆ‘” Ž‘™ ƒ† Š‹‰Š Ž‡˜‡Ž•Ȍ †‡–‡ –‡† L. monocytogenes in seven samples out of eight. In fact
characteristic L. monocytogenes ‘Ž‘‹‡•™‡”‡•‡‡„—–‘˜‡”‰”‘™„›L. innocua:–Š‹•†‹†‘–ƒŽŽ‘™‹•‘Žƒ–‹‘ƒ†’—”‹ϐ‹ ƒ–‹‘
ˆ‘”ˆ—”–Š‡” ‘ϐ‹”ƒ–‹‘Ǥ

Table F.3 — Results of data analysis obtained with ready-to-eat salads (mixture of fresh spinach
and iceberg lettuce)
Parameter 0 CFU/25g 9 CFU/ 25 g 90 CFU/ 25g
(blank) (low level (high level
contamination) contamination)
Number of participating collaborators 16 16 16
Number of collaborators retained after evaluation of the data 16 16 16
Number of samples 128 128 128
Number of samples retained after evaluation of the data 128 128 128
‡•‹–‹˜‹–›‹Ψ — 100 100
’‡ ‹ϐ‹ ‹–›‹Ψ 97,6a — —
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

a Three false positives have been obtained in three different laboratories (one sample out of eight). Possible reasons
ƒ› „‡ ȋ‹Ȍ ”‘••Ǧ ‘–ƒ‹ƒ–‹‘ ƒ– ‘”‰ƒ‹œ‡”ǯ• Ž‡˜‡Žǡ ȋ‹‹Ȍ ”‘••Ǧ ‘–ƒ‹ƒ–‹‘ ƒ– –Š‡ ’ƒ”–‹ ‹’ƒ–‹‰ Žƒ„‘”ƒ–‘”‹‡•ǯ Ž‡˜‡Žǡ ȋ‹‹‹Ȍ
˜‡”›Ž‘™ƒ†Š‡–‡”‘‰‡‡‘—• ‘–ƒ‹ƒ–‹‘‘ˆ–Š‡ƒ–”‹š—†‡–‡ –‡†„›’”‡Ž‹‹ƒ”›†‡–‡ –‹‘–‡•–•’‡”ˆ‘”‡†„‘–Š„›–Š‡
‘”‰ƒ‹œ‡”ƒ†„›–Š‡•ƒ’Ž‡’”‘†— ‡”ǡƒ†—Ž‹‡Ž›–‘ƒˆˆ‡ –“—ƒŽ‹–›”‡•—Ž–•‘ˆ–Š‡–”‹ƒŽǤ

Table F.4 — Results of data analysis obtained with powdered infant food formula
Parameter 0 CFU/25g 11 CFU/ 25 g 91 CFU/ 25g
(blank) (low level (high level
contamination) contamination)
Number of participating collaborators 16 16 16
Number of collaborators retained after evaluation of the data 16 16 16
Number of samples 128 128 128
Number of samples retained after evaluation of the data 128 128 128
‡•‹–‹˜‹–›‹Ψ — 99,2a 100
’‡ ‹ϐ‹ ‹–›‹Ψ 100 — —
ƒ‡Žƒ„‘”ƒ–‘”›Šƒ•†‡–‡ –‡†L. monocytogenes in seven samples out of eight.

32
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

ƒ„Ž‡ ǤͷȄ‡•—Ž–•‘ˆ†ƒ–ƒƒƒŽ›•‹•‘„–ƒ‹‡†™‹–Š Š‡‡•‡ȋ —”†‘ˆƒ”–‹ϐ‹ ‹ƒŽ Š‡‡•‡Ȍ

Parameter 0 CFU/25g 7 CFU/ 25 g 78 CFU/ 25g
(blank) (low level (high level
contamination) contamination)
Number of participating collaborators 15 15 15
Number of collaborators retained after evaluation of the data 14a 14a 14a
Number of samples 120 120 120
Number of samples retained after evaluation of the data 112 112 112
‡•‹–‹˜‹–›‹Ψ — 91,1b 100
’‡ ‹ϐ‹ ‹–›‹Ψ 100 — —
ƒ‡Žƒ„‘”ƒ–‘”›‡š Ž—†‡†ˆ‘”Šƒ˜‹‰”‡ ‡‹˜‡†•ƒ’Ž‡•–‘‘Žƒ–‡Ǥ
b Three laboratories have detected L. monocytogenes‹•‡˜‡•ƒ’Ž‡•‘—–‘ˆ‡‹‰Š–ƒ†‘‡Žƒ„‘”ƒ–‘”›‹‘‡•ƒ’Ž‡‘—–
of eight.

F.2 Values of LOD50 % from literature

LODͷͲΨ ‘—Ž†‘–„‡‡•–‹ƒ–‡†‹–Š‡ˆ”ƒ‡‘ˆ–Š‡‹–‡”Žƒ„‘”ƒ–‘”›•–—†›†‡• ”‹„‡†‹ F.1. However,
mean LODͷͲ Ψ values from ISO 11290-1:1996/Amd.12004, available from AFNOR validation studies
of alternative commercial methods, in accordance with ISO 16140:20032), are given in Table F.6 for
•‡˜‡”ƒŽˆ‘‘† ƒ–‡‰‘”‹‡•‘”‡˜‹”‘‡–ƒŽ•ƒ’Ž‡•Ǥ –‹• ‘•‹†‡”‡†–Šƒ––‡ Š‹ ƒŽ‘†‹ϐ‹ ƒ–‹‘•‘ˆ–Š‹•
†‘ —‡–†‘‘–Šƒ˜‡•‹‰‹ϐ‹ ƒ–‹’ƒ –‘–Š‡•‡˜ƒŽ—‡•ˆ‘”ͷͲΨ .

Table F.6 — LOD50 % (cfu/25g or 25ml)

Parameter Meat products Seafood Milk Vegetables Environmental
Rillettes Minced meat products products (n = 22) samples
(n = 19) (n = 2) (n = 22) (n = 23) (n = 22)
Mean 0,6 1 0,5 0,6 0, 6 0,5
Standard Devi-
0,2 0,9 0,2 0,2 0,2 0,1
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

ation
Extreme values 0,2 to 1,0 0,4 to 1,7 0,1 to 1,3 0,3 to 1,2 0,1 to 0,9 0,3 to 0,8
NOTE See Reference [20].

Table F.7 — AFNOR Validation reports providing LOD50 % values[20]

Proprietary method  ‡’‘”–‡ˆ‡”‡ ‡ȋ ‡”–‹ϐ‹ ƒ–‡—„‡”Ȁ†ƒ–‡‘ˆ
publication)
‡ƒ› AES 10/3 – 09/00
20 December 2012
ADIAFOOD Listeria monocytogenes AES 10/8 – 12/09
August 2010
Transia Plate Listeria monocytogenes TRA 02/11–03/08
2008 v01
ChromIDTM Lmono Agar BIO 12/31–05/11
ChromIDTM Ottaviani Agosti Agar BIO 12/24–03/08,
ͻ ‡„”—ƒ”›ʹͲͳʹ
Vidas Listeria monocytogenes 2 (30 °C) BIO 12/09–07/02
2007 v01

2) Withdrawn standard.

© ISO 2017 – All rights reserved

Impreso por: Generico
33
 ISO 11290-1:2017(E)

Table F.7 (continued)

Proprietary method  ‡’‘”–‡ˆ‡”‡ ‡ȋ ‡”–‹ϐ‹ ƒ–‡—„‡”Ȁ†ƒ–‡‘ˆ
publication)
Vidas Listeria monocytogenes 2 (37 °C) BIO 12/11–03/04
2013 v01
Vidas Listeria DUO BIO 12/18–03/06
2006 v01
VIDAS Listeria monocytogenes’”‡•• BIO 12/27–02/10
2012 v01
Rapid L’Mono (Detection) BRD 07/04–09/98
2010 v01
IQ Check Listeria monocytogenes II BRD 07/10 – 04/05
2013 v01
AL/Agar Detection BRD 07/16 – 01/09,
2013
CHROMagar™ Listeria CHR 21/1 – 12/01
7 October 2013
Lumiprobe 24 Listeria monocytogenes ͳͷȀͲ͵ΫͳʹȀͲͷ
‡„”—ƒ”›ʹͲͳͲ
Accuprobe Listeria monocytogenes BIO 12/4 - 02/95
͹ ‡„”—ƒ”›ʹͲͳͳ
MicroSEQ Listeria monocytogenes ABI 29/05–12/11
5 December 2011
Listeria Precis™ UNI 03/04–04/05
Ͷ ‡„”—ƒ”›ʹͲͳ͵
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Š‡”‘ ‹‡–‹ϐ‹ —”‡‡ –••ƒ› UNI 03/08–11/13

28 November 2013
® Listeria mono 24E QUA 18/05 – 07/08
ͻ —Ž›ʹͲͳʹ
GeneDisc Listeria monocytogenes GEN 25/08–07/10
31 March 2011
Compass Listeria Agar (Detection) BKR 23/02 – 11/02
28 June 2013

34
Impreso por: Generico
© ISO 2017 – All rights reserved
 ISO 11290-1:2017(E)

Bibliography

ȏͳȐ ISO 16140 (all parts), Microbiology of the food chain — Method validation
ȏʹȐ ISO 18593, Microbiology of food and animal feeding stuffs — Horizontal methods for sampling
techniques from surfaces using contact plates and swabs
ȏ͵Ȑ ISO/TS 17728, Microbiology of the food chain — Sampling techniques for microbiological analysis
of food and feed samples
ȏͶȐ BĆėėĔĜ G.I., & FĊđęčĆĒ  ǤǤǤ ‡†•Ǥ ‘™ƒ Ƭ –‡‡Žǯ• ƒ—ƒŽ ˆ‘” –Š‡ †‡–‹ϐ‹ ƒ–‹‘ ‘ˆ ‡†‹ ƒŽ
ƒ –‡”‹ƒǤƒ„”‹†‰‡‹˜‡”•‹–›”‡••ǡ ”‡ƒ–”‹–ƒ‹ǡŠ‹”††‹–‹‘ǡͳͻͻ͵
ȏͷȐ BĊėęĘĈč D., RĆĚ J., EĚČĘęĊė M.R., HĆĚČ M.C., LĆĜĘĔē P.A., LĆĈėĔĎĝ C.,, MĊĎđĊ L. Listeria
ϔŽ‡‹• Šƒ‹‹sp. nov., isolated from cheese. –Ǥ Ǥ›•–Ǥ˜‘ŽǤ‹ ”‘„‹‘ŽǤ 2013 Feb, 63 pp. 526–532
ȏ͸Ȑ BĊĚĒĊė R.R., & HĆğĊđĊČĊė W.C. Chromogenic media for the detection and/or enumeration
of Listeria monocytogenes ‡•—Ž–• ‘ˆ –”‹ƒŽ• ’‡”ˆ‘”‡† „› ƒ ™‘”‹‰ ‰”‘—’ ‘ˆ –Š‡ –‡”ƒ–‹‘ƒŽ
”‰ƒ‹œƒ–‹‘ˆ‘”–ƒ†ƒ”†‹œƒ–‹‘Ȃ Ȁ͵ͶȀͻǤ” ŠǤ‡„‡•‹––‡ŽŠ›‰ǤʹͲͲ͹ǡ58 pp. 47–50
ȏ͹Ȑ CĔėėĞ J.E.L., CĚėęĎĘ G.D.W., BĆĎėĉ R.M. eds. In: Handbook of Culture Media for Food and Water
Microbiology. (PĚćđĎĘčĎēČ R.S.C.) Third Edition, 2012
ȏͺȐ GĆėėĎęĞ G.M. (editor in chief), Bergey’s Manual of Systematic Bacteriology, 2nd edition, 2005
ȏͻȐ GėĊĊēĜĜĔĔĉ M., WĎđđđĎĘ C., DĔĘĜĊđđ P., AđđĊē G., PĆęĆĈĐ K. Evaluation of chromogenic
media for the detection of Listeria species in food. J. Appl. Microbiol. 2005, 99 pp. 1340–1345
ȏͳͲȐ GėĆěĊĘ L.M., HĊđĘĊđ L.O., SęĊĎČĊėĜĆđę A.G., MĔėĊĞ R.E., DĆēĊĘčěĆė M.I., RĔĔċ S.E. Listeria
marthii•’Ǥ‘˜Ǥǡ‹•‘Žƒ–‡†ˆ”‘–Š‡ƒ–—”ƒŽ‡˜‹”‘‡–ǡ ‹‰‡”ƒ‡•ƒ–‹‘ƒŽ ‘”‡•–Ǥ –Ǥ Ǥ›•–Ǥ
Evol. Microbiol. 2010, 60 pp. 1280–1288
ȏͳͳȐ JĔčēĘĔē J., JĎēēĊĒĆē K., ęĊđĒĆ G., ĒĎęč B.G., ĞĊ D., ĊĘĘĊė J., đĆĘğĊĐ J., EěĘĊē L.,
GĊēĉĊđ SǤǡ ĊēēĊęę R.W., SĜĆĒĎēĆęčĆē B., PėĚĈĐđĊė J., SęĊĎČĊėĜĆđę A., KĆęčĆėĎĔĚ S.,
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

ĎđĉĎėĎĒ S., VĔđĔĐčĔě D., RĆĘĔĔđĞ A.,CčĎğčĎĐĔě V., ĎĊĉĒĆēē M., FĔėęĊĘ E., DĚěĆđđ
Ǥ E., HĎęĈčĎēĘ  ǤǤ  ƒ–—”ƒŽ ƒ–›’‹ ƒŽ ‹•–‡”‹ƒ ‹‘ —ƒ •–”ƒ‹• ™‹–Š ‹•–‡”‹ƒ ‘‘ ›–‘‰‡‡•
’ƒ–Š‘‰‡‹ ‹–›‹•Žƒ†ͳ‰‡‡•Ǥ’’ŽǤ˜‹”‘Ǥ‹ ”‘„‹‘ŽǤʹͲͲͶǡ70 pp. 4256–4266
ȏͳʹȐ LĆēČ ĆđęĊė E., NĊĚčĆĚĘ K., SĈčĊėĊė S. Listeria weihenstephanensis sp. nov., isolated from
the water plant Lemna trisulca‘ˆƒ ‡”ƒˆ”‡•Š™ƒ–‡”’‘†Ǥ –Ǥ Ǥ›•–Ǥ˜‘ŽǤ‹ ”‘„‹‘ŽǤʹͲͳ͵
Feb, 63 pp. 641–647
ȏͳ͵Ȑ LĊĈđĊėĈĖǤ‘Ž‘‹ƒŽƒ–›’‹ ƒŽ‘”’Š‘Ž‘‰›ƒ†Ž‘™”‡ ‘˜‡”‹‡•‘ˆListeria monocytogenes strainson
Oxford, PALCAM, Rapid’L.mono and ALOA solid media. J. Microbiol. Methods. 2004, 57pp.
251–258
ȏͳͶȐ LĊĈđĊėĈĖ A., CđĊėĒĔēę D., BĎğĊę C., GėĎĒĔēę P.A., LĊ đijĈčĊǦĆęĴĔĘ A., RĔĈčĊ S.M. Listeria
rocourtiae•’Ǥ‘˜Ǥ –Ǥ Ǥ›•–Ǥ˜‘ŽǤ‹ ”‘„‹‘ŽǤʹͲͳͲǡ60 pp. 2210–2214
ȏͳͷȐ LĎĚ D., LĆĜėĊēĈĊ M.L., WĎĊĉĒĆēē M., GĔėĘĐĎ L., MĆēĉėĊđđ R.E., AĎēĘĜĔėęč A.J. et al. Listeria
monocytogenes •—„‰”‘—’• ǡ ǡ ƒ†  †‡Ž‹‡ƒ–‡ ‰‡‡–‹ ƒŽŽ› †‹•–‹ – ’‘’—Žƒ–‹‘• ™‹–Š
varied pathogenic potential. J. Clin. Microbiol. 2006, 44 pp. 4229–4233
ȏͳ͸Ȑ OęęĆěĎĆēĎ F. ęęĆěĎĆēĎ Ǥǡ ČĔĘęĎ Ǥ, Differential agar medium for Listeria monocytogenes.
—‹’‡” ”‘‹†›’‘•‹—”‘ ‡‡†‹‰•ǡ͸ —‹’‡”ǡ ”ƒ ‡ǡͳ͸Ǧͳͺ —‡ǡͳͻͻ͹
ȏͳ͹Ȑ OęęĆěĎĆēĎ F. ęęĆěĎĆēĎ Ǥǡ ČĔĘęĎ Ǥ Esperienza su un agar selettivo e differenziale per
Ǥ mono. Industrie Alimentari, 1997

© ISO 2017 – All rights reserved

Impreso por: Generico
35
 ISO 11290-1:2017(E)

[18] RĔćĊėęĘ A., NĎČčęĎēČĆđĊ K., JĊċċĊėĘ G., FĔėęĊĘ E., KĔēČĔ J.M., WĎĊĉĒĆēē M. Genetic and
’Š‡‘–›’‹  Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ Listeria monocytogenes Ž‹‡ƒ‰‡ Ǥ ‹ ”‘„‹‘Ž‘‰›Ǥ ʹͲͲ͸ǡ 152
pp. 685–693
[19] WĎđđĎĘ C., BĆĆđčĆĒ T., GėĊĊēĜĔĔĉ M., PėĊĘđĆēĉ F. Evaluation of a new chromogenic agar
for the detection of Listeria in food. J. Appl. Microbiol. 2006, 101 pp. 711–717
[20] AFNOR validation studies, available at http://nf-validation.afnor.org/en/?s=listeria
[21] AĚČĚĘęĎē J.-C., KĆđĒĔĐĔċċ M., EđđĘ T., FĆěėĊę S., DĊĘėĊĚĒĆĚĝ J., DĊĈĔĚėĘĊĚđđĊĘėĆĘĘĊĚė
E. Ćēĉ ēĆēĔĚ ĊĘĘĊ  Modeling the behavior of Listeria monocytogenes during enrichment
in half Fraser broth - Impact of pooling and the duration of enrichment on the detection of L.
monocytogenes in food. Food Microbiol. 2016, 60 pp. 131–136
[22] JĆČĆĉĊĊĘĆē B., BĆĘęĎĈĈčĒĎĉ V., KđĎďē A., MĈĆčĔē W. Validation of Test Portion Pooling for
the Detection of Listeria spp. and L. monocytogenes‹ƒ‹”›”‘†— –•Ǥ‘•–‡”’”‡•‡–‡†ƒ–ǣ  
2016, St. Louis, Jul 29 - Aug 3
[23] AēČĊđĎĉĎĘ A.S., KĆđĆĒĆĐĎ M.S., GĊĔėČĎĆĉĔĚǤǤ †‡–‹ϐ‹ ƒ–‹‘‘ˆ‘ǦListeria spp. bacterial
‹•‘Žƒ–‡•›‹‡Ž†‹‰ƒȾǦǦ‰Ž— ‘•‹†ƒ•‡Ǧ’‘•‹–‹˜‡’Š‡‘–›’‡‘‰ƒ”Listeria according to Ottaviani
and Agosti (ALOA). Int. J. Food Microbiol. 2015, 193 pp. 114–129
[24] CĆėĕĊēęĎĊė B, & BĆėėĊ L. Guidelines on Sampling the Food Processing Area and Equipment for
the Detection of Listeria monocytogenes. Listeria EURL, 2012
[25] BĆėėĊ L., AēČĊđĎĉĎĘ A.S., BĔĚĘĘĆĎĉ D., DĊĈĔĚėĘĊĚđđĊĘėĆĘĘĊĚė E., MĆēĘĔ E. ČēĆēĔĚ ćĊĘĘĊ
Ǥ’’Ž‹ ƒ„‹Ž‹–›‘ˆ–Š‡ ͳͳʹͻͲǦͳ–ƒ†ƒ”†‡–Š‘†ˆ‘”Listeria monocytogenes detection in the
presence of new Listeria species. Int. J. Food Microbiol. 2016, 238 pp. 281–287
[26] HĊēĐ C., WĆėĈčĔĈĐĎ S., EĒĎđĞ M., AĉĆĒ F., CđĞĉĊ M., KĊĕčĆė D ĆēĉĊĎĉĒĆēē Five new
species of Listeria (ǤϔŽ‘”‹†‡•‹• sp. nov., L. aquatic sp. nov., L. cornellensis sp. nov., L. riparia sp.
nov. and L. grandensis sp. nov.) from agricultural and natural environments in the United States.
–Ǥ Ǥ›•–Ǥ˜‘ŽǤ‹ ”‘„‹‘ŽǤʹͲͳͶǡ64 pp. 1882–1889
[27] WĊđđĊė D., AēĉėĚĘ A., WĎĊĉĒĆēē M., BĆĐĐĊė H. Listeria booriae sp. nov. and Listeria
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

newyorkensis •’Ǥ ‘˜Ǥǡ ˆ”‘ ˆ‘‘† ’”‘ ‡••‹‰ ‡˜‹”‘‡‡–• ‹ –Š‡ Ǥ –Ǥ Ǥ ›•–Ǥ ˜‘ŽǤ
Microbiol. 2015, 65 pp. 286–292
[28] ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment
or revision of a standardized reference method
[29] GēĆēĔĚĊĘĘĊ N., FĆěėĊę S., DĊĘėĊĚĒĆĚĝ J., DĊĈĔĚėĘĊĚđđĊĘėĆĘĘĊĚė E., KĆđĒĔĐĔċċ M.
˜ƒŽ—ƒ–‹‘‘ˆ”‡†— –‹‘‘ˆ ”ƒ•‡”‹ —„ƒ–‹‘„›ʹͶŠ‹–Š‡ ͳͳʹͻͲǦͳ•–ƒ†ƒ”†‘†‡–‡ –‹‘
ƒ††‹˜‡”•‹–›‘ˆListeria species. Int. J. Food Microbiol. 2016, 224 pp. 16–21
[30] ISO 16140:2003, 3)Microbiology of food and animal feeding stuffs — Protocol for the validation of
alternative methods

3) Withdrawn standard.

36
Impreso por: Generico
© ISO 2017 – All rights reserved
 Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

Impreso por: Generico

 ISO 11290-1:2017(E)
Ejemplar para uso exclusivo - UNIVERSIDAD DEL QUINDIO - 890.000.432--8

౧Ͳ͹ǤͳͲͲǤ͵Ͳ
Price based on 36 pages

© ISO 2017 – All rights reserved

Impreso por: Generico