EN ISO 11290-1:2017

SLOVENSKI STANDARD
01-september-2017
1DGRPHãþD
SIST EN ISO 11290-1:1997
SIST EN ISO 11290-1:1997/A1:2005
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Listeria monocytogenes in Listeria spp. - 1. del: Metoda za
ugotavljanje prisotnosti (ISO 11290-1:2017)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Listeria monocytogenes and of Listeria spp. - Part 1: Detection method (ISO 11290-
1:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die
Zählung von Listeria monocytogenes und von Listeria spp. - Teil 1: Nachweisverfahren
(ISO 11290-1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Listeria monocytogenes et de Listeria spp. - Partie 1: Méthode de
recherche (ISO 11290-1:2017)
Ta slovenski standard je istoveten z: EN ISO 11290-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 11290-1
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 11290-1:1996
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Listeria monocytogenes and
of Listeria spp. - Part 1: Detection method (ISO 11290-
1:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche et le dénombrement de Verfahren für den Nachweis und die Zählung von
Listeria monocytogenes et de Listeria spp. - Partie 1: Listeria monocytogenes und von Listeria spp. - Teil 1:
Méthode de recherche (ISO 11290-1:2017) Nachweisverfahren (ISO 11290-1:2017)
This European Standard was approved by CEN on 6 April 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11290-1:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 11290-1:2017) has been prepared by Technical Committee ISO/TC 34 “Food
products” in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
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 December 2017, and conflicting national standards
shall be withdrawn at the latest by December 2017.
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.
This document supersedes EN ISO 11290-1:1996.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11290-1:2017 has been approved by CEN as EN ISO 11290-1:2017 without any
modification.
INTERNATIONAL ISO
STANDARD 11290-1
Second edition
2017-05
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of Listeria
monocytogenes and of Listeria spp. —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Listeria monocytogenes et de
Listeria spp. —
Partie 1: Méthode de recherche
Reference number
ISO 11290-1:2017(E)
©
ISO 2017
ISO 11290-1:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

ISO 11290-1:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Primary enrichment in a selective liquid enrichment medium with reduced
concentration of selective agents (half-Fraser broth) . . 2
4.3 Secondary enrichment with a selective liquid enrichment medium with full
concentration of selective agents (Fraser broth) . 3
4.4 Plating out and identification . 3
4.5 Confirmation . 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
9.2 Primary enrichment . 4
9.3 Secondary enrichment . 5
9.4 Plating out and identification . 5
9.4.1 General . 5
9.4.2 Agar Listeria according to Ottaviani and Agosti . 5
9.4.3 Second selective medium . 6
9.5 Confirmation of Listeria monocytogenes or Listeria spp. . 6
9.5.1 Selection of colonies for confirmation . 6
9.5.2 Confirmation of L. monocytogenes .  . 6
9.5.3 Confirmation of Listeria spp. . 10
9.6 Interpretation of morphological and physiological properties and of the
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
11.2 Sensitivity .11
11.3 Specificity .11
11.4 Level of detection (LOD ) .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) Reactions for the identification of Listeria species .28
Annex E (informative) Listeria selective agars .30
ISO 11290-1:2017(E)
Annex F (informative) Results of the interlaboratory studies for detection of
Listeria monocytogenes .31
Bibliography .35
iv © ISO 2017 – All rights reserved

ISO 11290-1:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
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).
This second edition cancels and replaces the first edition (ISO 11290-1:1996), which has been technically
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 monocytogenes has been modified as listed below.
— Primary enrichment in half-Fraser broth: incubation for 25 h ± 1 h.
[29]
— Secondary enrichment in Fraser broth: incubation for 24 h ± 2 h.
— Half-Fraser and Fraser broths may be refrigerated before transfer or isolation on selective agar for
a maximum of 72 h.
— Storage of isolation plates: incubated plates can be refrigerated for a maximum of two days before
reading.
— Microscopic aspect for confirmation is optional if the isolation agar allows distinction between
pathogenic and non-pathogenic Listeria spp.
— CAMP test and catalase test are optional.
— Inclusion of new performance characteristics.
— Moreover, detection of Listeria spp. has been included in the scope and the title changed accordingly.
A list of parts in the ISO 11290 series can be found on the ISO website.
ISO 11290-1:2017(E)
Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO 11290-1:1996
[28]
are considered as major (see ISO 17468 ). The technical changes were assessed and were considered
to have no significant effect on the method performance characteristics or test results.
Because of the large variety of food and feed products, this horizontal method may not be appropriate
in every detail for certain products for which it may be necessary to use different or specific methods.
Nevertheless, in all cases, this horizontal method is intended to be applied as far as possible and
deviations from this only be made for justified technical reasons.
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
International Standards and/or national standards may already exist that do not comply with this
horizontal method. It is hoped that when such standards are reviewed, they will be changed to comply
with this document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.
vi © 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 Listeria monocytogenes
and of Listeria 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
This document specifies a horizontal method for
— the detection of L. monocytogenes, and
— 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.
It is possible that certain additionally described Listeria species may not be detected or confirmed by
[5],[10],[12],[14],[25],[26],[27]
this method.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 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 11290-1:2017(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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
microorganisms which form typical colonies on solid selective media and which display the
morphological, physiological and biochemical characteristics described when tests are carried out in
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
of product or a specified surface, when tests are carried out in accordance with this document
3.3
Listeria spp.
microorganisms which form typical colonies on solid selective media and which display the
morphological, physiological and biochemical characteristics described when tests are carried out in
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
or a specified surface, when tests are carried out in accordance with this document
4 Principle
4.1 General
Listeria spp. may be present in small numbers and are often accompanied by considerably larger
numbers of other microorganisms, therefore selective enrichment is necessary. It is also necessary to
detect injured and stressed Listeria spp. and the primary selective enrichment medium, with reduced
inhibitor concentration, fulfils at least part of this function.
NOTE Presence of L. monocytogenes can be masked by the presence of other 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
four successive stages, as described in the flowchart in Annex A.
4.2 Primary enrichment in a selective liquid enrichment medium with reduced
concentration of selective agents (half-Fraser broth)
Inoculation of a selective primary enrichment medium containing half the concentrations of
acriflavine and nalidixic acid (half-Fraser broth, see B.1), which is also used as a dilution fluid for the
test portion (9.1).
Incubation of the initial suspension at 30 °C for 24 h to 26 h.
2 © 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)
Inoculation of full-strength secondary liquid enrichment medium (Fraser broth) with a culture
obtained from 4.2.
[29]
Incubation of the Fraser broth at 37 °C for 24 h.
4.4 Plating out and identification
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);
— any other solid selective medium at the choice of the laboratory complementary to Agar 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 spp. are evident at 24 h the incubation may be stopped at this
stage. Incubation of the second selective medium at the appropriate temperature and examination after
the appropriate time.
4.5 Confirmation
Subculturing of the colonies of presumptive L. monocytogenes or Listeria spp., plated out as described
in 4.4, and confirmation by means of appropriate morphological and/or biochemical tests.
5 Culture media and reagents
For current laboratory practices, refer to ISO 11133.
Composition and performance testing of culture media and reagents and their preparation are
described in Annex B.
6 Equipment and consumables
Usual microbiological equipment (as specified in ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
As specified in ISO 7218.
6.2 Drying cabinet or incubator, capable of being maintained between 25 °C and 50 °C.
6.3 Incubators, capable of operating at 30 °C ± 1 °C, 37 °C ± 1 °C and at 25 °C ± 1 °C (optional).
6.4 Water bath, capable of operating at 47 °C to 50 °C.
6.5 Sterile loops approximately 3 mm in diameter or 10 µl, and inoculating 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
made which are accurate to ± 0,1 pH unit.
6.7 Graduated pipettes or automatic pipettes, of nominal capacities 1 ml and 10 ml.
ISO 11290-1:2017(E)
6.8 Petri dishes, for example of diameter 90 mm.
6.9 Microscope, preferably with phase-contrast, and with slides and cover slips.
6.10 Refrigerator, capable of operating at 5 °C ± 3 °C.
7 Sampling
Sampling is not part of the method specified in this document. If there is no specific International
Standard dealing with sampling of the product concerned, it is recommended that the parties
[3]
concerned come to an agreement on this subject. For food and feed samples, refer to ISO/TS 17728 .
[2]
For environmental samples, use ISO 18593 and see Reference [24].
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage (see ISO 7218).
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product
[2]
concerned [see ISO 6887 (all parts) and ISO 18593 ]. If there is no specific International Standard, it is
recommended that the parties concerned come to an agreement on this subject.
9 Procedure
9.1 Test portion and initial suspension
Refer to ISO 6887 (all parts) and any specific International Standard appropriate to the product
concerned.
For preparation of the initial suspension, use as dilution fluid the selective primary enrichment medium
specified in B.1 (half-Fraser broth).
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
selective primary enrichment medium (B.1), to obtain a tenfold dilution, and homogenize. Pre-warm
the selective primary enrichment medium to room temperature before use.
This document has been validated for test portions of 25 g or ml. A smaller test portion may be used,
without the need for additional validation/verification, providing that the same ratio between primary
enrichment broth and test portion is maintained. A larger test portion than that initially validated may
be used, if a validation/verification study has shown that there are no adverse effects on the detection
of L. monocytogenes or Listeria spp.
[1]
NOTE 1 Validation can be conducted in accordance with the appropriate document of ISO 16140 (all parts) .
Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017,
Annex D (verification protocol for pooling samples for qualitative tests). See References [21] and [22] as they
provide information on the particular case of Listeria pooling samples.
NOTE 2 For large quantities, it is recommended to pre-warm the selective primary enrichment medium to
30 °C ± 1 °C before mixing it with the test portion.
9.2 Primary enrichment
Incubate the primary enrichment medium (half-Fraser broth, see B.1), prepared in accordance with 9.1,
at 30 °C (6.3) for 25 h ± 1 h.
NOTE 1 A black coloration can develop during the incubation.
4 © 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 After incubation of the initial suspension (primary enrichment) for 25 h ± 1 h (9.2), transfer 0,1 ml
of the culture obtained in 9.2 (regardless of its colour) to a tube or bottle containing 10 ml of secondary
enrichment medium (Fraser broth) (B.2).
9.3.2 Incubate the inoculated medium (9.3.1) for 24 h ± 2 h at 37 °C (6.3).
NOTE In the case of Listeria spp. other than Listeria monocytogenes detection, additional 24 h incubation can
allow for recovery of more species.
9.4 Plating out and identification
9.4.1 General
9.4.1.1 From the primary enrichment culture (9.2) incubated for 25 h ± 1 h at 30 °C (6.3), inoculate,
by means of a loop (6.5), the surface of the first selective plating medium, Agar Listeria according to
Ottaviani and Agosti (B.3), to obtain well-separated colonies.
Proceed in the same way with the second selective plating-out medium of choice (B.4).
NOTE Half-Fraser broth and Fraser broth can be refrigerated at 5 °C (6.10) before isolation on selective agar
[20]
for a maximum of 72 h.
9.4.1.2 From the secondary enrichment medium incubated for 24 h ± 2 h at 37 °C (6.3) (9.3.2), repeat
the procedure described in 9.4.1.1 with the two selective plating-out media.
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 Listeria according to Ottaviani and Agosti incubate for a total of 48 h ± 2 h. If colonies of
presumptive L. monocytogenes or Listeria spp. are evident at 24 h ± 2 h the incubation may be stopped at
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 the blue-green colonies surrounded by an opaque halo
(typical colonies). Colonies of L. ivanovii are also blue-green and surrounded by an opaque halo.
Consider as presumptive Listeria spp. the blue-green colonies with or without opaque halo.
NOTE 1 Some strains of L. monocytogenes exposed to stress conditions, particularly acid stress, can show a
very weak halo (or even no halo).
NOTE 2 Some rare L. monocytogenes are characterized by a slow PIPLC (phosphatidyl inositol phospholipase C)
activity. Such bacteria are detected when the total duration of incubation is more than, for example, four days.
[13]
Some of these strains could be pathogenic. No L. monocytogenes strains have been described as PIPLC
negative.
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. monocytogenes, based on their characteristics for the type of medium
used (B.4). See Annex E for more information.
9.5 Confirmation of Listeria monocytogenes or Listeria spp.
9.5.1 Selection of colonies for confirmation
9.5.1.1 For confirmation of presumptive L. monocytogenes, take at least one colony presumed to be
L. monocytogenes (see 9.4.2 and 9.4.3). One confirmed isolate per sample is sufficient. If the first colony
is negative take further colonies presumed to be L. monocytogenes from selective medium (up to a
maximum of five colonies from each plate of each selective medium).
Streak the selected colonies onto the surface of pre-dried plates of a non-selective agar, for example
blood agar, nutrient agar, tryptone soya yeast extract agar (TSYEA) (B.14), in a manner which will allow
isolated colonies to develop.
Use of blood agar for pure culture enables interpretation of haemolysis, when positive, already at
that stage (see 9.5.2.5.2 and Annex D). If streaking on blood agar does not show haemolysis, then the
haemolysis test shall be done by stabbing (9.5.2.5.2) or in liquid medium (9.5.2.5.3).
Place the plates in the incubator set at 37 °C (6.3) for 18 h to 24 h or until growth is satisfactory.
If the colonies are not isolated, pick a typical L. monocytogenes colony onto another non-selective agar
plate. Carry out the following tests (9.5.2) from colonies of a pure culture on the non-selective agar.
9.5.1.2 For confirmation of presumptive Listeria spp., take at least one colony presumed to be
Listeria spp. (see 9.4.2 and 9.4.3). One confirmed isolate per sample is sufficient. If the first colony is
negative take further colonies presumed to be Listeria spp. from selective medium (up to a maximum of
five colonies from each plate of each selective medium).
For confirmation of Listeria spp. use plates of TSYEA.
Streak the selected colonies onto the surface of pre-dried plates of TSYEA (B.14), in a manner which
will allow isolated colonies to develop.
Place the plates in the incubator set at 37 °C (6.3) for 18 h to 24 h or until growth is satisfactory.
Typical colonies of Listeria spp. on TSYEA are 1 mm to 2 mm in diameter, convex, colourless and opaque
with an entire edge. When the plates are held to the light (artificial or natural) at about 45 degree angle,
colonies exhibit a blue-grey colour and a granular surface.
If the colonies are not isolated, pick a typical Listeria spp. colony onto another non-selective agar plate.
Carry out the following tests (9.5.3) from typical colonies of a pure culture on TSYEA.
9.5.2 Confirmation of L. monocytogenes
9.5.2.1 General
Carry out the confirmation tests for L. monocytogenes. Appropriate positive and negative control strains
for each of the confirmation tests shall be used.
Perform at minimum the mandatory tests as listed (in bold) in Table 1.
6 © ISO 2017 – All rights reserved

ISO 11290-1:2017(E)
Table 1 — Confirmation tests for L. monocytogenes
Tests L. monocytogenes confirmation tests Results
a
Mandatory Microscopic aspect (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) +
Motility at 25°C (9.5.2.3) +
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.
Details on results for confirmation tests can be found in Annex D.
NOTE An alternative procedure as mentioned in ISO 7218 can be used to confirm the isolate as Listeria
monocytogenes, providing the suitability of the relevant procedure is verified.
If shown to be reliable, miniaturized galleries for the biochemical identification of Listeria monocytogenes
may be used (see ISO 7218).
Rare strains of L. monocytogenes do not show beta-haemolysis or a positive reaction to the CAMP
test under the conditions described in this document. If typical colonies on Agar Listeria according to
Ottaviani and Agosti with PIPLC activity even if it is low, are negative for haemolysis, it is recommended
to perform additional tests (e.g. Gram stain, catalase, motility, CAMP test, PCR), in order to determine
whether this isolate is a non-haemolytic L. monocytogenes.
9.5.2.2 Catalase reaction (optional)
Take an isolated colony obtained in 9.5.1.1 and suspend it in a drop of hydrogen peroxide solution (B.6)
on a slide. The immediate formation of gas bubbles indicates a positive reaction.
NOTE A catalase reaction performed from a colony originating from a blood agar can sometimes lead to
false-positive results.
9.5.2.3 Motility test (optional)
Take an isolated colony obtained in 9.5.1.1 and suspend it in a tube containing a non-selective nutrient
liquid medium.
Incubate in the incubator (6.3) set at 25 °C for 8 h to 24 h until the medium turns cloudy.
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. monocytogenes) appear as slim, short rods with tumbling motility.
Cultures grown at temperatures above 25 °C may fail to exhibit this motion. Always compare them to
a known Listeria culture. Cocci, large rods, or rods with rapid swimming motility are not Listeria spp.
As an alternative test for motility, using an inoculating needle (6.5), dilute in sterile water (or other
appropriate diluent) a fragment of isolated colony obtained on non-selective agar. Listeria spp.
(including L. monocytogenes) appear as slim, short rods with tumbling motility.
As another alternative test for motility, using an inoculating needle (6.5), stab the motility agar (B.7)
with a culture taken from a typical colony obtained in 9.5.1.1. Incubate at 25 °C for 48 h ± 2 h.
ISO 11290-1:2017(E)
Examine for growth around the stab. Listeria spp. are motile, giving a typical umbrella-like growth
pattern. If growth is not sufficient, incubate for up to an additional five days and observe the stab again.
[5],[10],[12],[14],[25],[26],[27]
NOTE Some new Listeria species have been recently isolated. Most of them are not
mobile in the motility agar.
9.5.2.4 Microscopic aspect (optional in the case of use of agar specific for pathogenic
Listeria spp.)
Make a microscopic preparation (e.g. the Gram stain, wet microscopy) on a well-separated colony
obtained in 9.5.1.1. Listeria spp. (including L. monocytogenes) appear as Gram positive (if this stain
is performed), slim, short rods or coccobacilli, with tumbling motility when originating from a fresh
culture.
For Gram stain microscopic preparation see ISO 7218.
9.5.2.5 Haemolysis tests
9.5.2.5.1 General
Choose one of the haemolysis tests (9.5.2.5.2 or 9.5.2.5.3).
NOTE There exist rare strains of L. monocytogenes which do not show b-haemolysis or a positive reaction to
the CAMP test under the conditions described in this document.
9.5.2.5.2 Haemolysis on blood agar
If the morphological and physiological characteristics are indicative of Listeria spp., inoculate blood
agar plates (B.8) to determine the haemolytic reaction.
Dry the agar surface well before use. Take an isolated colony obtained in 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. innocua WDCM 00017 may be used.
After incubation at 37 °C (6.3) for 24 h ± 2 h, examine the test strains and controls. L. monocytogenes
show narrow, clear, light zones of haemolysis; L. innocua show no clear zone around the stab. L. seeligeri
show mostly a weak zone of haemolysis. L. ivanovii usually show wide, clearly delineated zones of
haemolysis. Examine the plates in a bright light to compare test cultures with controls.
NOTE 1 The haemolysis reaction is more readily seen by removing any colony growth on the surface of the
agar around the inoculum mark.
NOTE 2 The haemolysis test can be performed by stabbing the blood agar plate used for the CAMP test.
9.5.2.5.3 Haemolysis reaction using red blood corpuscles
The haemolytic reaction may also be carried out using red blood corpuscles as follows.
Disperse the colony in 150 µl of a non-selective liquid nutrient medium, incubate at 37 °C (6.3) for 2 h.
Add 150 µl of a suspension of red blood corpuscles (B.10). Incubate at 37 °C (6.3) for 15 min to 60 min,
then refrigerate at 5 °C (6.10) for approximately 2 h. Examine for haemolytic activity. If the reaction is
doubtful, leave at 5 °C (6.10) for up to 24 h ± 2 h. A sedimentation of red blood corpuscles (formation of
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 © ISO 2017 – All rights reserved

ISO 11290-1:2017(E)
9.5.2.6 CAMP test (optional)
If the result of the haemolysis test is difficult to interpret, the CAMP test is recommended to demonstrate
clearly that haemolysis is due to listeriolysin activity.
A β-haemolytic strain of Staphylococcus 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.11.4) so th
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