SIST EN ISO 11290-1:2017
(Main)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)
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)
This standard describes the detection of Listeria monocytogenes (Reference document: EN/ISO 11290 -1 incl./Amd. 1)
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)
Dieses Dokument legt ein horizontales Verfahren fest für
- den Nachweis von L. monocytogenes und
- den Nachweis von Listeria spp. (einschließlich L. monocytogenes).
Dieses Dokument gilt für
- Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind und
- Umgebungsproben aus dem Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln.
Es ist möglich, dass bestimmte, zusätzlich beschriebene Listeria Arten nicht unbedingt mit diesem Verfahren nachweisbar oder bestätigbar sind [5], [10], [12], [14], [25], [26], [27].
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)
ISO 11290-1:2017 spécifie une méthode horizontale pour:
- la recherche de L. monocytogenes; et
- la recherche de Listeria spp. (y compris L. monocytogenes).
ISO 11290-1:2017 s'applique aux:
- produits destinés à la consommation humaine ou à l'alimentation animale; et
- échantillons de l'environnement de production et de distribution des aliments.
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)
Ta standard opisuje način ugotavljanja prisotnosti Listeria monocytogenes (referenčni dokument: EN/ISO 11290 -1 vklj./dod. 1)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11290-1:2017
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
SIST EN ISO 11290-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11290-1:2017
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SIST EN ISO 11290-1:2017
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.
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SIST EN ISO 11290-1:2017
EN ISO 11290-1:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 11290-1:2017
EN ISO 11290-1:2017 (E)
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.
3
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SIST EN ISO 11290-1:2017
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SIST EN ISO 11290-1:2017
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
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 11290-1:2017
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
50
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 2017 – All rights reserved iii
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SIST EN ISO 11290-1:2017
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
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SIST EN ISO 11290-1:2017
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 2017 – All rights reserved v
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SIST EN ISO 11290-1:2017
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
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SIST EN ISO 11290-1:2017
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 2017 – All rights reserved 1
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SIST EN ISO 11290-1:2017
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
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SIST EN ISO 11290-1:2017
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.
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SIST EN ISO 11290-1:2017
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
...
SLOVENSKI STANDARD
oSIST prEN ISO 11290-1:2015
01-februar-2015
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Listeria monocytogenes in Listeria spp. - 2. del: 1. del:
Metoda za ugotavljanje prisotnosti (ISO/DIS 11290-1:2014)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Listeria monocytogenes and other Listeria spp. - Part 1: Detection method (ISO/DIS
11290-1:2014)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den
Nachweis und die Zählung von Listeria monocytogenes - Teil-1: Nachweisverfahren
(ISO/DIS 11290-1:2014)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Listeria monocytogenes et Listeria spp. - Partie 1: Méthode de
recherche (ISO/DIS 11290-1:2014)
Ta slovenski standard je istoveten z: prEN ISO 11290-1:2014
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 11290-1:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 11290-1:2015
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oSIST prEN ISO 11290-1:2015
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11290-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2014-10-30 2015-03-30
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Listeria monocytogenes
and other Listeria spp. —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de
Listeria monocytogenes et Listeria spp. —
Partie 1: Méthode de recherche
ICS: 07.100.30
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the European Committee for Standardization
(CEN), and processed under the CEN lead mode of collaboration as defined in the
Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member
bodies for a parallel five month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments
received, will be submitted to a parallel two-month approval vote in ISO and
THIS DOCUMENT IS A DRAFT CIRCULATED
formal vote in CEN.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 11290-1:2014(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2014
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oSIST prEN ISO 11290-1:2015
ISO/DIS 11290-1:2014(E)
Copyright notice
This ISO document is a Draft International Standard and is copyright-protected by ISO. Except as
permitted under the applicable laws of the user’s country, neither this ISO draft nor any extract
from it may be reproduced, stored in a retrieval system or transmitted in any form or by any means,
electronic, photocopying, recording or otherwise, without prior written permission being secured.
Requests for permission to reproduce should be addressed to either ISO at the address below or ISO’s
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Reproduction may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
ii © ISO 2014 – All rights reserved
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oSIST prEN ISO 11290-1:2015
ISO/DIS 11290-1
Contents Page
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle. 2
4.1 Primary enrichment in a selective liquid enrichment medium with reduced concentration
of selective agents (half-Fraser broth) . 2
4.2 Secondary enrichment with a selective liquid enrichment medium with full concentration
of selective agents (Fraser broth) . 2
4.3 Plating out and identification . 3
4.4 Confirmation . 3
5 Culture media and reagents . 3
6 Apparatus and glassware . 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 . 4
9.4 Plating out and identification . 5
9.5 Confirmation of Listeria monocytogenes and/or Listeria spp. . 5
9.6 Interpretation of morphological and physiological properties and of the biochemical
reactions . 11
9.7 Additional characterisation of isolated strains (optional) . 11
10 Expression of results . 11
11 Accuracy (precision) of the method . 11
11.1 Interlaboratory study . 11
11.2 Sensitivity . 11
11.3 Specificity . 12
11.4 Limit of Detection (LOD ). 12
50
12 Test report . 12
Annex A (normative) Diagram of procedure . 13
Annex B (normative) Composition and preparation of culture media and reagents . 14
B.1 Selective primary enrichment medium: Half-Fraser broth . 14
B.2 Selective secondary enrichment medium: Fraser broth . 16
B.3 Agar Listeria according to Ottaviani and Agosti . 17
B.4 Second selective solid plating-out medium . 19
B.5 Performance testing for the quality assurance of the culture media . 19
B.6 Hydrogen peroxide solution . 21
B.7 Motility agar . 21
B.8 Blood agar . 22
B.9 Phosphate-buffered saline (PBS) . 22
B.10 Red blood corpuscle suspension . 23
B.11 CAMP (Christie, Atkins, Munch-Petersen) medium and test strains . 23
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B.12 Carbohydrate utilization broth (L-Rhamnose and D-Xylose) . 23
B.13 Reagents for Voges-Proskauer (VP) Reaction . 24
B.14 Tryptone Soya Yeast Extract Agar (TSYEA) . 25
Annex C (informative) Distinction of Listeria spp. from other genera . 26
Annex D (informative) Reactions for the identification of species of Listeria . 27
Annex E (informative) Listeria selective agars . 29
Annex F (informative) Results of the interlaboratory studies . 31
F.1 Results of interlaboratory studies in the frame of CEN Mandate M381 . 31
F.2 Values of LOD from literature . 33
50%
Bibliography . 35
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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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 11290-1 was prepared by Technical Committee CEN/TC 275, Food analysis - Horizontal methods, and by
Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology in collaboration.
ISO 11290 consists of the following parts, under the general title Microbiology of the food chain — Horizontal
method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.:
Part 1: Detection method
Part 2: Enumeration method
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Introduction
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, every attempt should be made to apply this horizontal method as far as possible and that deviations
from this will only be made if absolutely necessary for justified technical reasons.
When this part of ISO 11290 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 part of ISO 11290 so
that eventually the only remaining departures from this horizontal method will be those necessary for
well-established technical reasons.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 11290-1
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 strongly recommended that
tests for detecting L. monocytogenes and Listeria spp. are undertaken in laboratories providing
biosafety level 2 conditions, under the control of a skilled microbiologist, and that great care is taken
in the disposal of all contaminated materials. In particular, 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. National legislation may involve more
specific demands. Only L. monocytogenes and L. ivanovii can give rise to listeriosis.
1 Scope
This part of ISO 11290 specifies a horizontal method for
the detection of L. monocytogenes;
the detection of Listeria spp.
This part of ISO 11290 is applicable to:
products intended for human consumption and for the feeding of animals;
environmental samples in the area of food production and food handling.
NOTE Certain new Listeria species may not be detected or confirmed by this method (see references [4, 9, 11, 13]).
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 6887, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination (all parts).
ISO 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations.
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media.
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Listeria monocytogenes
microorganism 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 part
of ISO 11290.
3.2
detection of Listeria monocytogenes
determination of the presence or absence 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 part of ISO 11290.
3.3
Listeria spp.
microorganism 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 part
of ISO 11290.
Certain new Listeria species may not be detected or confirmed by this method (see References [4, 9, 11, 13]).
3.4
detection of Listeria spp.
determination of the presence or absence 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 part of ISO 11290.
4 Principle
Listeria spp. may be present in small numbers and are often accompanied by considerably larger numbers of
other genera, therefore selective enrichment is necessary. It is also necessary to detect injured Listeria spp.
and the primary selective enrichment medium, with reduced inhibitor concentration, fulfils at least part of this
function.
Within the limits of this part of ISO 11290, the detection of L. monocytogenes and of Listeria spp. necessitates
four successive stages (see Annex A for a flowchart).
4.1 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), which is also used as a dilution fluid for the test portion (9.1).
Incubation of the initial suspension at 30 C ± 1 °C for 24 h ± 2 h.
4.2 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 4.1.
Incubation of the Fraser broth at 37 C ± 1 °C for 24 h ± 2 h.
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4.3 Plating out and identification
From the cultures obtained in 4.1 and 4.2, plating out on the two selective solid media:
Agar Listeria according to Ottaviani and Agosti (see Reference 1 and B.3);
any other solid selective medium at the choice of the laboratory complementary to Agar Listeria according
to Ottaviani and Agosti, such as for example Oxford, PALCAM, or another medium using a different
substrate than the one used in Listeria agar according to Ottaviani and Agosti (B.4). See Annex E for
some information about media.
Incubation of the Agar Listeria according to Ottaviani and Agosti at 37 °C ± 1 °C and examination after
24 h ± 2 h, and after a further 24 h ± 2 h, to check for the presence of characteristic colonies which are
presumed to be L. monocytogenes or Listeria spp. Incubation of the second selective medium at the
appropriate temperature and examination after the appropriate time.
4.4 Confirmation
Subculturing of the colonies of presumptive L. monocytogenes or Listeria spp., plated out as described in 4.3,
and confirmation by means of appropriate morphological and biochemical tests.
5 Culture media and reagents
For current laboratory practice, see ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
6 Apparatus and glassware
Usual microbiological equipment (see ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)
See ISO 7218.
6.2 Incubators, capable of operating at 30 C ± 1 C, 37 C 1 C and at 25 °C ± 1 °C.
6.3 Waterbath, capable of operating at 47 °C to 50 °C.
6.4 Drying cabinet or incubator, capable of being maintained between 25 C and 50 C.
6.5 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.
6.6 Loops, of platinum/iridium or nickel/chromium, approximately 3 mm in diameter or 10 µl, and wires of
the same material, or single-use loops.
6.7 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml and 1 ml, graduated
respectively in 0,5 ml and 0,1 ml divisions.
6.8 Microscope, preferably with phase-contrast, and with slides and coverslips.
6.9 Petri dishes, of diameter 90 mm.
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7 Sampling
Sampling is not part of the method specified in this part of ISO 11290. If there is no specific International
Standard dealing with sampling of the product concerned, it is recommended that the parties concerned come
to an agreement on this subject. For environmental samples, refer to ISO18593 [12].
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 appropriate to the product
concerned. 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
See ISO 6887 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 x g or x ml to 9x g or 9x ml of the selective
primary enrichment medium (B.1), to obtain a ratio of test portion to selective primary enrichment medium
of 1:9 (mass to volume or volume to volume).
9.2 Primary enrichment
Incubate the primary enrichment medium (half-Fraser broth), prepared in accordance with 9.1, at 30 C ± 1 °C
for 24 h 2 h.
NOTE 1 A black coloration may develop during the incubation.
NOTE 2 A minimum of 24 h incubation is recommended, in particular in the case of stress or low contamination levels
of Listeria spp. in the samples.
9.3 Secondary enrichment
9.3.1 After incubation of the initial suspension (primary enrichment) for 24 h ± 2 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 ± 1 °C.
NOTE 1 In the case of Listeria spp. detection, additional 24 h incubation may allow recovery of more species.
NOTE 2 Half-Fraser broth may be refrigerated before transfer to Fraser broth during a maximum of 72 h, if (i) a
validation study for such storage of the commercially available broth is available or (ii) the laboratory has performed a
verification study of the method using such storage, according to ISO 16140-3 [1].
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9.4 Plating out and identification
9.4.1 From the primary enrichment culture incubated for 24 h ± 2 h at 30 °C, inoculate, by means of a
loop (6.6), 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 may be refrigerated before isolation on selective agar during a maximum
of 72 h, if (i) a validation study for such storage of the commercially available broth is available or (ii) the laboratory has
performed a verification study of the method using such storage, according to ISO 16140-3 [1].
9.4.2 From the secondary enrichment medium incubated for 24 h ± 2 h at 37 C ± 1 °C (9.3.2), repeat the
procedure described in 9.4.1 with the two selective plating-out media.
9.4.3 Invert the Petri dishes obtained in 9.4.1 and 9.4.2 and place them in an incubator set at 37 °C ± 1 °C
for Agar Listeria according to Ottaviani and Agosti (B.3) and for the second selective medium (B.4), follow the
manufacturer's instructions.
9.4.4 After incubation for 24 h ± 2 h and for an additional 24 h ± 2 h for Agar Listeria according to Ottaviani
and Agosti or for the appropriate time (second selective agar), examine the dishes (9.4.3) for the presence of
colonies presumed to be L. monocytogenes and/or Listeria spp. Incubated plates can be refrigerated for a
maximum of 2 days before reading.
9.4.4.1 Agar Listeria according to Ottaviani and Agosti
Consider as 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.
NOTE 1 Some strains of L. monocytogenes show a very weak halo (even no halo) in cases of stress, in particular acid
stress.
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, 4 days. Some of these
strains could be pathogenic (see Reference [2]). No L. monocytogenes strains have been described as PIPLC negative.
Consider as Listeria spp. blue-green colonies with or without opaque halo.
NOTE 3 Some new Listeria species (such as L. rocourtiae), which have been recently characterized, may not grow on
Agar Listeria according to Ottaviani and Agosti (see Reference [13]).
NOTE 4 Some organisms other than Listeria spp. may produce blue colonies on this medium. See Annex C.
9.4.4.2 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.
9.5 Confirmation of Listeria monocytogenes and/or Listeria spp.
9.5.1 Selection of colonies for confirmation
9.5.1.1 For confirmation of presumptive L. monocytogenes, take from each plate of each selective
medium (see 9.4.4.1 and 9.4.4.2) at least one colony presumed to be L. monocytogenes and a further four
colonies if the first is negative. One confirmed isolate per sample is sufficient.
9.5.1.2 For confirmation of presumptive Listeria spp., take from each plate of each selective medium (see
9.4.4.1 and 9.4.4.2) at least one colony presumed to be Listeria spp. and a further four colonies if the first is
negative. One confirmed isolate per sample is sufficient.
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9.5.1.3 Streak the selected colonies (9.5.1.1 or 9.5.1.2) onto the surface of pre-dried plates of a non-
selective agar, for example blood agar, nutrient agar, tryptone soya yeast extract agar (TSYEA), in a manner
which will allow well-separated colonies to develop.
Place the plates in the incubator set at 37 C ± 1 °C for 18 h to 24 h or until growth is satisfactory.
Typical colonies of Listeria spp. are 1 mm to 2 mm in diameter, convex, colourless and opaque with an entire
edge. On TSYEA (B.14), 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 well separated, pick a typical
Listeria spp. colon
...
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