SIST EN ISO 11290-2:2017
(Main)Microbiology of the food chain - Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 2: Enumeration method (ISO 11290-2:2017)
Microbiology of the food chain - Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 2: Enumeration method (ISO 11290-2:2017)
This standard describes the enumeration of Listeria monocytogenes (Reference document: EN/ISO 11290 -2 incl./Amd. 1)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die Zählung von Listeria monocytogenes und von Listeria spp. - Teil 2: Zählverfahren (ISO 11290-2:2017)
Dieser Teil von ISO 11290 legt ein horizontales Verfahren fest für:
— die Zählung von L. monocytogenes;
— die Zählung von Listeria spp. (einschließlich L. monocytogenes).
Dieser Teil von ISO 11290 gilt für:
— Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind;
— Umgebungsproben im Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln.
Bestimmte zusätzlich beschriebene Listeria Arten sind mit diesem Verfahren möglicherweise nicht zählbar oder bestätigbar (siehe Literaturhinweise [3], [6], [9], [11]).
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes et de Listeria spp. - Partie 2: Méthode de dénombrement (ISO 11290-2:2017)
ISO 11290-2:2017 spécifie une méthode horizontale pour:
- le dénombrement de L. monocytogenes; et
- le dénombrement de Listeria spp. (y compris L. monocytogenes).
ISO 11290-2:2017 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. - 2. del: Metoda štetja (ISO 11290-2:2017)
Ta standard opisuje način ugotavljanja števila Listeria monocytogenes (referenčni dokument: EN/ISO 11290 -2 vklj./dod. 1)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11290-2:2017
01-september-2017
1DGRPHãþD
SIST EN ISO 11290-2:1999
SIST EN ISO 11290-2:1999/A1:2005
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Listeria monocytogenes in Listeria spp. - 2. del: Metoda štetja
(ISO 11290-2:2017)
Microbiology of the food chain - Horizontal method for the detection and enumeration of
Listeria monocytogenes and of Listeria spp. - Part 2: Enumeration method (ISO 11290-
2:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die
Zählung von Listeria monocytogenes und von Listeria spp. - Teil 2: Zählverfahren (ISO
11290-2:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Listeria monocytogenes et de Listeria spp. - Partie 2: Méthode de
dénombrement (ISO 11290-2:2017)
Ta slovenski standard je istoveten z: EN ISO 11290-2:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 11290-2:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11290-2:2017
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SIST EN ISO 11290-2:2017
EN ISO 11290-2
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 11290-2:1998
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Listeria monocytogenes and
of Listeria spp. - Part 2: Enumeration method (ISO 11290-
2:2017)
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 2: Listeria monocytogenes und von Listeria spp. - Teil 2:
Méthode de dénombrement (ISO 11290-2:2017) Zählverfahren (ISO 11290-2:2017)
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 11290-2:2017 E
worldwide for CEN national Members.
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SIST EN ISO 11290-2:2017
EN ISO 11290-2:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 11290-2:2017
EN ISO 11290-2:2017 (E)
European foreword
This document (EN ISO 11290-2:2017) 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 11290-2:1998.
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 11290-2:2017 has been approved by CEN as EN ISO 11290-2:2017 without any
modification.
3
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SIST EN ISO 11290-2:2017
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SIST EN ISO 11290-2:2017
INTERNATIONAL ISO
STANDARD 11290-2
Second edition
2017-05
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of Listeria
monocytogenes and of Listeria spp. —
Part 2:
Enumeration method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Listeria monocytogenes et de
Listeria spp. —
Partie 2: Méthode de dénombrement
Reference number
ISO 11290-2:2017(E)
©
ISO 2017
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(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.
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Tel. +41 22 749 01 11
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copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Initial suspension . 2
4.3 Surface plating . 2
4.4 Incubation . 2
4.5 Confirmation . 3
4.6 Enumeration . 3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 3
8 Preparation of test sample . 4
9 Procedure. 4
9.1 Test portion, initial suspension and dilutions . 4
9.2 Inoculation and incubation . 4
9.3 Enumeration of characteristic colonies . 4
9.4 Confirmation of L. monocytogenes or Listeria spp. . 5
9.4.1 Selection of colonies for confirmation . 5
9.4.2 Confirmation of L. monocytogenes . 6
9.4.3 Confirmation of Listeria spp. . 9
9.5 Interpretation of morphological and physiological properties and of the
biochemical reactions .10
9.6 Additional characterization of isolated strains (optional) .10
10 Expression of results .10
11 Performance characteristics of the method .10
11.1 Method validation study .10
11.2 Repeatability limit .11
11.3 Reproducibility limit .11
12 Test report .11
13 Quality assurance .12
Annex A (normative) Diagram of procedure .13
Annex B (normative) Composition and preparation of media and reagents .14
Annex C (informative) Distinction of Listeria spp. from other genera .22
Annex D (informative) Reactions for the identification of Listeria species .23
Annex E (informative) Results of interlaboratory studies for enumeration of
Listeria monocytogenes .25
Bibliography .28
© ISO 2017 – All rights reserved iii
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(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 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: www . i so .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 11290-2:1998), which has been technically
revised. It also incorporates the amendment ISO 11290-2:1998/Amd.1:2004.
The main changes, compared to ISO 11290-2:1998, are the following.
— The enumeration of Listeria monocytogenes has been modified as listed below.
— Primary suspension with buffered peptone water, half-Fraser broth with or without supplements,
and all appropriate diluents referred to in ISO 6887 (all parts).
— Resuscitation step deleted.
— Microscopic aspect, catalase and CAMP test for confirmation are optional.
— Inclusion of new performance characteristics.
— Moreover, enumeration 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.
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(E)
Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO 11290-2:1998
[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.
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SIST EN ISO 11290-2:2017
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SIST EN ISO 11290-2:2017
INTERNATIONAL STANDARD ISO 11290-2:2017(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Listeria monocytogenes
and of Listeria spp. —
Part 2:
Enumeration 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 enumeration of L. monocytogenes, and
— the enumeration 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 enumerated or confirmed
[3],[6],[9],[11]
by 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
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(E)
ISO 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
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 described and which display the
morphological, physiological and biochemical characteristics described when the analysis is carried
out in accordance with this document
3.2
enumeration of Listeria monocytogenes
determination of the number of colony-forming units (cfu) of Listeria monocytogenes, per gram, per
millilitre, per square centimetre, or per sampling device when the analysis is 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
enumeration of Listeria spp.
determination of the number of colony-forming units (cfu) of Listeria spp per gram, per millilitre, per
square centimetre, or per sampling device, when the analysis is carried out in accordance with this
document
4 Principle
4.1 General
Within the limits of this document, the enumeration of L. monocytogenes and of Listeria spp. requires
five successive steps, as described in the flowchart in Annex A.
4.2 Initial suspension
Preparation of the initial suspension in an appropriate diluent according to the sample type.
4.3 Surface plating
[13],[14]
Surface plating on Agar Listeria according to Ottaviani and Agosti of a specified quantity of the
test sample for liquid products or of the initial suspension for other products and/or decimal dilutions
if required.
4.4 Incubation
Incubation of the Petri dishes at 37 °C and examination after 24 h and after a further 24 h.
2 © ISO 2017 – All rights reserved
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(E)
4.5 Confirmation
Confirmation of presumptive colonies of L. monocytogenes and/or of presumptive Listeria spp. by means
of appropriate morphological and/or biochemical tests.
4.6 Enumeration
From the number of confirmed colonies, calculation of the number of L. monocytogenes and/or of
Listeria spp. per gram, per millilitre, per square centimetre, or per sampling device.
5 Culture media and reagents
For current laboratory practices, refer to ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
6 Equipment and consumables
Usual microbiological laboratory apparatus (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 37 °C ± 1 °C and 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 Glass or plastic spreaders, sterile.
6.7 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.
6.8 Total-delivery graduated pipettes or automatic pipettes, of nominal capacities 1 ml and 10 ml.
6.9 Petri dishes, of diameter 90 mm and/or 140 mm.
6.10 Microscope, preferably with phase-contrast, and with slides and cover slips.
6.11 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
concerned come to an agreement on this subject. For food and feed samples, refer to ISO/TS 17728. For
[23]
environmental samples, use ISO 18593 and see Reference .
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).
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SIST EN ISO 11290-2:2017
ISO 11290-2:2017(E)
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product
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, initial suspension and dilutions
Buffered peptone water, as well as other appropriate diluents referred to in ISO 6887 (all parts) and any
specific International Standard appropriate to the product concerned, may be used as diluent for the
initial suspension.
Half-Fraser broth (as specified in ISO 11290-1), supplemented with selective agents or not, may be used
as a diluent for the initial suspension when both the detection method (as specified in ISO 11290-1) and
this enumeration method are carried out on the same test sample. The selective agents should be added
(if required) to the suspension preferentially after enumeration, prior to the detection method.
If supplemented half-Fraser is used, inoculate the plates as soon as possible, up to 45 min.
9.2 Inoculation and incubation
9.2.1 Distribute, by means of a sterile pipette (6.8), 0,1 ml of the initial suspension (or sample if liquid)
and 0,1 ml of further decimal dilutions each inoculated onto the surface of a small dish (90 mm) of Agar
Listeria according to Ottaviani and Agosti (see B.2).
When, for certain products, it is desirable to estimate low numbers of L. monocytogenes and/or Listeria
spp., the limits of detection may be raised by a factor of 10 by examining 1 ml of the test sample if the
initial product is liquid, or 1 ml of the initial suspension for the other products. Distribute the 1 ml of
inoculum either on the surface of a large Petri dish (140 mm) or over the surface of three small dishes
(90 mm), dried beforehand if necessary in the incubator (6.2). If only the initial suspension is used, also
prepare duplicate plates using an additional three small Petri dishes or one large dish of medium (see
ISO 7218).
Repeat the procedure using 0,1 ml of the initial suspension (or sample if liquid) and 0,1 ml of further
decimal dilutions if necessary each inoculated onto the surface of a small dish (90 mm) of agar medium.
9.2.2 Carefully spread the inoculum as quickly as possible over the surface of the agar plate without
touching the sides of the dish with the spreader (6.6). Use a fresh sterile spreader for each dilution. Leave
the plates closed and upright for about 15 min at ambient temperature for the inoculum to be absorbed
into the agar.
It is possible to use the same spreader for all the dishes of a given sample, by beginning with the higher
dilution.
9.2.3 Incubate the Agar Listeria according to Ottaviani and Agosti plates prepared in 9.2.2 inverted at
37 °C (6.3) for 24 h ± 2 h and for an additional incubation at 37°C for 24 ± 2h.
9.3 Enumeration of characteristic colonies
9.3.1 After incubation for 24 h ± 2 h (bef
...
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