SIST EN ISO 6579-1:2017
(Main)Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. (ISO 6579-1:2017)
Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. (ISO 6579-1:2017)
This document specifies a horizontal method for the detection of Salmonella. It is applicable to the
following:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling;
— samples from the primary production stage such as animal faeces, dust, and swabs.
With this horizontal method, most of the Salmonella serovars are intended to be detected. For the
detection of some specific serovars, additional culture steps may be needed. For Salmonella Typhi and
Salmonella Paratyphi, the procedure is described in Annex D.
The selective enrichment medium modified semi-solid Rappaport-Vassiliadis (MSRV) agar is intended
for the detection of motile Salmonella and is not appropriate for the detection of non-motile Salmonella
strains.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis, zur Zählung und zur Serotypisierung von Salmonellen - Teil 1: Nachweis von Salmonella spp. (ISO 6579-1:2017)
Dieses Dokument legt ein horizontales Verfahren zum Nachweis von Salmonellen fest. Es gilt für Folgendes:
- Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind;
- Umgebungsproben im Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln;
- Proben aus der Primärproduktion, wie Tierkot, Staub und Tupfer.
Mit diesem horizontalen Verfahren sollen die meisten der Salmonella Serovare nachgewiesen werden. Für den Nachweis einiger spezifischer Serovare können zusätzliche Kultivierungsschritte notwendig sein. Für Salmonella Typhi und Salmonella Paratyphi ist die Durchführung in Anhang D beschrieben.
Das selektive Anreicherungsmedium modifizierter halbfester Rappaport Vassiliadis(MSRV) Agar ist für den Nachweis von beweglichen Salmonellen vorgesehen und ist für den Nachweis von unbeweglichen Salmonellen Stämmen nicht geeignet.
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le dénombrement et le sérotypage des Salmonella - Partie 1: Recherche des Salmonella spp. (ISO 6579-1:2017)
ISO 6759-1:2017 spécifie une méthode horizontale de recherche des Salmonella. Il s'applique aux:
- produits destinés à la consommation humaine et à l'alimentation animale.
- échantillons environnementaux dans le domaine de la production et de la manutention de denrées alimentaires.
- échantillons au stade de la production primaire, tels que des matières fécales, de la poussière ou des prélèvements de surface.
Cette méthode horizontale vise à rechercher la plupart des sérovars de Salmonella. Des étapes de culture supplémentaires peuvent être nécessaires pour la recherche de certains sérovars spécifiques. Pour Salmonella Typhi et Salmonella Paratyphi, le mode opératoire est décrit en Annexe D.
Le milieu d'enrichissement sélectif MSRV, gélose de Rappaport‐Vassiliadis semi‐solide modifiée, est conçu pour la recherche des Salmonella mobiles et n'est pas adapté à la détection des Salmonella immobiles.
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti, števila in serotipov Salmonella - 1. del: Ugotavljanje prisotnosti Salmonella spp. (ISO 6579-1:2017)
Ta dokument določa horizontalno metodo za ugotavljanje prisotnosti bakterij Salmonella. Uporablja se
za:
– izdelke, namenjene za prehrano ljudi in krmo živali;
– okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano;
– vzorce primarne stopnje proizvodnje (npr. živalski iztrebki, prah in brisi).
S to horizontalno metodo se predvidoma ugotovi prisotnost večine serovarjev Salmonella. Za ugotavljanje
prisotnosti nekaterih posebnih serovarjev bodo morda potrebi dodatni koraki kulture. Postopek za bakterije Salmonella Typhi in Salmonella Paratyphi je opisan v dodatku D.
Medij selektivnega bogatenja, spremenjen poltrd agar Rappaport–Vassiliadis (MSRV), je namenjen za odkrivanje gibljivih sevov bakterij Salmonella in ni primeren za odkrivanje negibljivih sevov bakterij Salmonella.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 6579-1:2017
01-maj-2017
1DGRPHãþD
SIST EN ISO 6579:2003
SIST EN ISO 6579:2003/A1:2007
SIST EN ISO 6579:2003/AC:2004
SIST EN ISO 6579:2003/AC:2006
SIST EN ISO 6785:2007
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti, števila in serotipov Salmonella - 1. del: Ugotavljanje prisotnosti
Salmonella spp. (ISO 6579-1:2017)
Microbiology of the food chain - Horizontal method for the detection, enumeration and
serotyping of Salmonella - Part 1: Detection of Salmonella spp. (ISO 6579-1:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis, zur
Zählung und zur Serotypisierung von Salmonellen - Teil 1: Nachweis von Salmonella
spp. (ISO 6579-1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le
dénombrement et le sérotypage des Salmonella - Partie 1: Recherche des Salmonella
spp. (ISO 6579-1:2017)
Ta slovenski standard je istoveten z: EN ISO 6579-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 6579-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 6579-1:2017
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SIST EN ISO 6579-1:2017
EN ISO 6579-1
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 6579:2002, EN ISO 6785:2007
English Version
Microbiology of the food chain - Horizontal method for the
detection, enumeration and serotyping of Salmonella - Part
1: Detection of Salmonella spp. (ISO 6579-1:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche, le dénombrement et le Verfahren zum Nachweis, zur Zählung und zur
sérotypage des Salmonella - Partie 1: Recherche des Serotypisierung von Salmonellen - Teil 1: Nachweis
Salmonella spp. (ISO 6579-1:2017) von Salmonella spp. (ISO 6579-1:2017)
This European Standard was approved by CEN on 3 February 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 6579-1:2017 E
worldwide for CEN national Members.
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SIST EN ISO 6579-1:2017
EN ISO 6579-1:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 6579-1:2017
EN ISO 6579-1:2017 (E)
European foreword
This document (EN ISO 6579-1:2017) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical
Committee ISO/TC 34 "Food products".
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 September 2017 and conflicting national standards
shall be withdrawn at the latest by September 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 6579:2002 and EN 6785:2007.
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 6579-1:2017 has been approved by CEN as EN ISO 6579-1:2017 without any
modification.
3
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SIST EN ISO 6579-1:2017
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SIST EN ISO 6579-1:2017
INTERNATIONAL ISO
STANDARD 6579-1
First edition
2017-02
Microbiology of the food chain —
Horizontal method for the detection,
enumeration and serotyping of
Salmonella —
Part 1:
Detection of Salmonella spp.
Microbiologie de la chaîne alimentaire — Méthode horizontale
pour la recherche, le dénombrement et le sérotypage des
Salmonella —
Partie 1: Recherche des Salmonella spp.
Reference number
ISO 6579-1:2017(E)
©
ISO 2017
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(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
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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 6579-1:2017
ISO 6579-1:2017(E)
Contents Page
Foreword .v
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Pre-enrichment in non-selective liquid medium . 2
4.3 Enrichment in/on selective media . 2
4.4 Plating out on selective solid media . 2
4.5 Confirmation . 3
5 Culture media, reagents, and antisera . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure (see diagrams in Annex A) . 4
9.1 Test portion and initial suspension . 4
9.2 Non-selective pre-enrichment . 4
9.3 Selective enrichment. 5
9.3.1 General. 5
9.3.2 Procedure for food, animal feed samples, and environmental samples
from the food production area . 5
9.3.3 Procedure for samples from the primary production stage . 5
9.4 Plating out . 6
9.4.1 General. 6
9.4.2 Procedure for food, animal feed samples, and environmental samples
from the food production area . 6
9.4.3 Procedure for samples from the primary production stage . 6
9.5 Confirmation . 7
9.5.1 General. 7
9.5.2 Selection of colonies for confirmation . 7
9.5.3 Biochemical testing . 8
9.5.4 Serological testing . . .11
9.5.5 Interpretation of biochemical and serological reactions .11
9.5.6 Serotyping.12
10 Expression of results .12
11 Performance characteristics of the method .12
11.1 Interlaboratory studies .12
11.2 Sensitivity .12
11.3 Specificity .12
11.4 LOD .
50 12
12 Test report .13
Annex A (normative) Diagrams of the procedures .14
Annex B (normative) Culture media and reagents .17
Annex C (informative) Method validation studies and performance characteristics .32
Annex D (normative) Detection of Salmonella enterica subspecies enterica serovars Typhi
and Paratyphi .38
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
Annex E (informative) Examples of selective plating-out media .43
Bibliography .48
iv © ISO 2017 – All rights reserved
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(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 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 TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This first edition of ISO 6579-1 cancels and replaces ISO 6579:2002 and ISO 6785:2001, which have been
technically revised. It also incorporates ISO 6579:2002/Amd 1:2007 and ISO 6579:2002/Cor 1:2004.
The main changes, compared to ISO 6579:2002, are the following.
— ISO 6785 has been incorporated in this document.
— Samples from the primary production stage have been added to the scope.
— Detection of Salmonella Typhi and Salmonella Paratyphi is described in Annex D.
— Descriptions of preparations of initial suspensions have been removed and references made to
relevant parts of ISO 6887, whenever possible.
— The temperature range for incubation of non-selective media has been extended from 37 °C ± 1 °C
to 34 °C to 38 °C without further tolerance.
— For selective enrichment, there is a choice between using the broth or the semi-solid agar of
Rappaport Vassiliadis medium (RVS or MSRV) for food, animal feed samples, and for environmental
samples from the food production area.
— The inoculation of the isolation medium has become less prescriptive; the objective is to obtain
well-isolated colonies after incubation.
— For confirmation, it is acceptable to perform the tests on only one suspect colony (instead of one
suspect colony of each medium combination). If this isolate tests negative for Salmonella, four more
suspect isolates from different media combinations shall be tested.
© ISO 2017 – All rights reserved v
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
— It is permitted to perform the biochemical confirmation directly on a suspect, well-isolated colony
from the selective plating medium. The purity check on the non-selective agar medium can then be
performed in parallel.
— Two confirmation tests have become optional ( ß-galactosidase test and indole reaction) and one
confirmation test has been deleted (Voges-Proskauer reaction).
— In this document, serological confirmation (to serogroup level) is described. For guidance on
serotyping (to serovar level), reference is made to ISO/TR 6579-3.
— Table 1 has been improved.
— Performance testing for the quality assurance of the culture media has been added to Annex B.
— Performance characteristics of MSRV have been added to Annex C.
A list of all parts in the ISO 6579 series can be found on the ISO website.
vi © ISO 2017 – All rights reserved
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
Introduction
This document describes a horizontal method for the detection of Salmonella spp. in food (including
milk and milk products, originally described in ISO 6785), in animal feed, in animal faeces, and in
environmental samples from the primary production stage (the latter two were originally described in
ISO 6579:2002/Amd 1:2007).
The main changes, listed in the foreword, introduced in this document compared to ISO 6579:2002, are
[37]
considered as minor (see ISO 17468 ).
[3]
A procedure for the enumeration of Salmonella spp. is described in ISO/TS 6579-2.
[24]
Guidance for serotyping of Salmonella spp. is described in ISO/TR 6579-3.
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SIST EN ISO 6579-1:2017
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SIST EN ISO 6579-1:2017
INTERNATIONAL STANDARD ISO 6579-1:2017(E)
Microbiology of the food chain — Horizontal method
for the detection, enumeration and serotyping of
Salmonella —
Part 1:
Detection of Salmonella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for detecting Salmonella 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 and to
ensure compliance with any national regulatory conditions.
1 Scope
This document specifies a horizontal method for the detection of Salmonella. It is applicable to the
following:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling;
— samples from the primary production stage such as animal faeces, dust, and swabs.
With this horizontal method, most of the Salmonella serovars are intended to be detected. For the
detection of some specific serovars, additional culture steps may be needed. For Salmonella Typhi and
Salmonella Paratyphi, the procedure is described in Annex D.
The selective enrichment medium modified semi-solid Rappaport-Vassiliadis (MSRV) agar is intended
for the detection of motile Salmonella and is not appropriate for the detection of non-motile Salmonella
strains.
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 food and animal feed — 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 11133:2014, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2017 – All rights reserved 1
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
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
Salmonella
microorganism which forms typical or less typical colonies on solid selective media and which displays
the characteristics described when confirmation tests are carried out in accordance with this document
3.2
detection of Salmonella
determination of Salmonella (3.1), in a particular mass or volume of product or surface area or object
(e.g. boot socks), when tests are carried out in accordance with this document
4 Principle
4.1 General
The detection of Salmonella requires four successive stages as specified in Annex A.
NOTE Salmonella can be present in small numbers and is often accompanied by considerably larger numbers
of other Enterobacteriaceae or bacteria of other families. Pre-enrichment is used to permit the detection of low
numbers of Salmonella or injured Salmonella.
4.2 Pre-enrichment in non-selective liquid medium
Buffered peptone water at ambient temperature is inoculated with the test portion, then incubated
between 34 °C and 38 °C for 18 h.
For large quantities (e.g. 1 l or more), it is recommended to pre-warm the BPW to 34 °C to 38 °C before
mixing it with the test portion.
4.3 Enrichment in/on selective media
Rappaport-Vassiliadis medium with soya (RVS broth) or Modified Semi-solid Rappaport-Vassiliadis
(MSRV) agar and Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn broth) are inoculated
with the culture obtained in 4.2.
The RVS broth or the MSRV agar is incubated at 41,5 °C for 24 h and the MKTTn broth at 37 °C for 24 h.
For some products, it may be necessary to incubate the selective enrichment medium/media for an
additional 24 h.
NOTE MSRV agar is intended for the detection of motile Salmonella strains and is not appropriate for the
detection of non-motile Salmonella strains.
4.4 Plating out on selective solid media
From the cultures obtained in 4.3, the following two selective solid media are inoculated:
— Xylose Lysine Deoxycholate agar (XLD agar);
— any other solid selective medium complementary to XLD agar (for examples, see Annex E).
The XLD agar is incubated at 37 °C and examined after 24 h. The second selective agar is incubated
according to the manufacturer’s instructions.
2 © ISO 2017 – All rights reserved
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
4.5 Confirmation
Colonies of presumptive Salmonella are subcultured and their identity is confirmed by means of
appropriate biochemical and serological tests.
5 Culture media, reagents, and antisera
For current laboratory practice, see ISO 7218 and ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see 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 oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating in the range 34 °C to 38 °C and at 37 °C ± 1 °C.
6.4 Incubator, capable of operating at 41,5 °C ± 1 °C or water bath capable of operating at
41,5 °C ± 1 °C.
6.5 Water bath, capable of operating at 47 °C to 50 °C.
6.6 Water bath, capable of operating at 37 °C ± 1 °C.
6.7 Water bath, capable of operating at 45 °C ± 1 °C.
It is recommended to use a water bath (6.4 to 6.7) containing an antibacterial agent because of the low
infective dose of Salmonella.
6.8 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.9 Freezer, capable of operating at -20 °C ± 5 °C.
6.10 Sterile loops, of approximate diameter, 3 mm (10 μl volume), and of 1 µl volume and inoculation
needle or wire.
6.11 pH-meter, having an accuracy of calibration of ±0,1 pH unit at 20 °C to 25 °C.
6.12 Sterile tubes, bottles, or flasks with caps of appropriate capacity.
6.13 Sterile graduated pipettes or automatic pipettes, of nominal capacities of 25 ml, 10 ml, 1 ml,
and 0,1 ml.
6.14 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
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SIST EN ISO 6579-1:2017
ISO 6579-1:2017(E)
7 Sampling
Sampling is not part of the method specified in this document (see the specific International Standard
dealing with the product concerned). If there is no specific International Standard, it is recommended
that the parties concerned come to an agreement on this subject.
[26] [27
A recommended sampling method is given in ISO/TS 17728 for food and animal feed, in ISO 707 ]
[28]
for milk and milk products, in ISO 13307 for sampling at the primary production stage, in
[29] [25]
ISO 17604 for sampling of carcasses, and in ISO 18593 for sampling of surfaces.
It is important that the laboratory receives a sample which is representative and has not been damaged
or changed during transport or storage.
8 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the produ
...
SLOVENSKI STANDARD
oSIST prEN ISO 6579-1:2014
01-september-2014
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti, števila in serotipov Salmonella - 1. del: Horizontalna metoda za
ugotavljanje prisotnosti Salmonella spp. (ISO/DIS 6579-1:2014)
Microbiology of the food chain - Horizontal method for the detection, enumeration and
serotyping of Salmonella - Part 1: Horizontal method for the detection of Salmonella spp.
(ISO/DIS 6579-1:2014)
Mikrobiolgie der Lebensmittelkette - Horizontales Verfahren zum Nachweis, zur Zählung
und zur Serotypisierung von Salmonellen - Teil 1: Nachweisverfahren (ISO/DIS 6579-
1:2014)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le
dénombrement et la sérotypie des Salmonella spp. - Partie 1: Méthode horizontale pour
la recherche des Salmonella spp. (ISO/DIS 6579-1:2014)
Ta slovenski standard je istoveten z: prEN ISO 6579-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 6579-1:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 6579-1:2014
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oSIST prEN ISO 6579-1:2014
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6579-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2014-06-05 2014-11-05
Microbiology of the food chain — Horizontal method
for the detection, enumeration and serotyping of
Salmonella —
Part 1:
Horizontal method for the detection of Salmonella spp.
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche, le dénombrement et la
sérotypie des Salmonella spp. —
Partie 1: Méthode horizontale pour la recherche des Salmonella spp.
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
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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
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©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2014
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Copyright notice
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oSIST prEN ISO 6579-1:2014
ISO/DIS 6579-1
Contents Page
Foreword . iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle. 2
4.1 General . 2
4.2 Pre-enrichment in non-selective liquid medium . 3
4.3 Enrichment in/on selective media . 3
4.4 Plating out and identification . 3
4.5 Confirmation of identity . 3
5 Culture media, reagents and sera . 3
5.1 General . 3
5.2 Culture media and reagents . 3
5.3 Antisera . 5
6 Apparatus and glassware . 5
7 Sampling. 6
8 Preparation of test sample . 6
9 Procedure (see diagrams in Annex A) . 6
9.1 Test portion and initial suspension . 6
9.2 Non-selective pre-enrichment . 7
9.3 Selective enrichment . 7
9.4 Plating out and identification . 8
9.5 Confirmation . 10
10 Expression of results . 13
11 Accuracy (precision) of the method . 14
11.1 Interlaboratory studies . 14
11.2 Sensitivity . 14
11.3 Specificity . 14
11.4 LOD . 14
50
12 Test report . 14
13 Quality assurance . 14
Annex A (normative) Diagrams of the procedures . 16
Annex B (normative) Composition and preparation of culture media and reagents . 18
Annex C (informative) Results of interlaboratory studies . 32
Annex D (normative) Detection of Salmonella enterica subspecies enterica serovars . 39
Annex E (informative) Information on some selective plating-out media[20] . 44
Bibliography . 47
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oSIST prEN ISO 6579-1:2014
ISO/DIS 6579-1
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 6579-1 was prepared by the European Committee for Standardization (CEN), in collaboration with
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).
ISO 6579 consists of the following parts, under the general title Microbiology of the food chain — Horizontal
method for the detection, enumeration and serotyping of Salmonella:
Part 1: Horizontal method for the detection of Salmonella spp.
Part 2: Enumeration by a miniaturized most probable number technique [Technical Specification]
Part 3: Guidelines for serotyping of Salmonella spp. [Technical Report]
This fifth edition cancels and replaces the fourth edition (ISO 6579:2002), which has been technically revised.
It also cancels and replaces ISO 6785:2007 [4] and Amendment 1 of ISO 6579 (2007) [2].
Annexes A and B and D form a normative part of this International Standard. Annexes C and E are for
information only.
Main changes in this fifth edition:
ISO 6785 has been incorporated in this version of ISO 6579-1;
samples from the primary production stage have been added to the scope;
detection of Salmonella Typhi and Salmonella Paratyphi is described in a normative annex;
for selective enrichment there is a choice between using the broth or the semi-solid agar of Rappaport
Vassiliadis medium (RVS or MSRV);
the inoculation of the isolation medium has become less prescriptive. The objective is to obtain well-
isolated colonies after incubation;
for confirmation it is acceptable to perform the tests on only one suspect colony (instead of one suspect
colony of each medium combination). If this isolate tests negative for Salmonella, 4 more suspect isolates
from different media combinations shall be tested;
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it is allowed to perform the biochemical confirmation directly on a suspect, well-isolated colony from the
selective plating medium. The purity check on the non-selective agar medium can then be performed in
parallel;
two confirmation tests have become optional (ß-galactosidase test and indole reaction), one confirmation
test has been deleted (Voges-Proskauer reaction).
in this part of ISO 6579, serological confirmation (to serogroup level) is described. For guidance on
serotyping (to serovar level) reference is made to part 3 of ISO 6579;
Table 1 (Interpretation of biochemical tests) has been improved;
performance testing for the quality assurance of the culture media has been added to Annex B;
performance characteristics of MSRV have been added to Annex C.
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oSIST prEN ISO 6579-1:2014
ISO/DIS 6579-1
Introduction
This part of ISO 6579 describes a horizontal method for the detection of Salmonella spp. in food (including
milk and milk products, originally described in ISO 6785), in animal feed, in animal faeces and in
environmental samples from the primary production stage (the latter two were originally described in
Amendment 1 of ISO 6579:2002).
With this horizontal method, most of the Salmonella serovars will be detected. For the detection of some
specific serovars additional culture steps may be needed. For Salmonella Typhi and Salmonella Paratyphi this
is described in a separate annex.
A procedure for the enumeration of Salmonella spp. is described in part 2 of ISO/TS 6579 [3].
Guidance for serotyping of Salmonella spp. is described in part 3 of ISO/TR 6579.
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oSIST prEN ISO 6579-1:2014
DRAFT INTERNATIONAL STANDARD ISO/DIS 6579-1
Microbiology of the food chain — Horizontal method for the
detection, enumeration and serotyping of Salmonella — Part 1:
Horizontal method for the detection of Salmonella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting Salmonella 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 International Standard should be familiar with normal laboratory practice. This
standard 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 and to ensure
compliance with any national regulatory conditions.
1 Scope
This International Standard specifies a horizontal method for the detection of Salmonella. Additional culture
steps for the detection of Salmonella Typhi and Salmonella Paratyphi are specified in an Annex of this
International Standard.
Subject to the limitations discussed in the Introduction, this International Standard is applicable to:
products intended for human consumption and the feeding of animals;
environmental samples in the area of food production and food handling.
Samples from the primary production stage, such as animal faeces, dust, swabs.
The selective enrichment medium for detection of Salmonella in samples from the primary production stage
(MSRV) is intended for the detection of motile Salmonellae and is not appropriate for the detection of non-
motile Salmonellae and/or brilliant-green sensitive Salmonella strains.
2 Normative references
The following referenced documents are indispensable for the application 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 707, Milk and milk products: guidance on sampling
ISO/TR 6579-3, Microbiology of the food chain — Horizontal method for the detection, enumeration and
serotyping of Salmonella — Part 3 Guidelines for serotyping of Salmonella spp.
ISO 6887-1, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension
and decimal dilutions
ISO 6887-2, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 2: Specific rules for the preparation of meat and meat
products
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ISO 6887-3, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and fishery
products
ISO 6887-4, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 4: Specific rules for the preparation of miscellaneous products
ISO 6887-5, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 5: Specific rules for the preparation of milk and
milk products
ISO 6887-6, Microbiology of food and animal feed — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 6: Specific rules for the preparation of samples taken
at the primary production stage
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 13307, Microbiology of food and animal feed — Primary production stage — Sampling techniques
ISO 17604, Microbiology of the food chain — Carcass sampling for microbiological analysis
ISO/TS 17728, Microbiology of the food chain — Sampling techniques for microbiological analysis of food and
feed samples
ISO 18593, Microbiology of food and animal feeding stuffs — Horizontal method for sampling techniques from
surfaces using contact plates and swabs
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Salmonella
microorganisms which form typical or less typical colonies on solid selective media and which display the
biochemical and serological characteristics described when tests are carried out in accordance with this
International Standard
3.2
detection of Salmonella
determination of Salmonella (3.1), in a particular mass or volume of product or surface area or object (e.g.
bootsocks), when tests are carried out in accordance with this International Standard
4 Principle
4.1 General
The detection of Salmonella necessitates four successive stages (see also Annex A).
NOTE Salmonella can be present in small numbers and is often accompanied by considerably larger numbers of
other Enterobacteriaceæ or other families. Furthermore, pre-enrichment is used to permit the detection of low numbers of
Salmonella or injured Salmonella.
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4.2 Pre-enrichment in non-selective liquid medium
Buffered peptone water at ambient temperature, is inoculated with the test portion, then incubated between
34 °C and 38°C for 18 h.
For certain foodstuffs the use of other pre-enrichment procedures is necessary. See 9.1.2.
For large quantities (e.g. 1 litre or more), it is recommended to pre-heat, the buffered peptone water to 34 °C
to 38 °C before inoculating it with the test portion.
4.3 Enrichment in/on selective media
Rappaport-Vassiliadis medium with soya (RVS broth) or Modified semi-solid Rappaport-Vassiliadis (MSRV)
agar and Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn broth) are inoculated with the culture
obtained in 4.2.
The RVS broth or the MSRV agar is incubated at 41,5 °C for 24 h and the MKTTn broth at 37 °C for 24 h.
For some products it may be necessary to incubate the selective enrichment medium/media for an additional
24 h.
NOTE MSRV is intended for the detection of motile Salmonellae and is not appropriate for the detection of non-motile
Salmonellae and/or brilliant-green sensitive Salmonella strains.
4.4 Plating out and identification
From the cultures obtained in 4.3, two selective solid media are inoculated:
Xylose lysine deoxycholate agar (XLD agar);
any other solid selective medium complementary to XLD agar (see Annex E).
The XLD agar is incubated at 37 °C and examined after 24 h. The second selective agar is incubated
according to the manufacturer's recommendations.
4.5 Confirmation of identity
Colonies of presumptive Salmonella are subcultured and their identity is confirmed by means of appropriate
biochemical and serological tests.
5 Culture media, reagents and sera
5.1 General
For current laboratory practice, see ISO 7218.
5.2 Culture media and reagents
Because of the large number of culture media and reagents, it is considered preferable, for clarity, to give their
compositions and preparations in Annex B. Alternatively, dehydrated complete media or diluents may be
used. Follow, in that respect, the manufacturer’s instructions.
5.2.1 Non-selective pre-enrichment medium: Buffered peptone water
See B.1.
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5.2.2 First selective enrichment medium: Rappaport-Vassiliadis medium with soya (RVS broth) or
Modified semi-solid Rappaport-Vassiliadis (MSRV) agar
See B.2 or B.3.
5.2.3 Second selective enrichment medium: Muller-Kauffmann tetrathionate novobiocin broth
(MKTTn broth)
See B.4.
5.2.4 Solid selective plating-out media
5.2.4.1 First medium: Xylose lysine deoxycholate agar (XLD agar)
See B.5.
5.2.4.2 Second medium
The choice of the second appropriate medium is left to the discretion of the testing laboratory (see Annex E).
The manufacturer's instructions should be followed regarding its preparation for use.
5.2.5 Non-selective agar medium
The choice of the non-selective agar medium for purity check is left to the discretion of the testing laboratory.
The manufacturer’s instructions should be followed regarding its preparation for use. An example of a
non-selective agar medium is Nutrient agar (see B.6).
5.2.6 Triple sugar/iron agar (TSI agar)
See B.7.
NOTE As an alternative, a double sugar/iron agar can be used (Kligler-Hajna).
5.2.7 Urea agar (Christensen)
See B.8.
5.2.8 L-Lysine decarboxylation medium
See B.9.
5.2.9 Reagent for detection of -galactosidase (or prepared paper discs used in accordance with the
manufacturer's instructions) – Optional
See B.10.
Additional toluene is needed.
5.2.10 Medium and reagents for indole reaction - Optional
See B.11.
5.2.11 Saline solution
See B.12.
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5.3 Antisera
Several types of agglutinating sera containing antibodies for one or several O-antigens are available
commercially; i.e. anti-sera containing one or more “O” groups (called monovalent or polyvalent anti-O sera),
anti-Vi sera, and anti-sera containing antibodies for one or several H-factors (called monovalent or polyvalent
anti-H sera).
Every attempt should be made to ensure that the anti-sera used are adequate to provide for the detection of
all Salmonella serotypes. Assistance towards this objective may be obtained by using only anti-sera prepared
by a supplier recognized as competent (for example, by an appropriate government agency).
6 Apparatus and glassware
Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory 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 Drying cabinet or oven, ventilated by convection, capable of operating between 25 °C and 50 °C.
6.3a Incubator, capable of operating in the range 34 °C to 38 °C.
6.3b Incubator, capable of operating at 37 °C 1 °C.
6.4 Incubator, capable of operating at 41,5 °C 1 °C, or water bath, capable of operating at
41,5 °C 1 °C.
6.5 Water bath, capable of operating at 47°C to 50 °C.
6.6 Water bath, capable of operating at 37 °C 1 °C.
6.7 Water bath, capable of operating at 45 °C 1 °C.
It is recommended to use a water bath (6.4 - 6.7) containing an antibacterial agent because of the low
infective dose of Salmonella.
6.8 Refrigerator, capable of operating at 5 °C 3 °C
6.9 Sterile loops, of approximate diameter 3 mm (10 l volume), and of 1µl volume, and inoculation needle
or wire.
6.10 pH-meter, having an accuracy of calibration of 0,1 pH unit at 20 °C to 25 °C.
6.11 Tubes or flasks, of appropriate capacity.
Bottles or flasks with non-toxic metallic or plastic screw-caps may be used.
6.12 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml, 1 ml and 0,1 ml.
6.13 Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter approximately
140 mm).
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7 Sampling
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
For sampling of food and animal feed, see ISO/TS 17728. For sampling at the primary production stage, see
ISO 13307. For sampling of carcasses, see ISO 17604.
Sampling is not part of the method specified in this International Standard. See the specific International
Standard dealing with the product concerned. If there is no specific International Standard, it is recommended
that the parties concerned come to an agreement on this subject.
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with 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 (see diagrams in Annex A)
9.1 Test portion and initial suspension
9.1.1 General
For the preparation of test samples and initial suspensions of specific products, see the procedures as
described in ISO 6887 (all parts).
For preparation of the initial suspension, in the general case, use as diluent the pre-enrichment medium
specified in 5.2.1 (buffered peptone water). Pre-warm the BPW to room temperature before use.
In general, an amount of sample (mass or volume) is added to a quantity of BPW (mass or volume) to yield a
1/10 dilution (generally this concerns 25 g of sample added to 225 ml of BPW). However, for some type of
samples it may be necessary to use another ratio (e.g. bootsocks).
NOTE When more than one 25 g test portion from a specified lot of product has to be examined, and when evidence
is available that combining test portions does not affect the result for that particular food, the test portions can be pooled.
More information on pooling of samples, as well as a procedure to test the influence of pooling on the sensitivity of the
method, can be found in ISO 6887-1.
9.1.2 Specific preparations of the initial suspension for certain foodstuffs
The following preparations of the initial suspension are specific for Salmonella. Specific preparations
applicable for the determination of several microorganisms, including Salmonella, are described in
ISO 6887-2, ISO 6887-3, ISO 6887-4, ISO 6887-5 and ISO 6887-6.
9.1.2.1 Cocoa and cocoa-containing products
See ISO 6887 part 4.
9.1.2.2 Acidic and acidifying foodstuffs
See ISO 6887 part 1 and part 4.
9.1.2.3 Raw milk, heat-treated milk and liquid milk products
Pipette 25 ml of the test sample into a flask (6.11) containing 225 ml of BPW (5.2.1) and mix.
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9.1.2.4 Dried milk products
Prepare a stoppered flask (6.11) with 225 ml of B
...
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