SIST EN ISO 6579-1:2017

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
ISO 6579-1:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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
ISO 6579-1:2017(E)
Annex E (informative) Examples of selective plating-out media .43
Bibliography .48
iv © ISO 2017 – All rights reserved

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 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

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.
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 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

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).
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 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
For preparation of the initial suspension, in the general case, use as diluent the pre-enrichment medium
specified in B.2 (buffered peptone water). Pre-warm the BPW to room temperature before use.
In general, an amount of test portion (mass or volume) is added to a quantity of BPW (mass or volume)
to yield a tenfold dilution. For this, a 25 g test portion is mixed with 225 ml of BPW. However, for some
type of samples (e.g. boot socks, dust), it may be necessary to use another ratio.
For specific products, follow the procedures specified in ISO 6887 (all parts).
This document has been validated for test portions of 25 g. A smaller test portion may be used without
the need for additional validation/verification provided that the same ratio between (pre-)enrichment
broth and test portion is maintained. A larger test portion than that initially validated may be used
if a validation/verification study has shown that there are no negative effects on the detection of
Salmonella spp.
NOTE 1 Validation can be conducted according to the appropriate parts of ISO 16140. Verification for pooling
[38]
samples can be conducted according to the protocol described in ISO 6887-1:2017, Annex D .
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.
NOTE 2 When more than one 25 g test portion from a specified lot of product is 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
[38]
pooling on the sensitivity of the method can be found in ISO 6887-1 .
9.2 Non-selective pre-enrichment
Incubate the initial suspension (9.1) between 34 °C and 38 °C (6.3) for 18 h ± 2 h.
It is permissible to store the pre-enriched sample after incubation at 5 °C (6.8) for a maximum of 72 h
(see References [30] to [34]).
4 © ISO 2017 – All rights reserved

ISO 6579-1:2017(E)
9.3 Selective enrichment
9.3.1 General
Allow the selective enrichment media, RVS broth or MSRV agar (B.3 or B.4), and MKTTn broth (B.5) to
equilibrate at room temperature if they were stored at a lower temperature.
Minimize the transfer of particulate material from the pre-enrichment into the selective enrichment
media.
After incubation, it is permissible to store the selective enrichment at 5 °C (6.8) for a maximum of 72 h
(see References [30] to [34]).
NOTE MSRV agar is intended for the detection of motile Salmonella strains and is not appropriate for the
detection of non-motile Salmonella strains.
9.3.2 Procedure for food, animal feed samples, and environmental samples from the food
production area
Transfer 0,1 ml of the culture obtained in 9.2 to a tube containing 10 ml of the RVS broth (B.3) or to the
surface of a MSRV agar plate (B.4). Inoculate the MSRV agar with one to three equally spaced spots on
the surface of the medium.
Transfer 1 ml of the culture obtained in 9.2 to a tube containing 10 ml of MKTTn broth (B.5).
Incubate the inoculated RVS broth at 41,5 °C (6.4) for 24 h ± 3 h.
Incubate the inoculated MSRV agar plates at 41,5 °C (6.4) for 24 h ± 3 h. Do not invert the plates.
Incubate the inoculated MKTTn broth at 37 °C (6.3) for 24 h ± 3 h.
Suspect MSRV plates will show a grey-white, turbid zone extending out from the inoculated drop.
In dried milk products and cheese, Salmonella may be sublethally injured. Incubate the selective
enrichment media from these products for an additional 24 h ± 3 h (see Reference [35]).
For some other products, e.g. when investigating outbreak samples, this additional incubation time may
also be beneficial.
9.3.3 Procedure for samples from the primary production stage
Inoculate the MSRV agar (B.4) with 0,1 ml of the pre-enriched culture (9.2) as one to three equally
spaced spots on the surface of the medium.
Incubate the inoculated MSRV plates at 41,5 °C (6.4) for 24 h ± 3 h.
Do not invert the plates.
Suspect MSRV plates will show a grey-white, turbid zone extending out from the inoculated drop.
If the plates are negative after 24 h, re-incubate for a further 24 h ± 3 h.
NOTE Sensitivity can be improved by using a second selective enrichment procedure, e.g. MKTTn broth
[36]
incubated at 41,5 °C for 24 h.
ISO 6579-1:2017(E)
9.4 Plating out
9.4.1 General
From the selective enriched cultures (9.3), inoculate two selective isolation agar media. The first
isolation medium is Xylose Lysine Deoxycholate (XLD) agar. The second isolation medium is chosen by
the testing laboratory.
Choose a second selective plating medium which is complementary to XLD agar and is based on different
diagnostic characteristics to those of XLD agar to facilitate detection of, for instance, lactose positive or
H2S-negative Salmonella. For examples of isolation media, see Annex E.
Allow the XLD agar (B.6) plates and the second selective plating medium to equilibrate at room
temperature if they were stored at a lower temperature. If necessary, dry the surface of the plates
before use (see ISO 11133).
9.4.2 Procedure for food, animal feed samples, and environmental samples from the food
production area
From the culture obtained in the RVS broth (9.3.2), inoculate by means of a 10 µl loop (6.10) the surface
of an XLD plate (B.6) so that well-isolated colonies will be obtained. Proceed in the same way with the
second selective plating-out medium.
From the positive growth obtained on the MSRV agar (9.3.2), determine the furthest point of opaque
growth from the inoculation points and dip a 1 µl loop (6.10) just inside the border of the opaque
growth. Withdraw the loop ensuring that no large lumps of MSRV agar are extracted. Inoculate the
surface of an XLD plate (B.6) so that well-isolated colonies will be obtained. Proceed in the same way
with the second selective plating-out medium.
From the culture obtained in the MKTTn broth (9.3.2), inoculate by means of a 10 µl loop (6.10) the
surface of an XLD plate (B.6) so that well-isolated colonies are obtained. Proceed in the same way with
the second selective plating-out medium.
NOTE 1 To obtain well-isolated colonies, large size Petri dishes with plating-out media (diameter
approximately 140 mm) or two normal size plates (diameter approximately 90 mm) can be used.
Incubate the XLD plates inverted at 37 °C (6.3) for 24 h ± 3 h.
Incubate the second selective plating-out medium in accordance with the manufacturer’s instructions.
If the selective enrichment media have been incubated for an additional 24 h, follow the same plating-
out procedure as described above.
Typical colonies of Salmonella on XLD agar have a black centre and a lightly transparent zone of reddish
colour due to the colour change of the indicator.
NOTE 2 Salmonella H2S-negative variants grown on XLD agar are pink with a darker pink centre. Lactose-
positive Salmonella grown on XLD agar are yellow with or without blackening. The occurrence of these
phenotypes is summarized in Table 1.
Check the second selective plating medium after the appropriate incubation time for the presence of
colonies which, from their characteristics, are considered to be presumptive Salmonella.
9.4.3 Procedure for samples from the primary production stage
From the positive growth obtained on the MSRV agar (9.3.3), determine the furthest point of opaque
growth from the inoculation points and dip a 1 μl loop (6.10) just inside the border of the opaque
growth. Withdraw the loop ensuring that no large lumps of MSRV agar are extracted. Inoculate the
surface of an XLD plate so that well-isolated colonies will be obtained. Proceed in the same way with
the second selective plating medium.
6 © ISO 2017 – All rights reserved

ISO 6579-1:2017(E)
Incubate the XLD plates inverted at 37 °C (6.3) for 24 h ± 3 h.
Incubate the second selective plating medium in accordance with the manufacturer’s instructions.
Return negative MSRV plates to the 41,5 °C incubator and incubate for a further 24 h ± 3 h. Perform the
selective plating procedure if, after 48 h of incubation, these MSRV plates become positive.
Typical colonies of Salmonella on XLD agar have a black centre and a lightly transparent zone of reddish
colour due to the colour change of the indicator.
NOTE Salmonella H2S-negative variants grown on XLD agar are pink with a darker pink centre. Lactose-
positive Salmonella grown on XLD agar are yellow with or without blackening. The occurrence of these
phenotypes is summarized in Table 1.
Check the second selective plating medium after the appropriate incubation time for the presence of
colonies which, from their characteristics, are considered to be presumptive Salmonella.
9.5 Confirmation
9.5.1 General
The combination of biochemical and serological test results indicate whether an isolate belongs to the
genus Salmonella. For characterization of Salmonella strains, full serotyping is needed. Guidance for
[24]
serotyping is described in ISO/TR 6579-3 .
For some of the confirmation media as specified in 9.5.3 and in B.8 to B.12, alternative (commercial)
formulations exist which may also be applicable for biochemical confirmation of Salmonella. These
alternative formulations are allowed, provided that the performance for the biochemical confirmation
of Salmonella is verified before use.
For a clear distinction between positive and negative biochemical reactions, it is helpful to verify the
reactions of the media of each biochemical test with well-characterized positive and negative control
strains.
NOTE 1 The recognition of colonies of Salmonella is, to a large extent, a matter of experience and their
appearance can vary somewhat, not only from serovar to serovar, but also from batch to batch of the selective
culture medium used.
If shown to be reliable, miniaturized galleries for the biochemical identification of Salmonella may be
used (see ISO 7218).
NOTE 2 Alternative procedures can be used to confirm the isolate as Salmonella spp. providing the suitability
of the alternative procedure is verified (see ISO 7218).
9.5.2 Selection of colonies for confirmation
Mark suspect colonies on each plate (9.4). Select at least one typical or suspect colony for subculture
and confirmation. If this is negative, select up to four more suspect colonies ensuring that these
colonies are subcultured from different selective enrichment/isolation medium combinations showing
suspect growth.
Streak the selected colonies onto the surface of a pre-dried non-selective agar medium (B.7) in a manner
which will allow well-isolated colonies to develop. Incubate the inoculated plates between 34 °C and
38 °C (6.3) for 24 h ± 3 h.
Alternatively, if well-isolated colonies (of a pure culture) are available on the selective plating media
(9.4), the biochemical confirmation can be performed directly on a suspect, well-isolated colony
from the selective plating medium. The culture step on the non-selective agar medium can then be
performed in parallel with the biochemical tests for purity check of the colony taken from the selective
agar medium.
ISO 6579-1:2017(E)
Use pure cultures for biochemical and serological confirmation.
NOTE For epidemiological purposes or during outbreak investigations, confirmation of additional colonies,
e.g. five typical or suspect colonies from each selective enrichment/isolation medium combination, can be
beneficial.
9.5.3 Biochemical testing
9.5.3.1 General
Inoculate the biochemical confirmation media with each of the cultures obtained from the colonies
selected in 9.4 or 9.5.2. For confirmation of Salmonella spp., at least the tests specified in 9.5.3.2 to
9.5.3.4 shall be performed. The tests specified in 9.5.3.5 and 9.5.3.6 can also be performed when the
results of the other confirmation tests do not give a clear identification.
9.5.3.2 TSI agar (B.8)
Streak the agar slant surface and stab the butt. Incubate at 37 °C (6.3) for 24 h ± 3 h.
Interpret the changes in the medium as follows:
a) butt
— yellow: glucose positive (glucose fermentation);
— red or unchanged: glucose negative (no fermentation of glucose);
— black: formation of hydrogen sulphide;
— bubbles or cracks: gas formation from glucose;
b) slant surface
— yellow: lactose and/or sucrose positive (lactose and/or sucrose fermentation);
— red or unchanged: lactose and sucrose negative (no fermentation of lactose or sucrose).
The majority of the typical Salmonella cultures show alkaline (red) slants and acid (yellow) butts with
gas formation (bubbles) and (in about 90 % of the cases) formation of hydrogen sulfide (blackening of
the agar) (see Table 1).
When a lactose-positive Salmonella is isolated, the TSI slant is yellow. Thus, preliminary confirmation of
Salmonella cultures shall not be based on the results of the TSI agar test only (see 9.5.3.1).
NOTE Kligler-Hajna medium gives similar results as TSI agar.
9.5.3.3 Urea agar (B.9)
Streak the agar slant surface. Incubate at 37 °C (6.3) for up to 24 h.
If the reaction is positive, urea is hydrolyzed, liberating ammonia. This changes the colour of phenol red
to rose-pink and later to deep cerise. The reaction is often apparent after 2 h to 4 h.
Typical Salmonella cultures do not hydrolyze urea so that the colour of the urea agar will remain
unchanged (see Table 1).
9.5.3.4 L-Lysine decarboxylation medium (LDC, B.10)
Inoculate just below the surface of the liquid medium. Incubate at 37 °C (6.3) for 24 h ± 3 h.
Turbidity and a purple colour after incubation indicate a positive reaction. A yellow colour indicates a
negative reaction.
8 © ISO 2017 – All rights reserved

ISO 6579-1:2017(E)
The majority of the typical Salmonella cultures show a positive reaction in LDC (see Table 1).
9.5.3.5 Detection of β -galactosidase (B.11) (optional)
The β-galactosidase test can be used to distinguish Salmonella enterica subspecies arizonae and
diarizonae and other members of the Enterobacteriaceae (all give a positive reaction) from other
subspecies of Salmonella enterica (in general these give a negative reaction, see Table 1).
Several procedures to perform the β-galactosidase test exist. An example is given below.
Suspend a loopful of the s
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