ISO/TR 6579-3:2014

TECHNICAL ISO/TR
REPORT 6579-3
First edition
2014-07-15
Microbiology of the food chain —
Horizontal method for the detection,
enumeration and serotyping of
Salmonella —
Part 3:
Guidelines for serotyping of
Salmonella spp.
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche, le dénombrement et la sérotypie des Salmonella —
Partie 3: Lignes directrices pour la sérotypie des Salmonella spp.
Reference number
©
ISO 2014
© ISO 2014
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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Culture media and sera . 2
5.1 General . 2
5.2 Culture media and reagents . 2
5.3 Antisera . 2
6 Apparatus . 2
7 Sample . 3
8 Taxonomy of Salmonella . 3
8.1 General . 3
8.2 Nomenclature. 3
8.3 Biochemical characteristics . 4
8.4 Antigenic characteristics . 5
9 Procedure for Salmonella serotyping . 7
9.1 General . 7
9.2 Example procedure for serotyping five public health-related Salmonella serovars . 7
10 Quality control .11
11 Reporting .11
Annex A (informative) Composition and preparation of culture media and reagents .13
Annex B (informative) Examples of procedures for serotyping an unknown Salmonella isolate .20
Annex C (informative) Biochemical tests .24
Annex D (informative) Schematic overview for serotyping five important public-health related
Salmonella serovars .26
Annex E (informative) Microtitre plate method for serotyping Salmonella spp.27
Annex F (informative) Examples of procedures for phase inversion .29
Bibliography .32
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 WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
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:
1)
— Part 1: Horizontal method for the detection of Salmonella spp.
2)
— Part 2: Enumeration by a miniaturized most probable number technique [Technical Specification]
— Part 3: Guidelines for serotyping of Salmonella spp. [Technical Report]
1) Under preparation. (Revision of ISO 6579:2002)
2) The main element of the series title has been changed since Part 2 was published. It is intended that upon
revision, the main element of the title will be aligned with Part 3.
iv © ISO 2014 – All rights reserved

Introduction
This part of ISO 6579 gives information on the taxonomy of Salmonella spp. and it gives guidance on
serotyping of Salmonella serovars, based on the White–Kauffmann–Le Minor scheme (see Reference [9]).
TECHNICAL REPORT ISO/TR 6579-3:2014(E)
Microbiology of the food chain — Horizontal method
for the detection, enumeration and serotyping of
Salmonella —
Part 3:
Guidelines for serotyping of Salmonella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting and typing Salmonella, be undertaken only in properly equipped laboratories, under
the control of a skilled microbiologist, and that great care is taken in the disposal of all incubated
materials.
IMPORTANT — 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 part of ISO 6579 gives guidance on the procedure for serotyping Salmonella serovars and is
applicable to the serotyping of pure cultures of Salmonella spp., independent of the source from which
they are isolated.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 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.
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Salmonella
gram-negative, oxidase-negative, facultatively anaerobic, non-spore-forming, rod-shaped bacteria which
generally form colonies of 2 mm to 4 mm in diameter on solid selective media and display biochemical
and serological characteristics described when tests are carried out in accordance with this part of
ISO 6579
3.2
serotyping of Salmonella
determination of the presence or absence of specific O-antigens, H-antigens and Vi-antigens in an isolate
confirmed as Salmonella (3.1)
3.3
antigenic formula
combination of numbers and letters representing the O-, H-, and Vi-antigens of an isolate confirmed as
Salmonella (3.1)
4 Principle
For the serotyping of Salmonella spp. the following antigens are determined for isolates biochemically
confirmed as Salmonella spp.:
O-antigens, H-antigens and Vi-antigens.
NOTE Alternative procedures can be used to confirm the isolate being Salmonella spp. provided the suitability
of the alternative procedure is verified (see ISO 7218).
5 Culture media and sera
5.1 General
For current laboratory practice, apply ISO 7218.
For the performance testing of media, follow the recommendations of ISO 11133.
5.2 Culture media and reagents
See Annex A.
5.3 Antisera
O-antisera, H-antisera and Vi-antisera are available from various commercial suppliers. Information on
relevant polyvalent antisera and monovalent antisera can be found in Annex B.
6 Apparatus
Disposable supplies are an acceptable alternative to reusable glassware if they have similar specifications.
Usual microbiological laboratory equipment and, in particular, the following.
6.1 Incubator, to grow Salmonella isolates, capable of operating in the range 34 °C to 38 °C.
NOTE In this part of ISO 6579, the incubation temperature is not a differential parameter. Isolates are
cultured to obtain sufficient material to perform the tests on a pure culture. Therefore, culture step is performed
at an optimal growth temperature. For Salmonella this is generally a temperature between 34 °C and 38 °C.
6.2 Oven (for dry sterilization) or autoclave (for wet sterilization). See ISO 7218.
6.3 Refrigerator (for storage of prepared media), capable of operating at 5 °C ± 3 °C.
6.4 Glass slides.
6.5 Sterile inoculation instrument, e.g. needles, wires, wooden sticks, loops (e.g. of 1 µl).
2 © ISO 2014 – All rights reserved

6.6 Sterile test tubes and flasks, of appropriate capacity. Flasks or bottles and test tubes with non-
toxic metallic or plastic (screw) caps may be used.
6.7 Sterile Petri dishes, with diameters of approximately 55 mm and 90 mm.
6.8 Water bath, capable of operating at 47-50 °C.
6.9 Water bath (or incubator), capable of operating at 50 °C ± 2 °C.
7 Sample
It is important that the laboratory works with a pure culture which has been biochemically confirmed
as Salmonella spp.
8 Taxonomy of Salmonella
8.1 General
Approximately every 7 years, the WHO Collaborating Centre for Reference and Research on Salmonella
(Institut Pasteur, Paris) publishes an update of the “Antigenic formulae of the Salmonella serovars”,
which is the basis for assigning serovar names and formulas to isolates of Salmonella spp. At the time of
publication, the latest version of the White–Kauffmann–Le Minor scheme is that of 2007 (Reference [9]).
NOTE Supplements to the White-Kauffmann-Le Minor scheme are published in Research in Microbiology,
a publication of the Institut Pasteur (formerly called Annales de l’Institut Pasteur/Microbiologie). For instance,
supplement no. 47 was published in 2010 and characterises new serovars found between 2003 and 2007
(Reference [10]).
This part of ISO 6579 provides guidance on the serotyping of Salmonella serovars.
8.2 Nomenclature
Different nomenclatures have been used (or are still in use) for Salmonella strains:
— originally, Kauffmann (Reference [12]) considered each Salmonella serovar as a separate species;
— different type species have been used: S. enterica vs. S. choleraesuis, each having another type strain;
— some “important” Salmonella strains (like Salmonella Typhi and Salmonella Paratyphi) were
considered to be species and not being “only” serovars of a species.
The Judicial Commission of the International Committee on Systematics of Procaryotes indicated that
many synonyms can be used in Salmonella nomenclature (Reference [22]). In this part of ISO 6579,
the widely accepted current nomenclature is used, which is also approved by the WHO Collaborating
Centre for Reference and Research on Salmonella (Reference [9]), the American Society for Microbiology
(Reference [20]), the Centers for Disease Control and Prevention (Reference [3]) and Bergey’s manual
(Reference [17]). According to the current nomenclature, the genus Salmonella belongs to the family
of Enterobacteriaceae and consists of only two species: S. enterica and S. bongori. S. enterica is divided
into six subspecies: S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S.
enterica subsp. diarizonae, S. enterica subsp. houtenae, and S. enterica subsp. indica.
Salmonella serovars belonging to S. enterica subsp. enterica are isolated most frequently (more than
99,5 % of isolated Salmonella strains) and they are designated by a name, usually related to the
geographical place where the serovar was first isolated. Serovars belonging to other subspecies of
S. enterica and those of S. bongori are designated by their antigenic formula.
Due to combinations of subspecies and many serovars, the full names are long (e.g. Salmonella enterica
subsp. enterica serovar Typhimurium). It has therefore generally been accepted to use a shorter
way to indicate the names of the serovars of subspecies enterica. The White–Kauffmann–Le Minor
scheme suggests the following shortened names: S. enterica serovar Typhimurium or Salmonella ser.
Typhimurium. According to Reference [3], at the first citation of a serovar in a text the genus name
should be given followed by the word “serovar” or the abbreviated term “ser”. and then the serovar
name. Subsequently, the name may be written with the genus followed directly by the serovar name (e.g.
Salmonella Typhimurium). This way of indicating Salmonella serovars is also accepted in the majority of
journals [e.g. journals of the American Society for Microbiology (ASM)] and is also used in this part of
ISO 6579.
In summary, the nomenclature of Salmonella:
family: Enterobacteriaceae (first letter capitalized, italicized)
genus: Salmonella (first letter capitalized, italicized)
species: enterica (not capitalized, italicized)
subspecies: enterica (not capitalized, italicized)
serovar (serotype or ser.): e.g. Typhimurium (first letter capitalized, not italicized)
subspecies: salamae
arizonae
diarizonae
houtenae
indica
species: bongori
In the 47th Supplement to the White–Kauffmann–Le Minor scheme (Reference [10]) more than 2600
Salmonella serovars are mentioned and the numbers increase regularly, as summarized in Table 1.
Table 1 — Number of Salmonella serovars through the years
Supplement
a b c
Species/subspecies 1998 2001 2007
Number of serovars
Salmonella enterica 2 443 2 502 2 587
subsp. enterica 1 454 1 492 1 547
subsp. salamae 489 500 513
subsp. arizonae 94 95 100
subsp. diarizonae 324 331 341
subsp. houtenae 70 71 73
subsp. indica 12 13 13
Salmonella bongori 20 21 23
Total no. of serovars (genus Salmonella) 2 463 2 523 2 610
a
Reference [18].
b
Reference [19].
c
Reference [10], covering 2003–2007.
8.3 Biochemical characteristics
The Salmonella species and subspecies are identified based on different biochemical tests. In Table 2, the
differential characteristics are listed. See Reference [9] and Annex C for further details.
4 © ISO 2014 – All rights reserved

Table 2 — Biochemical characteristics of Salmonella species and subspecies (Reference [9])
Species S. enterica
S. bongori
Subspecies enterica salamae arizonae diarizonae houtenae indica
Dulcitol + + − − − d +
a
ONPG (2 h) − − + + − d +
Malonate − + + + − − −
Gelatinase − + + + + + −
Sorbitol + + + + + − +
b
Growth with KCN − − − − + − +
c
l(+)-tartrate + − − − − − −
Galacturonate − + − + + + +
e
γ-Glutamyltransferase + + − + + + +
β-Glucuronidase d d − + − d −
Mucate + + + − (70 %) − + +
Salicin − − − − + − −
Lactose − − − (75 %) + (75 %)+ − d −
Lysis by phage O1 + + − + − + d
+ = 90 % or more positive reaction
− = 90 % or more negative reaction
d = different reactions given by different serovars
a
o-Nitrophenyl-β-d-galactopyranoside (test for β-galactosidase).
b
Potassium cyanide.
c
= d-Tartrate, Paratyphi B: −, Paratyphi B biovar Java: +
e
= Typhimurium: d, Dublin: −.
8.4 Antigenic characteristics
8.4.1 General
The important antigenic characteristics of Salmonella for serological tests are divided into three main
types, being:
— the O-antigen, also called the somatic antigen;
— the H-antigen, also called the flagellar antigen;
— the Vi-antigen, also called the capsular antigen.
The antigenic formula of Salmonella spp. exists of these three types of antigens, reported in the following
way: O-antigens, Vi-antigen (if present): H-antigens of first phase: H-antigens of second phase. For
instance, the antigenic formula of Salmonella Paratyphi C is: 6,7,[Vi]:c:1,5; with O-antigens O:6 and O:7;
with the Vi-antigen, which can be present or absent (indicated by the square brackets); with H-antigen
H:c for the first phase; with H-antigens H:1 and H:5 for the second phase.
8.4.2 The O-antigen (somatic antigen)
This antigen consists of a cell wall component and the main substances are polysaccharide, protein,
and phospholipid. The O-antigen is very robust and can resist temperatures up to 100 °C for 150 min,
treatment with 95 % volume fraction ethanol or dilute acid (Reference [16]).
The reaction of the O-antigen with antisera results in granular agglutination. Historically, the O-antigens
were classified in individual O-antigen groups in the Kauffmann–White scheme (Reference [9]). The
groups were named with Roman letters beginning with group A, which include antigen O:2, up to group
Z which contain antigen O:50. As there were more O-antigens than letters, the remaining antigens were
not given as group, but were named by the O-antigens O:51 to O:67. Nowadays it is preferred to designate
each O-group using the characteristic O-factor. The letters have been kept and are shown inside brackets,
e.g. O:4 (B) (Reference [9]). In Table 3, the old and new designations are summarized.
Table 3 — Salmonella serogroups (old designation) and relevant O-antigens (new designation)
Group O-antigen Group O-antigen Group O-antigen
A 2 G -G 13 Q 39
1 2
B 4 H 6,14 R 40
a
C (, C ) 6,7 I 16 S 41
1 4
C , C 8 J 17 T 42
2 3
D 9 K 18 U 43
D 9,46 L 21 V 44
D 9,46,27 M 28 W 45
b
E (, E , E ) 3,10 N 30 X 47
1 2 3
E 1,3,19 O 35 Y 48
F 11 P 38 Z 50
a
C has been merged into C
4 1.
b
E and E have been merged into E
2 3 1.
8.4.3 The H-antigen (flagellar antigen)
This antigen is located on the flagellum and the main component is protein. It is less robust than
O-antigens. It can easily be decomposed by alcohol, acid, and temperature above 60 °C, but it is resistant
to a formalin solution with a volume fraction of 0,5 % (Reference [16]).
The reaction of the H-antigen with antisera results in floccular agglutination. Many Salmonella spp.
possess two phases of the H-antigen, but monophasic and triphasic variants are also known. The first
phase is called the specific phase and the second phase is called the non-specific phase. The first phase
is indicated by a lower case letter, a to z. However, since the identification of the z-antigen, many new
H-antigens have been detected and are named z , z , z . z
1 2 3 91.
Examples of monophasic serovars are:
— Salmonella Paratyphi A: 1,2,12:a:[1,5]; with H:a for the first phase and where the square brackets
indicate that the second phase (H:1,5) can be present or absent;
— Salmonella Typhi: 9,12,[Vi]:d:-; with H:d for the first phase;
— Salmonella Derby: 1,4,[5],12:f,g:[1,2]; with H:f,g for the first phase and where the second phase
(H:1,2) can be present or absent;
— Salmonella Enteritidis: 1,9,12: g,m:-; with H:g,m for the first phase. In addition to factors H:g,m, some
strains may have factor H:p, or H:f, or H:t. Exceptional strains can have antigen H:1,7 as second
phase;
— Salmonella Dublin: 1,9,12,[Vi]:g,p:-; with H:g,p for the first phase.
NOTE 1 Underlined O-factors are determined by phage conversion. They are only present if the culture is
lysogenized by the corresponding converting phage (Reference [9]).
6 © ISO 2014 – All rights reserved

NOTE 2 O- or H-factors indicated in square brackets can be present or absent, without relation to phage
conversion (Reference [9]).
NOTE 3 Diphasic strains of Salmonella Derby and Salmonella Enteritidis are very rare. It is possible that phase
inversion is required to detect these rare strains. However, this is only necessary for certain (special) cases (e.g.
in case of deviating sources and/or in the case of (special) travel-related cases).
8.4.4 Vi-antigen (capsular antigen)
This antigen is a surface (capsular) antigen and can mask the O-antigens so that the bacteria are not
agglutinated with O-antisera. The main component of the Vi-antigen is polysaccharide. The Salmonella
strains which possess a Vi-antigen are more virulent than the strains without Vi-antigen. The Vi-antigen
can be present in only three Salmonella serovars:
— Salmonella Typhi : 9,12,[Vi]:d:-;
— Salmonella Paratyphi C : 6,7,[Vi]:c:1,5;
— Salmonella Dublin: 1,9,12,[Vi]:g,p:-.
The square brackets indicate that the Vi-antigen can be present or absent.
NOTE The presence of Vi-antigens in Salmonella isolates from food or veterinary samples is very rare. If Vi is
present, it masks the detection of O-antigens. To detect the O-antigens, it can prove necessary to heat a suspension
of the isolate (e.g. in physiological saline solution) at 100 °C for 60 min, or at 120 °C for 15 min.
9 Procedure for Salmonella serotyping
9.1 General
Before starting the serotyping, it is important to confirm biochemically that the isolate belongs to
the genus Salmonella (as specified in ISO 6579-1). Although the H-antigens are specific for Salmonella,
several O-antigens are common in different genera of the Enterobacteriaceae (e.g. Salmonella, Citrobacter,
Hafnia).
NOTE Alternative procedures can be used to confirm that the isolate belongs to the genus Salmonella.
provided the suitability of the alternative procedure is verified (see ISO 7218).
Each supplier of antisera produces its own sets of antisera, with its own unique instructions for use. It
is therefore not possible to provide here one general set of instructions for serotyping, as it is always
important to follow the instructions of the supplier to obtain optimal results. Some manufacturers
supply pools of antisera (mixtures of several O-antisera or H-antisera), which are very useful at the
beginning of serotyping an unknown type. When the strain agglutinates with an antisera pool, it can be
further tested with group antisera and/or single factor antisera relevant to the positive pool. When the
focus is on typing only certain serovars and it is sufficient to indicate the other serovars as Salmonella
spp., the agglutination can immediately be performed with only the specific monofactor antisera of the
relevant serovars.
In Annex B, the general procedure is given for serotyping an unknown Salmonella isolate.
9.2 Example procedure for serotyping five public health-related Salmonella serovars
9.2.1 General
In the following example, the procedure is described for serotyping five important public health-related
Salmonella serovars (see Annex D). In Table 4 these strains are shown with their antigenic formula.
In the following sections, slide agglutination of Salmonella isolates is described, which is the procedure
most often performed. However, others also exist, such as the microtitre plate method (see Annex E).
Table 4 — Antigenic formula of five important public health-related Salmonella serovars
H-antigens
b
Name O-antigens
phase I phase II
Salmonella Typhimurium 1,4,[5]12 i 1,2
a
Salmonella Enteritidis 1,9,12 g,m -
Salmonella Infantis 6,7,14 r 1,5
Salmonella Virchow 6,7,14 r 1,2
Salmonella Hadar 6,8 z e,n,x
a
In addition to factors H:g,m, some strains may have factor H:p or H:f or H:t. Exceptional strains can have antigen H:1,7 as
second phase (Reference [9]).
b
Underlined O factors are determined by phage conversion. They are present only if the culture is lysogenized by the
corresponding converting phage (Reference [9]).
9.2.2 Selection of a colony suspected for Salmonella
Culture a colony from a pure culture which is suspected to be Salmonella according to the biochemical
characterization. Use the culture media and methods as prescribed by the manufacturer of the antisera
used. If no information is given, a non-selective agar medium like nutrient agar (e.g. see A.2) can be used.
Incubate the inoculated nutrient agar plate(s) between 34 °C and 38 °C (6.1) “overnight” (approximately
18 h).
9.2.3 Investigation for auto-agglutination
An example of the test for auto-agglutination is described in the following. Other methods may also be
used. Follow, in that respect, the manufacturer’s instructions.
— Add one drop of saline (this may vary from 8,5 g/l NaCl to 35 g/l NaCl, where the higher concentration
may be more sensitive) on a glass slide (6.4).
— Transfer a small amount of bacterial culture (e.g. the amount which can be taken with a 1 µl
disposable loop) on to the glass slide and mix with the drop of saline.
— Gently tilt the slide back and forth. Depending on the manufacturer and/or on the saline concentration,
this should take 5 s to 60 s.
— Assess the suspension. The presence of granules in the suspension indicates auto-agglutination.
Strains with a positive reaction in the auto-agglutination test are hard to investigate further for
serotyping. For auto-agglutinating strains, it is not possible to test for the O-antigens. However, it
may sometimes still be possible to investigate for the H-antigens.
If a strain shows auto-agglutination, either one or both of the following can be tried on the same colony
and/or on additional colonies.
— Suspend the colony in sterile water instead of in a saline solution and follow the procedure for auto-
agglutination as described in the foregoing.
— Grow the strain on a semi-solid agar medium like modified semi-solid Rappaport Vassiliadis (MSRV)
agar (as specified in ISO 6579-1) and use colony material from the semi-solid agar to perform the
procedure for auto-agglutination as described in the foregoing.
NOTE Auto-agglutinating strains are also called “rough” strains.
9.2.4 Agglutination with O-antisera
The instructions for the use of antisera may differ per manufacturer. Therefore it is important to always
follow the manufacturer’s instructions.
8 © ISO 2014 – All rights reserved

Most manufacturers use a slide agglutination method for the detection of O-antigens. In this method,
one drop of antiserum is mixed with a bacterial suspension (directly from a plate, tube or broth) on a
slide. Gently tilt the slide back and forth. Subsequently observe the slide for agglutination. The presence
of granules indicates a positive reaction.
For different manufacturers, the following variations in the procedure can be found:
— the size of the drop on the slide (e.g. 25 µl or a “drop”);
— the way the antiserum and bacterial material are mixed on the slide (bacterial material directly
from the agar or via a suspension, antiserum directly on the slide or added to a bacterial suspension);
— time of tilting the slide back and forth (can vary from 5 s to 60 s);
— the way of observing agglutination (magnification or not, dark background or normal lighting);
— interpretation of the results (read the footnotes with regard to the limitations of the procedure).
9.2.5 Agglutination with H-antisera
After agglutination with the O-antisera, perform the agglutination with H-antisera. Salmonella often
possesses two types of H-antigens (phase 1 and phase 2). If one H-phase is found negative for biphasic
strains, a phase inversion method is required. The dominant H-phase is repressed with a phase inversion
method. By repressing the dominant H-phase the second H-phase can be expressed and identified.
A frequently used method for phase inversion is that of Sven Gard, see 9.2.7. For this, specific phase
inversion antiserum is added to a swarming agar medium and the Salmonella strain is spot inoculated
on the plate. The agar medium shall be sufficiently soft for motile Salmonella to swarm over the medium.
The agar concentration in the medium may vary from a mass fraction of 0,5 % to 1 % (depending on
the gel strength of the agar). Examples of “swarming” media are given in A.3. Other examples for phase
inversion are given in Annex F.
For the investigation for agglutination, follow the manufacturer’s instructions.
9.2.6 Agglutination tests for serotyping five important public health-related Salmonella sero-
vars
9.2.6.1 General
For the detection of the five Salmonella serovars mentioned in Table 4, the following O-antisera and
H-antisera are needed:
O:4, O:5, O:6 (O:6 or O:6,7 or O:6,14,24), O:7, O:8, O:9 and O:46.
H:i, H:2, H:G or H:g (monovalent), H:m, H:q, H:s, H:t, H:r, H:5, H:z and H:x.
For the serotyping of the Salmonella strains in the order shown in Table 4, follow the procedure specified
in 9.2.6.2 to 9.2.6.5 (also see the scheme in Annex D).
For more information on sequential tests of the different antisera, see Annex B.
9.2.6.2 If O:4 is positive
Agglutinate with O:5. The result can be either positive or negative for Salmonella Typhimurium, still the
information may be relevant for epidemiological purposes.
Agglutinate with H:i and H:2 antisera (phase inversion may be necessary).
The serovar of the strain is Typhimurium if both reactions are positive.
Agglutination with O:12 is not necessary to indicate the isolate to be Salmonella Typhimurium, as
the positivity of O:4 implicates the presence of O:12. Likewise, the detection of antigen H:1 has no
discriminatory power and is not needed to be tested additionally.
9.2.6.3 If O:9 is positive
Agglutinate with H:G(complex) or with H:g (monovalent antibody) antiserum.
If this reaction is positive, subsequently agglutinate with H:m antiserum.
If this is also positive, agglutinate with H:q and H:s antisera as negative controls.
If these latter reactions are negative, agglutinate subsequently with O:46 and, if wanted, with O:12. If
O:46 is negative (and O:12 is positive), the serovar of the strain is Enteritidis.
NOTE In addition to factors H:g,m, some Salmonella Enteritidis strains can give a positive reaction with H:p,
or H:f, or H:t antiserum. However, these strains are very rare. Exceptional strains can have antigen H:1,7 as a
second phase.
9.2.6.4 If O:7 is positive
Agglutinate with H:r, H:2 and H:5 antisera (phase inversion may be necessary).
If H:r and H:2 are positive, the serovar of the strain is: Virchow.
If H:r and H:5 are positive, the serovar of the strain is: Infantis.
Detection of antigen H:1 has no discriminatory power and is not needed to be tested additionally.
9.2.6.5 If O:8 is positive
Agglutinate with H:z and H:x antisera.
If both are positive, subsequently agglutinate with O:6 or O:6,7 or O: 6,14,24 antiserum.
If O:6 or O:6,7 or O:6,14,24 is positive, the serovar of the strain is: Hadar.
Some batches of O:6,7 antiserum do not react with O:6,8 strains. When purchasing this antiserum, make
sure it also reacts with O:6,8 strains (ask the manufacturer).
NOTE Colonial form variation can occur with the expression of the O:6 antigen by some serogroup C
1 2
serovars (Reference [11]). For that reason, it is not always possible to indicate, e.g. Salmonella Hadar and Salmonella
Istanbul as distinct serovars.
9.2.7 Example of phase inversion using the Sven Gard method
The example described here for phase inversion is intended for the serotyping of Salmonella Typhimurium.
In general, the method is also applicable to other Salmonella serovars.
When O:4 and H:i are positive, but H:2 is negative, prepare a swarm agar plate (see A.3 or F.1), with anti-
H:i (e.g. phase inversion serum-mix containing H:i). Inoculate the strain by one spot on the centre of the
plate. Incubate the plate between 34 °C and 38 °C (6.1) overnight (approximately 18 h).
After incubation, agglutinate the strain again with H:2 antisera.
If H:2 is again negative, subsequently agglutinate with H:i again.
If H:i gives a negative or weak reaction, the strain is not Salmonella Typhimurium.
10 © ISO 2014 – All rights reserved

If H:i gives a (strong) positive reaction, repeat the phase inversion. Again prepare an agar plate with
anti-H:i and inoculate this plate with bacterial material from the furthest point of spread of the opaque
growth of the first phase inversion agar plate. Repeat the phase inversion procedure as described above.
NOTE 1 Sometimes it is necessary to add more H:i antiserum into the swarm agar (of the repeated phase
inversion plate) to get a better reaction.
NOTE 2 Monophasic variants of Salmonella Typhimurium also exist, e.g. lacking, or not expressing, the second
H phase: 1,4,[5]12:i:-. While serotyping this variant, phase inversion can be repeated once or more, to exclude the
presence of the second phase. Alternatively, a molecular method can be used to confirm whether the strain is a
variant of Salmonella Typhimurium.
10 Quality control
The sera used for agglutination should be clear (unless the antisera are used for latex tests). Always
inspect the sera before use. In case of turbidity, follow the instructions of the manufacturer.
Possible procedures for quality control of the serotyping are given in the following.
— Two strains are selected (or the number of strains that represents approximately 2 % of the
workload) from the incoming work per week. Each of the selected strains is cultured twice. The
duplicate strains are then treated as two new isolates received for serotyping. On completion of
the work, the results for the two strains and their duplicates are compared for any discrepancies. If
there are any discrepancies, these are then further investigated.
— The laboratory keeps fully characterized Salmonella serovars in stock (e.g. from a culture collection
or from interlaboratory comparison studies). From this stock, regularly (e.g. weekly) one or two
serovars of the 10 serovars most frequently identified in the laboratory are selected to check the
serotyping procedure. The serovar(s) used to perform the quality control may vary weekly to
ensure that different antisera are tested over the course of time.
— It is important to test the ability of serotyping against other laboratories. For this it is advisable to
regularly participate in interlaboratory comparison studies, whenever available.
11 Reporting
For isolates belonging to S. enterica subsp. enterica, report the (full) name and, whenever possible/needed,
also the antigenic formula. For the other (sub)species report the antigenic formula as found.
The notation of the antigenic formula is as follows:
O-antigens (separated by commas), Vi-antigen (if present): H-antigens of first phase (separated by
commas): H-antigens of second phase (if present; separated by commas); H-antigens of third phase (if
present).
For instance, according to the White-Kauffmann-Le Minor scheme (Reference [9]), the full antigenic
formula of Salmonella Typhimurium is: 1,4,[5],12:i:1,2.
With O-antigens O:1,4,[5],12; where the underlined O factor is determined by phage conversion and
is only present if the culture is lysogenized by the corresponding converting phage and where the
square brackets indicate that the factor may be present or absent without a relation to phage conversion
(Reference [9]);With H-antigens H:i for the first phase and H:1,2 for the second phase.
For the final reporting, the antigenic formula of an isolate is preferably given without square brackets or
underlining. Report the antigenic formula that has been determined, e.g. 4,12:i:1,2.
For other subspecies of S. enterica, the subspecies to which the serovar belongs is indicated by the
following symbol (Reference [9]):
II for serovar of S. enterica subsp. salamae;
IIIa for serovar of S. enterica subsp. arizonae;
IIIb for serovar of S. enterica subsp. diarizonae;
IV for serovar of S. enterica subsp. houtenae;
VI for serovar of S. enterica subsp. indica.
An example of reporting for a subspecies other than S. enterica subsp. enterica is: S. II 30:z :e,n,x,z
10 15.
12 © ISO 2014 – All rights reserved

Annex A
(informative)
Composition and preparation of culture media and reagents
A.1 General
The culture media as described in this annex are related to biochemical tests (see Annex C). The
composition of the media as described here can be considered as examples. Media with same names may
be described in literature or are commercially available in slightly different compositions. Therefore,
it is important for each biochemical test to verify the reactions of the media with well characterized
positive and negative control strains.
IMPORTANT — If media or reagents are prepared from dehydrated complete media or reagents,
or if ready-to-use media and reagents are used, follow the manufacturer’s instructions regarding
preparation, storage conditions, expiry date, and use.
The shelf-lives of the media indicated in this annex have been shown in some studies. The user should
verify this under their own storage conditions (see ISO 11133).
A.2 Nutrient agar
A.2.1 Composition
Meat extract 3,0 g
a
Peptone 5,0 g
Sodium chloride (NaCl) 5,0 g
(optional)
b
Agar 9 g to 18 g
Water 1 000 ml
a
For example enzymatic digest of casein.
b
Depending on the gel strength of the agar.
A.2.2 Preparation
Dissolve the components in the water by heating.
Adjust the pH, if necessary, so that after sterilization it corresponds to 7,0 ± 0,2 at 25 °C.
Transfer the culture medium into flasks (6.6) of appropriate capacity.
Sterilize in the autoclave (6.2) maintained at 121 °C for 15 min.
Transfer approximately 20 ml of the melted medium into sterile Petri dishes with a diameter of 90 mm
(6.7). Allow to solidify.
Store the poured plates (upside down), protected for desiccation and in the dark, at 5 °C (6.3) for up to
2 months.
If necessary, dry the plates before use.
A.3 Swarm agar media — Sven Gard (examples)
A.3.1 Example 1 (Reference [16])
Use of nutrient agar with amended agar concentration
It is necessary to determine the agar concentration of the nutrient agar intended for H-phase inversion
in a Petri dish. It can vary from a mass fraction of 0,5 % to 1 % depending on the batch of agar used. The
agar medium is required to be sufficiently soft for a spot-inoculated motile Salmonella to swarm over the
medium after overnight incubation between 34 °C and 38 °C (6.1). Preliminary trials, without addition
of antiserum, are therefore necessary, using either a nutrient agar (pH 7,4) prepared for that purpose
or ordinary laboratory nutrient agar made semi-solid by the addition of broth in a proportion allowing
overnight swarming. The volume required for a 10 cm Petri dish is 30 ml.
Swarming on the surface is eas
...