Iso TS 06579-2-2012

TECHNICAL ISO/TS
SPECIFICATION 6579-2
First edition
2012-11-01
Microbiology of food and animal feed —

Horizontal method for the detection,
enumeration and serotyping of
Salmonella —
Part 2:
Enumeration by a miniaturized most
probable number technique
Microbiologie des aliments — Méthode horizontale pour la recherche, le
dénombrement et le sérotypage des Salmonella —
Partie 2: Dénombrement par une technique miniaturisée du nombre le
plus probable
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Reference number
ISO/TS 6579-2:2012(E)
Copyright International Organization for Standardization © ISO 2012

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ISO/TS 6579-2:2012(E)
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ii
Copyright International Organization for Standardization © ISO 2012 – All rights reserved
ISO/TS 6579-2:2012(E)
Contents Page
Foreword ............................................................................................................................................................................ iv
Introduction ........................................................................................................................................................................ v
1 Scope ...................................................................................................................................................................... 1
2 Normative references ......................................................................................................................................... 1
3 Terms and definitions ......................................................................................................................................... 2
4 Principle ................................................................................................................................................................. 2
4.1 General ................................................................................................................................................................... 2
4.2 Pre-enrichment in non-selective liquid medium ......................................................................................... 2
4.3 Enrichment on a selective semi-solid medium ........................................................................................... 2
4.4 Selective plating and identification ................................................................................................................ 2
4.5 Confirmation ......................................................................................................................................................... 2
4.6 Calculation of most probable number ........................................................................................................... 3
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5 Culture media and sera ...................................................................................................................................... 3

5.1 General ................................................................................................................................................................... 3
5.2 Culture media ....................................................................................................................................................... 3
6 Apparatus and glassware ................................................................................................................................. 3
7 Sampling ................................................................................................................................................................ 4
8 Preparation of test sample ................................................................................................................................ 4
9 Procedure .............................................................................................................................................................. 4
9.1 Test portion, initial suspension ....................................................................................................................... 4
9.2 Dilution and pre-enrichment in non-selective liquid medium ................................................................. 5
9.3 Selective enrichment on a semi-solid medium ........................................................................................... 5
9.4 Selective plating .................................................................................................................................................. 5
9.5 Biochemical and serological confirmation................................................................................................... 6
10 Expression of results ......................................................................................................................................... 8
11 Test report ............................................................................................................................................................. 8
Annex A (informative) Composition and preparation of culture media and reagents ..................................... 9
Annex B (informative) Diagram of procedure ........................................................................................................... 17
Bibliography ..................................................................................................................................................................... 18
Copyright International Organization for Standardization © ISO 2012 – All rights reserved iii
ISO/TS 6579-2:2012(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.
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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a technical
committee may decide to publish other types of document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee
casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further
three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed,
it is reviewed again after a further three years, at which time it must either be transformed into an International
Standard or be withdrawn.
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/TS 6579-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
ISO 6579 consists of the following part, under the general title Microbiology of food and animal feed —
Horizontal method for the detection, enumeration and serotyping of Salmonella:
— Part 2: Enumeration·by·a·miniaturized·most·probable·number·technique [Technical Specification]

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Additional parts, dealing with a detection method and guidance for serotyping are planned. ISO 6579:2002 is
to be withdrawn at a later date.
iv
ISO/TS 6579-2:2012(E)
Introduction
The procedure described is based on the method reported in Reference [1].
The enumeration procedure described here concerns a miniaturized most probable number (MPN) technique.
For this mini-MSRV (modified semi-solid Rappaport–Vassiliadis) MPN technique, the volume of primary
dilution tested is less than the volume in the detection method specified in ISO 6579:2002 + Cor.1:2004 +
Amd.1:2007.[5] For this reason, the sensitivity of the mini-MSRV technique is lower than in these detection
methods (Reference [1]). The detection limit of the mini-MSRV method is approximately 1 cfu/g, but can vary
according to Salmonella serovar and per matrix. For the previously mentioned detection methods, this is typically
1 cfu/25 g (0,04 cfu/g). For samples with (very) low numbers of Salmonella spp. (<1 cfu/g), it is possible that
the mini-MSRV procedure is not sufficiently sensitive. If a quantitative result is requested for samples likely to
contain such low numbers (and tested negative with this mini-MSRV technique, for example), it is advisable to
enumerate with a “conventional” MPN technique (not miniaturized). For other samples, the mini-MSRV method
can have an advantage over the conventional MPN technique because the performance of the miniaturized
MPN technique can take less time and need fewer resources (due to small amounts).
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© ISO 2012 – All rights reserved

Copyright International Organization for Standardization v
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Copyright International Organization for Standardization

TECHNICAL SPECIFICATION ISO/TS 6579-2:2012(E)
Microbiology of food and animal feed — Horizontal method for

the detection, enumeration and serotyping of Salmonella —
Part 2:
Enumeration by a miniaturized most probable number technique
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting Sal-
monella, 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 condi-
tions.
1 Scope
This part of ISO 6579 gives a method for the enumeration of Salmonella spp. present in:
— products intended for human consumption and for the feeding of animals;
— environmental samples in the area of food production and food handling;
— animal faeces;
— environmental samples from the primary production stage;
by calculation of the most probable number (MPN).

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The method is based on miniaturization of the dilution, pre-enrichment and selective enrichment steps. The
selective enrichment medium, modified semi-solid Rappaport–Vassiliadis (MSRV), is intended for the detection
of motile salmonellae and is not appropriate for the detection of non-motile salmonellae.
It is possible that the method is less appropriate to enumerate Salmonella ser. Typhi and Salmonella ser. Paratyphi.
The method is not appropriate for the enumeration of Salmonella spp. in (very) low contaminated samples (<1 cfu/g).
NOTE The number of non-motile salmonellae is generally low in most of the matrices relevant for this method. In
this note, examples are given for samples from primary production. The non-motile Salmonella biovars of Salmonella
Gallinarum (Salmonella Gallinarum biovar gallinarum and Salmonella Gallinarum biovar pullorum) do not seem to survive
long in environmental samples and are therefore rarely detected in faecal or environmental (such as dust) samples
(regardless of the method). The number of other non-motile Salmonella serovars in faecal samples seems to be generally
low. For example, in Reference [4] in which approximately 1 000 faecal samples of poultry layer flocks and approximately
900 faecal samples of broiler flocks were analysed, less than 1 % of the total number of samples were positive in a
selective broth and at the same time negative on MSRV medium (and likely to be non-motile). Similar results were found
in a Dutch study with ca 3 200 faecal samples of pigs (unpublished data). On the other hand, in the case of the study
reported in Reference [4], up to almost 40 % of positive samples would not have been detected (i.e. false negatives) if only
a selective broth (in this case Rappaport–Vassiliadis) had been used instead of a semi-solid medium.
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 6887 (all parts), Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination
Copyright International Organization for Standardization © ISO 2012 – All rights reserved 1
ISO/TS 6579-2:2012(E)
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO/TS 11133-1, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production of
culture media — Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory
ISO/TS 11133-2, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production
of culture media — Part 2: Practical guidelines on performance testing of culture media
3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
3.1
Salmonella
microorganism which forms typical or less typical colonies on solid selective media and which displays specific
biochemical and serological characteristics
NOTE Suitable tests for the specific biochemical and serological characteristics are specified in this part of ISO 6579.
3.2
count of Salmonella
number of Salmonella spp. found per millilitre or per gram of a test sample or per surface area or on an object
(e.g. bootsocks)
4 Principle
4.1 General
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The enumeration of Salmonella spp. in the MPN format necessitates four successive stages (4.2 to 4.5).
4.2 Pre-enrichment in non-selective liquid medium

Preparation of a 10 -1 dilution of the sample in buffered peptone water (BPW) (initial suspension).
Addition of the initial suspension to the first (empty) row of three wells of a 12-well microtiter plate.
Inoculation of the second row of three wells containing non-selective pre-enrichment broth (BPW) with a
specified quantity, from the first row in a 12-well microtiter plate.
Inoculation of the third, fourth and if necessary more rows of three wells containing BPW.
Incubation of the 12-well microtiter plates at 37 °C for 18 h.
4.3 Enrichment on a selective semi-solid medium

Subculturing of each well obtained in 4.2 in a well containing a semi-solid agar (MSRV).
Incubation at 41,5 °C (MSRV) for 24 h. If MSRV is negative after 24 h, the plate is incubated for a further 24 h.
4.4 Selective plating and identification

From the (suspect) cultures (the highest dilutions) obtained in 4.3, a selective solid medium xylose–lysine–
deoxycholate (XLD) agar is inoculated and incubated at 37 °C for 24 h.
4.5 Confirmation
Colonies of presumptive Salmonella obtained in 4.4 are confirmed by means of appropriate biochemical and
serological tests.
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ISO/TS 6579-2:2012(E)
4.6 Calculation of most probable number

From the number of confirmed positive wells, the MPN of Salmonella spp. per millilitre or per gram of the test
sample is calculated.
5 Culture media and sera
5.1 General
For current laboratory practice, see ISO 7218.
For the performance testing of media, follow the recommendations of ISO/TS 11133-1, ISO/TS 11133-2 and
the information as given in A.8.
All media and reagents needed are described in Annex A. Alternatively, dehydrated complete media or diluents
can be used. In the latter case, follow the manufacturer’s instructions.
5.2 Culture media
5.2.1 Non-selective pre-enrichment medium: Buffered peptone water (BPW). See A.1.
5.2.2 Semi-solid selective enrichment agar: Modified semi-solid Rappaport–Vassiliadis medium

(MSRV). See A.2.
5.2.3 Xylose–lysine–deoxycholate (XLD) agar. See A.3.
5.2.4 Nutrient agar. See A.4.
5.2.5 Triple sugar–iron agar (TSI agar). See A.5.
As an alternative, a double sugar–iron agar (Kligler–Hajna) may be used.

5.2.6 Urea agar (Christensen). See A.6.
5.2.7 l-Lysine decarboxylation medium (LDC). See A.7.
5.2.8 Antisera. Several types of agglutinating sera containing antibodies for one or several O-antigens and
for one or several H-antigens are commercially available.
Every attempt should be made to ensure that the anti-sera used are adequate to provide for the detection of
all Salmonella serotypes.
6 Apparatus and glassware

Disposable supplies are an acceptable alternative to reusable glassware if it has similar specifications.
Usual microbiological laboratory equipment and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). See ISO 7218.
6.2 Drying cabinet or ventilated oven, capable of being maintained at between 25 °C and 50 °C or a
laminar air flow cabinet.
6.3 Incubators, capable of operating at 37 °C ± 1 °C and 41,5 °C ± 1 °C.

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ISO/TS 6579-2:2012(E)
6.4 Water bath, capable of operating at 47 °C to 50 °C.
6.5 Refrigerator (for storage of prepared media), capable of operating at 5 °C ± 3 °C.
6.6 pH-meter, having a resolution of 0,01 pH and accurate to within ± 0,1 pH units at 25 °C. See ISO 7218.
6.7 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.
6.8 Sterile loops of 1 µl.
6.9 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml (with 0,5 ml division),
2 ml (with 0,1 ml division), 0,1 ml (with 0,01 ml division). Multi-channel pipettes of 0,5 ml and 0,02 ml nominal
capacity for pipetting three wells at a time.
6.10 Sterile Petri dishes, of approximately 90 mm diameter.
6.11 Sterile 12-well microtiter plates with wells of approximately 25 mm diameter and 20 mm deep (5 ml)
with a flat bottom with lid.
6.12 Homogenizer. See ISO 7218.
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7 Sampling
Sampling is not part of the method specified in this part of ISO 6579. See the specific International Standard
dealing with the product concerned.
It is important that the laboratory receive a truly representative sample which has not been damaged or changed
during transport or storage.
If there is no specific International Standard dealing with sampling of the product concerned, it is recommended
that the parties concerned come to an agreement on the subject.
8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard appropriate to the product concerned.
If there is no specific International Standard, it is recommended that the parties concerned come to an
agreement on the subject.
9 Procedure
9.1 Test portion, initial suspension

See ISO 6887 and any specific International Standard appropriate to the product concerned. Prepare an initial
suspension by diluting the test portion 10-fold in BPW (5.2.1). For example, add 25 g of sample to 225 ml of
BPW and homogenize, e.g. in a stomacher (6.12), for 1 min.
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ISO/TS 6579-2:2012(E)
9.2 Dilution and pre-enrichment in non-selective liquid medium

Take a 12-well microtiter plate (6.11) with empty wells in the first row of three wells and containing 2 ml BPW
(5.2.1) in the other wells (second, third and fourth row with each 3 wells; see Annex B).
NOTE 1 As a general case, the described procedure specifies dilutions for one 12-well microtiter plate. When a higher
number than 500 cfu/g of Salmonella is suspected, it is necessary to proceed with a second 12-well microtitre plate
containing 2 ml BPW (5.2.1) in each well. Prepare a sufficient number of plates (dilutions) to ensure that the final well in the
12-well microtiter plate yields a negative result.
Transfer to each well of the first (empty) row of three wells, using a pipette (6.9), 2,5 ml of the initial suspension (9.1).
Transfer 0,5 ml (e.g. by using a multi-channel pipette; 6.9) of each well from the first row into the 2 ml BPW in
the successive wells in the second row (first 5 -1 dilution).
Transfer 0,5 ml (e.g. by using a multi-channel pipette; 6.9, with new tips) of each well from the second row into
the 2 ml BPW in the successive wells in the third row (second 5 -1 dilution).
Before transferring the 0,5 ml of the second row into the third row, mix the suspensions in the wells by repeatedly
(carefully) sucking up and blowing out of the suspension in the pipette and in the wells.
Proceed in the same way for the other rows.
Incubate the 12-well microtiter plate at 37 °C (6.3) for 18 h ± 2 h.
NOTE 2 As the contamination level of the tested samples is generally unknown and often low, it can prove worthwhile to
check for the presence of Salmonella spp. in the sample by also culturing the initial suspension. For this purpose, incubate
(6.3) the initial suspension (9.1) at 37 °C for 18 h± 2 h. For the next culture steps, follow the procedures as described in
9.3 to 9.5. In 9.3: inoculate a Petri dish containing MSRV with 1–3 equally spaced spots with a total volume of 0,1 ml of the
incubated BPW culture of the initial suspension (9.1).
9.3 Selective enrichment on a semi-solid medium

Allow the MSRV (5.2.2) in the 12-well microtiter plates to equilibrate at room temperature if they were stored at
a lower temperature.
Inoculate each well containing 2 ml MSRV with 20 µl of the BPW culture (9.2), e.g. using a multi-channel pipette
(6.9). Use new tips for each row of three wells.
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Place the 20 µl BPW culture at the margin of the well and on the surface of the medium (see Annex B).
When taking a subculture from BPW, try not to disturb particulate samples. Therefore, move the microtitre
plates carefully. Avoid pipetting particulate matter on to the MSRV plates.
Incubate the inoculated MSRV plates at 41,5 °C (6.3) for 24 h ± 3 h.
Do not invert the plates.
Suspect wells show a grey-white, turbid zone extending out from the inoculated drop. The turbid zone is
characterized by a white halo with a clearly defined edge.
If the wells are negative after 24 h, reincubate for a further 24 h ± 3 h.
9.4 Selective plating

Allow the XLD agar (5.2.3) plates to equilibrate at room temperature if they were stored at lower temperature.
If necessary, dry the surface of the plates before use.
Subculture suspect MSRV wells (9.3) by dipping a 1 µl loop (6.8) just inside the border of the opaque growth
and by inoculating this culture material on the surface of one normal size XLD plate, so that well-isolated
colonies are obtained.
ISO/TS 6579-2:2012(E)
Subculture at least the highest dilutions: the ones which still show three suspect MSRV wells as well as the
subsequent dilutions showing two or one suspect MSRV wells.
Incubate the inverted XLD plates at 37 °C (6.3) for 24 h ± 3 h.
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 additional wells become suspect.
Typical colonies of Salmonella spp. grown on XLD agar have a black centre and a lightly transparent zone of
reddish colour due to the colour change of the indicator.
NOTE Hydrogen sulfide-negative variants of Salmonella spp. grown on XLD agar are pink with a darker pink centre.
Lactose-positive variants of Salmonella spp. grown on XLD agar are yellow with or without blackening.
9.5 Biochemical and serological confirmation
9.5.1 General
Perform confirmation on at least one well-isolated suspect colony from each XLD plate (9.4). If no well-isolated
colony can be obtained, it may be necessary to perform an extra culture step on XLD agar or on a non-
selective agar, e.g. nutrient agar (5.2.4), to obtain well-isolated colonies.
If all wells selected fail to confirm as Salmonella spp., presumptive MSRV wells not already subcultured (in
lower dilutions) should be checked.
If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella
spp. can be used. The use of identification kits concerns the biochemical confirmation of colonies. These kits
should be used following the manufacturer’s instructions.
NOTE The recognition of colonies of Salmonella spp. 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.
9.5.2 Selection of colonies for confirmation
For confirmation, take from each XLD plate (9.4) at least one colony considered to be typical or suspect.
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, like nutrient agar (5.2.4) can then be performed in parallel
with the biochemical tests for purity check of the colony taken from the selective agar medium. Incubate
inoculated nutrient agar plates at 37 °C (6.3) for 24 h ± 3 h.
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9.5.3 Biochemical confirmation
9.5.3.1 General
By means of an inoculating wire, inoculate the media specified in 9.5.3.2, 9.5.3.3 and 9.5.3.4 with each of the
cultures obtained from the colonies selected in 9.5.2.
9.5.3.2 TSI agar (5.2.5)
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.
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ISO/TS 6579-2:2012(E)
yellow glucose positive (glucose used)

red or unchanged glucose negative (glucose not used)
Butt
black formation of hydrogen sulfide
bubbles or cracks gas formation from glucose
yellow lactose and/or sucrose positive (lactose and/or sucrose used)
Slant surface
red or unchanged lactose and sucrose negative (neither lactose nor sucrose used)
Typical cultures of Salmonella spp. show alkaline (red) slant 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.
A lactose-positive variant of Salmonella spp. gives a yellow slant on TSI. Thus, preliminary confirmation of
Salmonella cultures shall not be based on the results of the TSI agar test only.
9.5.3.3 Urea agar (5.2.6)
Streak the agar slant surface. Incubate at 37 °C (6.3) for 24 h ± 3 h and examine at intervals.
If the reaction is positive, decomposition of urea liberates ammonia, which 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.
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Typical Salmonella cultures do not hydrolyse urea, so that the colour of the urea remains unchanged (yellow;
see Table 1).
9.5.3.4 l-Lysine decarboxylation medium (5.2.7)
Inoculate just below the surface of the liquid medium. Incubate at 37 °C (6.3) for 24 h ± 3 h.
Turbidity and purple colour after incubation indicates a positive reaction. A yellow colour indicates a negative reaction.
Most typical Salmonella cultures show a positive reaction (see Table 1).
9.5.3.5 Interpretation of the biochemical tests
Salmonella generally show the reactions given in Table 1. If these reactions are found it can be considered that
the sample may contain Salmonella spp.
Table 1 — Interpretation of biochemical tests; typical reactions of most Salmonella serovars

(percentages indicated in brackets)
TSI agar
yellow glucose positive (100 %)
Butt black formation of hydrogen sulfide, H2S (91,6 %)
bubbles and/or cracks gas formation from glucose (91,9 %)
Slant surface red or unchanged lactose and/or sucrose negative (respectively 99,2 % and 99,5 %)
Urea agar
— yellow, no colour changing of the medium negative (100 %)
LDC
— purple colour and turbidity positive (94,6 %)
9.5.4 Serological confirmation and serotyping
Biochemical test results can indicate whether an isolate belongs to the genus Salmonella. For a full typing of
Salmonella strains, serotyping has also to be performed. Serological confirmation gives additional information
ISO/TS 6579-2:2012(E)
to which serogroup the isolate belongs. With serotyping it is possible to further type the isolate to serovar level.
For further details, see ISO 6579[5] and Reference [3].
10 Expression of results
Count the number of wells giving a positive confirmed reaction for each dilution. Calculate the MPN from the
number of positive confirmed wells at each dilution.
If all wells are negative, but the initial suspension (10 -1 dilution) was found to be positive for Salmonella spp.
(after confirmation), the result can be reported as: Salmonella spp. present in the amount of sample tested (e.g.
25 g), but less than the lower limit of detection of the mini-MPN method (<1 cfu/g).
For the calculation of the MPN, the formulas given in ISO 7218 can be used, as no “standard” MPN tables are
known to be available for the dilutions used. A software program for use in Excel1) has been written that can
handle up to 10 levels of serial dilutions. It is highly recommended that this program be used rather than other
programs, since the results for any specific combination of results derived with three dilutions will be the same
as those in the tables published in ISO 7218. Details of the calculations are described in Reference [2] and the
software is freely available for download from:
http://standards.iso.org/iso/ts/6579/-2/
11 Test report
The test report shall contain at least the following information:
a) all information necessary for the complete identification of the sample;
b) the sampling method used, if known;
c) the test method used, with reference to this part of ISO 6579 (ISO/TS 6579-2:2012);
d) all operating detail not specified in this part of ISO 6579, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);
e) the test result(s) obtained;
f) if repeatability has been checked, the final quoted result obtained.
1) Excel is the trade name of a product supplied by Microsoft. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they
can be shown to lead to the same results.
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ISO/TS 6579-2:2012(E)
Annex A
(informative)
Composition and preparation of culture media and reagents
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/TS 11133-1 and ISO/TS 11133-2).
Performance testing for the quality assurance of the culture media is described in A.8.
A.1 Buffered peptone water (BPW)
A.1.1 Composition
Peptonea 10,0 g
Sodium chloride 5,0 g
Disodium hydrogenphosphate dodecahydrate (Na2HPO4⋅12H2O) 9,0 g
Potassium dihydrogenphosphate (KH2PO4) 1,5 g
Water 1 000 ml
a For example, enzymatic digest of casein.
A.1.2 Preparation
Dissolve the components in the water, by heating (without boiling) if necessary.
Adjust the pH, if necessary, so that after sterilization it corresponds to 7,0 ± 0,2 at 25 °C.
Dispense the medium into flasks (6.7) of suitable capacity to obtain the portions necessary for the test.
Sterilize in the autoclave (6.1) maintained at 121 °C for 15 min.
Store airtight, closed flasks in the dark at 5 °C (6.5) for up to 6 months.
Transfer aseptically 2 ml, e.g. by using a multi-channel pipette (6.9), into each well from the 12-well microtitre
plates (6.11). Prepare one 12-well microtitre plate with an empty first row of three wells.
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Copyright International Organization for Standardization 9
ISO/TS 6579-2:2012(E)
A.2 Modified semi-solid Rappaport–Vassiliadis (MSRV) medium
A.2.1 Base medium
A.2.1.1 Composition
Enzymatic digest of animal and plant tissue 4,6 g
Acid hydrolysate of casein 4,6 g
Sodium chloride (NaCl) 7,3 g
Magnesium chloride anhydrous (MgCl2) 10,9 g
Malachite green oxalate 0,04 g
Agar 2,7 g
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Water 1 000 ml
A.2.1.2 Preparation
Dissolve the components in the water.
Heat to boiling with agitation.
IMPORTANT Do not autoclave.
Do not hold the medium at high temperatures longer than necessary.
Cool the medium to 47 °C to 50 °C.
A.2.2 Novobiocin solution
A.2.2.1 Composition
Novobiocin sodium salt 0,05 g
Water 10 ml
A.2.2.2 Preparation
Dissolve the novobiocin sodium salt in the water.
Sterilize by filtration through a filter with a pore size of 0,22 µm.
The solution may be stored at 5 °C (6.5) for up to 4 weeks, or in small portions (e.g. of 2 ml) at −20 °C for up to 1 year.
A.2.3 Complete medium
A.2.3.1 Composition
Base medium (A.2.1) 1 000 ml
Novobiocin solution (A.2.2) 2 ml
10
ISO/TS 6579-2:2012(E)
A.2.3.2 Preparation
Aseptically add 2 ml of the novobiocin solution (A.2.2) to 1 000 ml of base medium (A.2.1) at 47 °C to 50 °C.
Mix carefully.
The final concentration of novobiocin is 10 mg/l MSRV medium.
The final pH should be 5,2 (5,1 to 5,4) at 20 °C to 25 °C.
Transfer 2 ml (e.g. by using a multi-channel pipette, 6.9) into each well from the 12-well microtitre plates (6.11).
If necessary, also pour into plates up to a final volume of 15 ml to 20 ml in Petri dishes with a diameter of
90 mm (6.10).
Allow the medium to solidify before moving and handle with care.
Store the plates, with surface upwards, at 5 °C (6.5) in the dark for up to 2 weeks.
Do not invert the plates, as the semi-solid agar is too liquid.
Any plates in which the semi-solid agar has liquefied or fragmented should not be used.
Immediately before use, and only if visible moisture is apparent, dry the surface of the agar plates carefully,
e.g. by placing them with the lids off and the agar surface upwards in a laminar air flow cabinet. Take care not
to overdry the medium.
A.3 Xylose–lysine–deoxycholate (XLD) agar
A.3.1 Composition
Yeast extract 3,0 g
d(+)-Xylose 3,75 g
Lactose 7,5 g
Sucrose (saccharose) 7,5 g
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l(+)-lysine hydrochloride 5,0 g
Sodium thiosulfate (Na2S2O3) 6,8 g
Iron(III) ammonium citrate 0,8 g
Phenol red 0,08 g
Sodium deoxycholate 1,0 g
Agar 9 g to 18 ga
Water 1 000 ml
a Depending on the gel strength of the agar.
A.3.2 Preparation
Dissolve the components in the water.
Heat with frequent agitation, until the medium starts to boil. Avoid overheating.
IMPORTANT — Do not autoclave.
It is important to avoid preparing large volumes which causes prolonged heating.

ISO/TS 6579-2:2012(E)
Adjust the pH, if necessary, so that it corresponds to 7,4 ± 0,2 at 25 °C.
Transfer the medium to a water bath (6.4) at 47 °C to 50 °C.
Pour into Petri dishes (6.10) as soon as the medium has cooled. Allow to solidify.
Store the poured plates (upside down) at 5 °C (6.5), in the dark for up to 4 weeks. Protect them from drying.
Immediately before use and only if necessary, dry the surface of the agar plates carefully. Take care not to
overdry the medium.
A.4 Nutrient agar
A.4.1 Composition
Meat extract 3,0 g
Peptonea 5,0 g
Sodium chloride (NaCl) (optional) 5,0 g
Agar 9 g to 18 gb
Water 1 000 ml
b Depending on the gel strength of the agar.
A.4.2 Preparation
Dissolve the components in the water by heating.
Transfer the culture medium into flasks (6.7) of appropriate capacity.
Transfer about 15 ml of the melted medium into sterile Petri dishes (6.10). Allow to solidify.
Store the poured plates (upside down) at 5 °C (6.5) in the dark for up to 4 weeks. Protect them from drying.
Alternatively, other appropriate non-selective agar media may be used.
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12
ISO/TS 6579-2:2012(E)
A.5 Triple sugar–iron agar (TSI agar)
A.5.1 Composition
Meat extract 3,0 g
Yeast extract 3,0 g
Peptone 20,0 g
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Lactose 10,0 g
Sucrose 10,0 g
Glucose (dextrose) 1,0 g
Iron(III) citrate (ferric citrate) 0,3 g
Sodium thiosulfate (Na2S2O3) 0,3 g
Phenol red 0,024 g
Agar 9 g to 18 ga
Water 1 000 ml
a Depending on the gel strength of the agar.
A.5.2 Preparation
Dissolve the components in the water, by heating if necessary.
Dispense the medium in quantities of 10 ml into tubes, preferably with screw caps.
Allow to set in a sloping position to give a butt of depth 2,5 cm to about 5 cm.
Tubes closed with screw caps can be stored at 5 °C (6.5), in the dark for up to 3 months.

ISO/TS 6579-2:2012(E)
A.6 Urea agar (UA) Christensen
A.6.1 Base medium
A.6.1.1 Composition
Peptonea 1,0 g
Glucose (dextrose) 1,0 g
Phenol red 0,012 g
Agar 9 g to 18 gb
Water 1 000 ml
b Depending on the gel strength of the agar.
A.6.1.2 Preparation
Store airtight, closed flasks at 5 °C (6.5), in the dark for up to 3 months.
A.6.2 Urea solution
A.6.2.1 Composition
Urea 400 g
Water, to a final volume of 1 000 ml
A.6.2.2 Preparation
Dissolve the urea in the water and sterilize by filtration.
Store airtight, closed flasks at 5 °C (6.5), in the dark for up to 3 months.
A.6.3 Complete medium
A.6.3.1 Composition
Base (A.6.1) 950 ml
Urea (A.6.2) 50 ml
A.6.3.2 Preparation
Add, under aseptic conditions, the urea solution to the base, previously melted and then cooled to 47 °C to 50 °C.
Dispense the complete medium in quantities of 10 ml into sterile tubes.
14
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ISO/TS 6579-2:2012(E)
Allow to set in a sloping position.
A.7 l-Lysine decarboxylation medium (LDC)
A.7.1 Composition
l-Lysine monohydrochloride 5,0 g
Yeast extract 3,0 g
Glucose 1,0 g
Bromocresol purple 0,015 g
Water 1 000 ml
A.7.2 Preparation
Dispense the medium in quantities of 2-5 ml into sterile narrow culture tubes (6.7), preferably with screw caps.
A.8 Performance testing for the quality assurance of the culture media
Information on the performance testing of the culture media indicated in this part of ISO 6579 is given in Table
A.1. For the definitions of selectivity and productivity, see ISO/TS 11133-1 and ISO/TS 11133-2.
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ISO/TS 6579-2:2012(E)
Table A.1 — Performance testing of culture media
Medium Function Incubation Control strainsa Criterionc

Salmonella Typhimurium WDCM 00031b
BPW Productivity 18 h at 37 ºC Turbidity (1 to 2)
Salmonella Enteritidis WDCM 00030
Grey-white, turbid zone
extending out from
(2x) 24 h at Salmonella Typhimurium WDCM 00031b inoculated drop(s). After
Productivity 24 h–48 h, the turbid
41,5 ºC Salmonella Enteritidis WDCM 00030 zone(s) will be (almost)
fully migrated over the
plate
MSRV
Possible growth at the
Escherichia coli WDCM 00012b place of the inoculated
Escherichia coli WDCM 00013 drop(s) without a turbid
(2x) 24 h at
Selectivity zone
41,5 ºC
Enterococcus faecalis WDCM 00009b
No growth
Enterococcus faecalis WDCM 00087
Good growth (2) of
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colonies with black

Salmonella Typhimurium WDCM 00031b centre and a lightly
Productivity 24 h at 37 ºC transparent zone of
Salmonella Enteritidis WDCM 00030 reddish colour due to
the colour change of
XLD the medium
Escherichia coli WDCM 00012b Growth or partial
inhibition (0 to 1) of
Escherichia coli WDCM 00013 yellow colonies
Selectivity 24 h at 37 ºC
Enterococcus faecalis WDCM 00009b
Total inhibition (0)
Enterococcus faecalis WDCM 00087
Nutrient
Productivity 24 h at 37 ºC Salmonella Typhimurium WDCM 00031 Good growth (2)
agar
aFor information on culture collection strain numbers and contact details, refer to the reference strain catalogue available on http://
refs.wdcm.org/home.htm (WDCM: World Data Centre for Microorganisms).
bStrain to be used to be chosen by the end user laboratory (minimum).
c Growthis categorized as: 0 — no growth; 1 — weak growth (partial inhibition); 2 — good growth (see ISO/TS 11133-1 and
ISO/TS 11133-2).
16
ISO/TS 6579-2:2012(E)
Annex B
(informative)
Diagram of procedure
Serial dilutions and Transfer of Selective

pre-enrichment pre-enrichment enrichment
in BPW BPW (20 µl) on MSRV
on to MSRV
500 µl in 2 ml
0,20 g 0,04 g 0,008 g 0,002 g
2 ml MSRV
Initial suspension
Suspected MSRV
2 ml BPW after incubation
Inoculum
Incubation at 41,5 °C ± 1 °C
Incubation at 37 °C ± 1 °C
for 24 h ± 3 h
for 18 h ± 2 h
Negative wells: further
incubation up to 48 h
Plating out suspect wells of (at

least) highest dilution(s) on
selective isolation medium XLD
at 37 °C ± 1 °C for 24 h ± 3 h
From each plate test a characteristic

colony biochemically and for
definitive confirmation
Generation of the MPN
Figure B.1 — Schematic presentation of the mini-MSRV protocol

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ISO/TS 6579-2:2012(E)
Bibliography
[1] Fravalo, P., Hascoet, Y., le Fellic, M., Queguiner, s., Petton, J., salvat, G. Convenient method
for rapid and quantitative assessment of Salmonella enterica contamination: The mini-MSRV MPN
technique. J. Rapid Meth. Automat. Microbiol. 2003, 11, pp. 81–88
[2] Jarvis, B., WilricH, c., WilricH, P.-t. Reconsideration of the derivation of most probable numbers, their
standard deviations, confidence bounds and rarity values. J. Appl. Microbiol. 2010, 109, pp. 1660–1667
[3] griMont, P.a.d., Weill, F.-X. Antigenic formulae of the Salmonella serovars, 9th edition, WHO Collaborating
Centre for Reference and Research on Salmonella. Paris: Institut Pasteur, 2007. 166 p. Available (viewed
2012-10-15) at: http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-089
[4] voogt, n., r aes, M., Wannet, W.J.B., H enken, a.M., van de giessen, A.W. Comparison of
selective enrichment media for the detection of Salmonella in poultry faeces. Lett. Appl. Microbiol.
2001, 32, pp. 89–92
[5] ISO 6579:2002 + Cor.1:2004 + Amd.1:2007, Microbiology of food and animal feeding stuffs — Horizontal
method for the detection of Salmonella spp.
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18
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ISO/TS 6579-2:2012(E)
ICS 07.100.30
Price based on 18 pages
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TECHNICAL ISO/TS

Microbiology of food and animal feed —

Copyright International Organization for Standardization © ISO 2012

COPYRIGHT PROTECTED DOCUMENT

5 Culture media and sera ...................................................................................................................................... 3

— Part 2: Enumeration·by·a·miniaturized·most·probable·number·technique [Technical Specification]

The procedure described is based on the method reported in Reference [1].

© ISO 2012 – All rights reserved

Copyright International Organization for Standardization

Microbiology of food and animal feed — Horizontal method for

— environmental samples in the area of food production and food handling;

— environmental samples from the primary production stage;

by calculation of the most probable number (MPN).

3 Terms and definitions

4.2 Pre-enrichment in non-selective liquid medium

Incubation of the 12-well microtiter plates at 37 °C for 18 h.

4.3 Enrichment on a selective semi-solid medium

4.4 Selective plating and identification

4.6 Calculation of most probable number

5 Culture media and sera

5.2 Culture media

5.2.2 Semi-solid selective enrichment agar: Modified semi-solid Rappaport–Vassiliadis medium

5.2.3 Xylose–lysine–deoxycholate (XLD) agar. See A.3.

5.2.4 Nutrient agar. See A.4.

5.2.5 Triple sugar–iron agar (TSI agar). See A.5.

As an alternative, a double sugar–iron agar (Kligler–Hajna) may be used.

5.2.7 l-Lysine decarboxylation medium (LDC). See A.7.

6 Apparatus and glassware

Usual microbiological laboratory equipment and, in particular, the following.

6.3 Incubators, capable of operating at 37 °C ± 1 °C and 41,5 °C ± 1 °C.

6.4 Water bath, capable of operating at 47 °C to 50 °C.

6.5 Refrigerator (for storage of prepared media), capable of operating at 5 °C ± 3 °C.

6.8 Sterile loops of 1 µl.

6.10 Sterile Petri dishes, of approximately 90 mm diameter.

6.12 Homogenizer. See ISO 7218.

8 Preparation of test sample

9.1 Test portion, initial suspension

9.2 Dilution and pre-enrichment in non-selective liquid medium

Proceed in the same way for the other rows.

Incubate the 12-well microtiter plate at 37 °C (6.3) for 18 h ± 2 h.

9.3 Selective enrichment on a semi-solid medium

Incubate the inoculated MSRV plates at 41,5 °C (6.3) for 24 h ± 3 h.

Do not invert the plates.

If the wells are negative after 24 h, reincubate for a further 24 h ± 3 h.

9.4 Selective plating

Incubate the inverted XLD plates at 37 °C (6.3) for 24 h ± 3 h.

9.5 Biochemical and serological confirmation

9.5.2 Selection of colonies for confirmation

Use pure cultures for biochemical and serological confirmation. --`,`,,,,,```,````,`,,`,`````-`-`,,`,,`,`,,`---

9.5.3 Biochemical confirmation

9.5.3.2 TSI agar (5.2.5)

Interpret the changes in the medium as follows.

yellow glucose positive (glucose used)

9.5.3.3 Urea agar (5.2.6)

9.5.3.4 l-Lysine decarboxylation medium (5.2.7)

9.5.3.5 Interpretation of the biochemical tests

Table 1 — Interpretation of biochemical tests; typical reactions of most Salmonella serovars

9.5.4 Serological confirmation and serotyping

a) all information necessary for the complete identification of the sample;