ISO 19250:2010

INTERNATIONAL ISO
STANDARD 19250
First edition
2010-07-15

Water quality — Detection of Salmonella
spp.
Qualité de l'eau — Recherche de Salmonella spp.




Reference number
ISO 19250:2010(E)
©
ISO 2010

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ISO 19250:2010(E)
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ii © ISO 2010 – All rights reserved

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ISO 19250:2010(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 in selective liquid media .2
4.4 Plating out and recognition.3
4.5 Confirmation .3
5 Apparatus.3
6 Sampling.4
7 Culture media and reagents .4
8 Procedure.5
8.1 Preparation of the sample .5
8.2 Non-selective pre-enrichment.5
8.3 Selective enrichment.5
8.4 Plating out .6
8.5 Confirmation .6
9 Expression of results.9
10 Test report.9
Annex A (normative) Diagram of procedure .10
Annex B (normative) Composition and preparation of culture media and reagents.11
Annex C (informative) Results of the interlaboratory trial.18
Bibliography.23

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ISO 19250:2010(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.
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 19250 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This edition cancels and replaces ISO 6340:1995, which has been technically revised.

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ISO 19250:2010(E)
Introduction
Salmonella species are bacteria which are widely distributed all over the world. They are usually classified as
pathogens, although their virulence and pathogenesis vary widely. The natural hosts of Salmonella include
humans, agricultural and domestic livestock, and wild animals including birds. Humans and animals can
excrete these bacteria while carrying them asymptomatically as well as during disease. It is therefore
impossible to eliminate them from the environment. Following the infection of humans, the transmission of
Salmonella can cause severe disease.
Since water is a recognized vehicle of infection, the presence or absence of Salmonella is monitored in water
where there is perceived to be a risk of infection. Salmonella can be present in all types of domestic and
agricultural waste water, freshwaters, including ground and drinking waters, as well as sea water.
The detection of Salmonella in water usually requires a concentration step. Since Salmonella cells can be
present in low numbers and injured in the aqueous environment, their detection in water usually requires a
pre-enrichment step.

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INTERNATIONAL STANDARD ISO 19250:2010(E)

Water quality — Detection of Salmonella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting Salmonella, and especially S. enterica subsp. enterica ser. Typhi (Salmonella ser. Typhi) and
S. enterica subsp. enterica ser. Paratyphi (Salmonella ser. Paratyphi), be undertaken only in properly
equipped laboratories, under the control of a skilled microbiologist, and that great care be 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 problems, 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.
IMPORTANT — It is absolutely essential that tests conducted according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the detection of Salmonella spp. (presumptive or
confirmed) in water samples. It is possible that, for epidemiological purposes or during outbreak investigations,
other media are also required.
WARNING — It is possible that the method does not recover all Salmonella ser. Typhi and ser.
Paratyphi.
NOTE For a semi-quantitative approach, most probable number (MPN) tests can be performed using appropriate
sample volumes. For these cases, the volume of the buffered peptone water is adjusted accordingly.
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 6579, Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Salmonella
spp.
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses
ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 19458, Water quality — Sampling for microbiological analysis
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ISO 19250:2010(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
presumptive Salmonella spp.
bacteria which grow in the selective enrichment medium specified, and form typical or atypical colonies on the
solid selective media
3.2
confirmed Salmonella spp.
bacteria which grow in the selective enrichment medium specified, and form typical and suspicious colonies
on the solid selective media, and which display specfic biochemical and serological characteristics
NOTE The specific biochemical and serological characteristics are determined by tests specified in this International
Standard.
3.3
Salmonella detection
determination of the presence or absence of Salmonella (3.4)
3.4
Salmonella spp.
Salmonella
microorganisms which form typical or atypical colonies on solid selective media and which display specific
biochemical and serological characteristics
4 Principle
4.1 General
The detection of Salmonella necessitates four successive stages (see also Annex A).
Pre-enrichment is often necessary to permit detection of low numbers of Salmonella or injured Salmonella.
Some Salmonella and those which are sublethally injured may require additional incubation time (4.3).
Furthermore, Salmonella can be present in small numbers and are often accompanied by considerably Iarger
numbers of other members of Enterobacteriaceae or of other families. Therefore, selective enrichment is
necessary.
4.2 Pre-enrichment in non-selective liquid medium
Buffered peptone water (B.1) is inoculated at ambient temperature with a known volume of the sample or its
dilutions, then incubated at (36 ± 2) °C for (18 ± 2) h. Larger volumes can be concentrated using membrane
filtration and the membrane filter is then added to buffered peptone water.
NOTE For waste water it has been shown that shorter incubation times or direct inoculation of the sample in selective
medium (4.3) produce better results.
For a semi-quantitative approach, MPN tests can be performed using appropriate sample volumes. In these
cases, adjust the volumes of the buffered peptone water accordingly.
4.3 Enrichment in selective liquid media
Rappaport-Vassiliadis medium with soya (RVS broth) and Muller-Kauffmann tetrathionate-novobiocin broth
(MKTTn) are inoculated with the culture obtained in 4.2.
The RVS broth is incubated at (41,5 ± 1) °C for (24 ± 3) h and the MKTTn broth at (37 ± 1) °C for (24 ± 3) h.
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ISO 19250:2010(E)
To detect slow-growing Salmonella spp., incubate the enrichment broth for a further (24 ± 3) h to a total of
(48 ± 4) h at (41,5 ± 1,0) °C.
NOTE Salmonella Typhi and Salmonella Paratyphi A are usually not important in routine water quality monitoring, but
can be relevant in epidemiological investigations. MKTTn broth is used for enrichment with incubation at (36 ± 2) °C for up
to (24 ± 3) h and recovers most strains of Salmonella, including some strains of Salmonella Paratyphi, but is not thought to
be able to recover strains of Salmonella Paratyphi C. MKTTn broth is not used if Salmonella Typhi is suspected after the
use of selenite cystine broth.
4.4 Plating out and recognition
From the cultures obtained in 4.3, two selective solid media are inoculated:
a) xylose lysine deoxycholate agar (XLD agar);
b) any other solid selective medium complementary to XLD agar and, if applicable, appropriate for the
isolation of lactose-positive Salmonella and Salmonella Typhi and Salmonella Paratyphi strains — the
laboratory may choose which medium to use.
Incubate the XLD agar at (36 ± 2) °C and examine after (24 ± 3) h to check for the presence of colonies which
are considered to be presumptive Salmonella. Incubate the second selective agar according to the
manufacturer's recommendations.
NOTE For information, brilliant green agar (BGA), bismuth sulfite agar, etc., can be used as the second plating-out
medium.
4.5 Confirmation
Subculture colonies of presumptive Salmonella, then plate out as described in 4.4 and confirm their identity by
means of appropriate biochemical (8.5.3) and serological (8.5.4) tests.
5 Apparatus
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
5.1 General. Except for disposable glassware which is delivered sterile, sterilize glassware as specified in
ISO 8199. Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable
specifications.
5.2 Autoclave, capable of being maintained at (121 ± 3) °C and at (115 ± 3) °C.
5.3 Water bath or incubator, capable of being maintained at (36 ± 2) °C.
5.4 Water bath or incubator, capable of being maintained at (41,5 ± 1,0) °C.
5.5 Water baths, capable of operating at (70 ± 1) °C and at 50 °C to 55 °C.
5.6 Membrane filtration apparatus, as specified in ISO 8199.
5.7 Sterile membrane filters, with a nominal pore size of 0,45 µm.
The quality of membrane filters may vary from brand to brand or even from batch to batch. It is therefore
advisable to check the quality on a regular basis, as specified in ISO 7704.
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ISO 19250:2010(E)
5.8 pH-meter, with an accuracy of calibration of ± 0,1 pH at 20 °C to 25 °C.
5.9 Sterile forceps.
5.10 Sterile loops, approximate diameter 3 mm (10 µl volume), and inoculation needle or wire.
6 Sampling
Sampling is not part of the method specified in this International Standard. Samples should be taken in
accordance with ISO 19458.
It is important the laboratory receive a truly representative sample which has not been damaged or changed
during transport or storage.
7 Culture media and reagents
[2] [3]
NOTE For guidelines on quality assurance and performance testing, see ISO/TS 11133-1 and ISO/TS 11133-2 .
7.1 Basic materials. For uniformity of results, in the preparation of media, either use a dehydrated
complete medium or use constituents of uniform quality and reagents of recognized analytical grade.
Other grades of reagents may be used provided they can be shown to produce comparable results.
[1]
7.2 Water, ISO 3696 , grade 3.
7.3 Culture media, prepared in accordance with Annex B.
7.3.1 Buffered peptone water, non-selective pre-enrichment medium buffered peptone water (BPW, B.1).
7.3.2 Rappaport-Vassiliadis broth with soya (RVS broth, B.2), selective enrichment medium.
7.3.3 Xylose lysine deoxycholate agar (XLD agar, B.3).
7.3.4 Second solid selective plating-out medium, whose choice is left to the discretion of the testing
laboratory. Follow the manufacturer's instructions precisely regarding its preparation for use.
7.3.5 Nutrient agar (B.4), or other appropriate non-selective agar.
7.3.6 Triple sugar and iron agar (TSI agar, B.5).
As an alternative, iron and two sugar agar may be used.
7.3.7 Urea agar, Christensen (B.6).
7.3.8 L-Lysine decarboxylation medium (B.7).
7.3.9 Selenite cystine broth (B.8).
7.3.10 Muller-Kaufmann tetrathionate-novobiocin broth (MKTTn, B.9).
7.3.11 Filter aid (B.10).
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ISO 19250:2010(E)
8 Procedure
See Figure A.1.
8.1 Preparation of the sample
For the preparation of the sample, filtration and inoculation on isolation media, follow the instructions as
specified in ISO 8199 and ISO 6887-1. Start the examination preferably immediately after taking the samples.
If the samples are kept at ambient temperatures, start the examination within 12 h after sampling. Under
exceptional circumstances, it is allowable for the samples to be kept at (5 ± 3) °C for up to 24 h prior to
examination.
The volume of the sample to be analysed depends on the type of water. Usual volumes for bathing water and
drinking water are 1 000 ml to 5 000 ml. For polluted surface waters and waste water, smaller volumes are
usually analysed.
If sample dilutions are necessary (e.g. for waste water samples), prepare these dilutions as specified in
ISO 8199.
8.2 Non-selective pre-enrichment
8.2.1 Non-selective pre-enrichment for volumes less than 10 ml
Inoculate 50 ml of BPW (B.1) at room temperature with the sample or dilutions thereof and incubate at
(36 ± 2) °C for (18 ± 2) h.
8.2.2 Non-selective pre-enrichment for volumes greater than 10 ml
Filter a volume of water appropriate for the water being examined.
Immerse the membrane filter in 50 ml of BPW (B.1).
Alternatively, add the sample to the same volume of double strength BPW.
Note that the latter procedure is not suitable for mineral waters with high salt content or sea water.
Incubate the cultures at (36 ± 2) °C for (18 ± 2) h.
8.2.3 Recommendation for turbid or polluted water
For turbid or polluted waters, sterile filter aid (B.10) can be added and the sample filtered through a sterile
absorbent pad acting as a supporting base instead of using the membrane.
In this case, filter an aliquot of filter aid, typically 15 ml, to form an initial layer on the absorbent pad. Mix a
second aliquot, typically 15 ml, with the volume of sample and filter. For turbid or dirty waters, additional
aliquots may be filtered. When filtration is complete, remove the funnel and carefully transfer the absorbent
pad and filter aid to BPW (B.1). If necessary, retain a small volume of BPW to rinse the funnel so that the
final volume of BPW is 100 ml. Incubate for presence or absence, or dispense as an MPN series for a
semi-quantitative count.
8.3 Selective enrichment
Allow the enrichment broth(s) to equilibrate to room temperature if they were stored at a lower temperature.
Transfer 0,1 ml of the culture obtained in 8.2 to a tube containing 10 ml of the RVS broth (B.2). When
MKTTn (B.9) is also used, transfer 1 ml of the culture obtained in 8.2 to a tube containing 10 ml of the MKTTn
broth.
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ISO 19250:2010(E)
Incubate the inoculated RVS broth at (41,5 ± 1,0) °C for (24 ± 3) h and, if necessary (see 4.3), for (48 ± 4) h.
Care should be taken that the maximum allowed incubation temperature (42,5 °C) is not exceeded. Incubate
the inoculated MKTTn broth at (36 ± 2) °C for (24 ± 3) h.
NOTE For RVS broth, the magnesium chloride concentration and incubation temperature have been optimized to
yield good recovery without losing selectivity according to Reference [5].
8.4 Plating out
8.4.1 General
Allow the XLD agar plates and the second selective plating-out medium (see ISO 6579:2002, 5.2.4.2) to
equilibrate at room temperature if they were stored at a lower temperature. If necessary, dry the surface of the
plates before use.
8.4.2 Plating from RVS broth
Using the culture obtained in the RVS broth, inoculate, after incubation for (24 ± 3) h and, if necessary
(see 4.3), for (48 ± 4) h, by means of a sterile loop (5.10), the surface of the following enrichment media so
that well-isolated colonies are obtained:
a) XLD agar (B.3);
b) an additional selective medium (7.3.4).
Invert the dishes so that the bottom is uppermost, and place them in the incubator (5.3) set at (36 ± 2) °C for
(24 ± 3) h for the XLD agar. The manufacturer's instructions shall be followed for the second selective
plating-out medium.
8.4.3 Plating from MKTTn broth
After incubation at (36 ± 2) °C for (24 ± 3) h using the culture obtained, repeat the procedure specified in 8.4.2
with the two selective plating-out media.
8.5 Confirmation
8.5.1 General
If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella
may be used. Use these kits according to the manufacturer's instructions.
8.5.2 Selection of colonies for confirmation
For routine monitoring purposes, take, for confirmation, from each Petri dish of each selective medium (8.4),
at least one discrete colony considered to be typical or presumptive Salmonella. If the first colony is not
confirmed as Salmonella, then take a further four colonies.
On XLD agar, typical Salmonella colonies usually have a black centre and a lightly transparent zone of
reddish colour due to the colour change of the indicator. It is recommended that at least five colonies be
identified for epidemiological studies. If on one dish there are fewer than five typical or suspect colonies, take
all the typical or suspect colonies for confirmation.
NOTE The recognition of Salmonella colonies 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 medium used. Shigella,
Providencia and H S-negative Salmonella spp.(e.g. Salmonella Paratyphi A) appear as pink with a darker pink centre;
2
lactose-positive Salmonella grown on XLD are yellow with or without blackening; Enterobacteriaceae e.g. Escherichia coli,
Enterobacter, Klebsiella, Citrobacter, Proteus, and Serratia appear as yellow, opaque colonies.
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ISO 19250:2010(E)
Streak the selected colonies on to the surface of pre-dried “non-selective” agar plates (e.g. nutrient agar, B.4)
to allow the development of well-isolated colonies.
Incubate the inoculated plates at (36 ± 2) °C for (24 ± 3) h.
If the plating fails to produce distinct colonies, repeat in a manner which ensures that single discrete colonies
are produced. Use single isolated colonies for biochemical and, if appropriate, serological confirmation. If
biochemical kits are used for identification, follow the manufacturer's instructions.
8.5.3 Biochemical confirmation
8.5.3.1 General
By means of an inoculating wire, inoculate the media specified in 8.5.3.2 to 8.5.3.4 with each of the cultures
obtained from the colonies selected in 8.5.2.
8.5.3.2 TSI agar
Streak the TSI agar (B.5) slant surface and stab the butt. Incubate at (36 ± 2) °C for (24 ± 3) h. Interpret the
appearance of the medium as in Table 1.
Table 1 — Interpretation of changes in medium
Appearance Interpretation
Butt
Yellow (acid) Glucose positive (fermentation of glucose)
Red or unchanged (alkaline) Glucose negative (no fermentation of carbohydrates)
Black Formation of hydrogen sulfide
Bubbles or cracks Gas formation from glucose
Slant surface
Yellow Lactose or sucrose positive (lactose or sucrose used)
Red or unchanged (alkaline) Lactose and sucrose negative (neither lactose nor sucrose used)
Typical Salmonella cultures show alkaline (red) slants, gas formation (bubbles) and acid (yellow) butts, with (in
about 90 % of the cases) formation of hydrogen sulfide (blackening of the agar).
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.
8.5.3.3 Urea agar
Streak the urea agar (B.6) slant surface. Incubate at (36 ± 2) °C for up to 24 h and examine at intervals. lf the
reaction is positive, hydrolysis 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. Typical Salmonella cultures show a
negative reaction, i.e. no colour production.
8.5.3.4 L-Lysine decarboxylation medium
Inoculate well below the surface of the liquid L-lysine decarboxylation medium (B.7). Incubate at (36 ± 2) °C
for (24 ± 3) h. Typical Salmonella cultures show a purple colour after incubation.
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ISO 19250:2010(E)
8.5.3.5 Interpretation of the biochemical tests
Salmonella generally show the reactions given in Table 2.
Table 2 — Biochemical reactions of Salmonella
Salmonella strains showing the reaction
a
Test Subclause Reaction
b
%
TSI glucose (acid formation) 8.5.3.2 100,0
+
c
TSI glucose (gas formation) 8.5.3.2 +
91,9
d
TSI lactose 8.5.3.2 −
99,2
TSI sucrose 8.5.3.2 99,5
−
e
TSI hydrogen sulfide 8.5.3.2 +
91,6
Urea hydrolysis 8.5.3.3 − 99,0
f
L-Lysine decarboxylation 8.5.3.4 +
94,6
a
See Reference [8].
b
These figures indicate only that not all strains of Salmonella show the reactions marked + or −. They may vary between
geographical areas and from water source to water source.
c
Salmonella Typhi is anaerogenic.
d
The Salmonella enterica subsp. arizonae gives positive or negative lactose reactions but is always β-galactosidase positive. The
Salmonella enterica subsp. diarizonae gives a negative lactose reaction, but gives a positive β-galactosidase reaction. In addition, it is
possible that these strains do not produce H S. For the study of these strains, it can be useful to carry out complimentary biochemical
2
tests (References [9][10]).
e
Acid formation is sometimes difficult to recognize due to strong blackening.
f
Salmonella Paratyphi A is negative.

The biochemical reactions in Table 3 are typical of Salmonella spp.
Table 3 — Typical biochemical reactions of Salmonella spp. to tests
Test Subclause Reaction
TSI lactose 8.5.3.2 −
TSI glucose 8.5.3.2 +
TSI sucrose 8.5.3.2 −
TSI hydrogen sulfide 8.5.3.2 +
Urea splitting 8.5.3.3 −
L-Lysine decarboxylase 8.5.3.4 +
Isolates which only vary from the typical parameters listed in Table 3 by one or two reactions can still be
Salmonella and should be further investigated and sent to a recognized reference laboratory for confirmation.
8.5.4 Serological confirmation and serotyping
Isolates which are typical according to the biochemical reactions listed in Table 3 are presumptive Salmonella
spp. and should, if need be, be investigated further by a reference laboratory. The presence of Salmonella O-,
Vi-, and H-antigens is detected by slide agglutination as specified in ISO 6579 with the appropriate sera, from
pure colonies (8.5.2) and after auto-agglutinating strains have been eliminated.
Isolates which give positive serological reactions are confirmed as Salmonella spp.
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ISO 19250:2010(E)
9 Expression of results
In accordance with the results of the biochemical tests (8.5.3) and serological confirmation (8.5.4), indicate
whether presumptive or confirmed Salmonella were detected in the test portion ex
...

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Qualité de l'eau - Recherche de Salmonella spp.Water quality - Detection of Salmonella spp.07.100.20Mikrobiologija vodeMicrobiology of waterICS:Ta slovenski standard je istoveten z:ISO 19250:2010SIST ISO 19250:2011en,fr01-junij-2011SIST ISO 19250:2011SLOVENSKI
STANDARDSIST ISO 6340:19981DGRPHãþD



SIST ISO 19250:2011



2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Qualité de l'eau - Recherche de Salmonella spp.Water quality - Detection of Salmonella spp.07.100.20Mikrobiologija vodeMicrobiology of waterICS:Ta slovenski standard je istoveten z:ISO 19250:2010SIST ISO 19250:2011en,fr01-junij-2011SIST ISO 19250:2011SLOVENSKI
STANDARDSIST ISO 6340:19981DGRPHãþDSIST ISO 19250:2011



SIST ISO 19250:2011SIST ISO 19250:2011



Reference numberISO 19250:2010(E)© ISO 2010
INTERNATIONAL STANDARD ISO19250First edition2010-07-15Water quality — Detection of Salmonella spp. Qualité de l'eau — Recherche de Salmonella spp.
SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
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ISO 2010 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing 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.
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© ISO 2010 – All rights reserved
SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) © ISO 2010 – All rights reserved
iii 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 in selective liquid media.2 4.4 Plating out and recognition.3 4.5 Confirmation.3 5 Apparatus.3 6 Sampling.4 7 Culture media and reagents.4 8 Procedure.5 8.1 Preparation of the sample.5 8.2 Non-selective pre-enrichment.5 8.3 Selective enrichment.5 8.4 Plating out.6 8.5 Confirmation.6 9 Expression of results.9 10 Test report.9 Annex A (normative)
Diagram of procedure.10 Annex B (normative)
Composition and preparation of culture media and reagents.11 Annex C (informative)
Results of the interlaboratory trial.18 Bibliography.23
SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) iv
© ISO 2010 – All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 19250 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. This edition cancels and replaces ISO 6340:1995, which has been technically revised.
SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) © ISO 2010 – All rights reserved
v Introduction Salmonella species are bacteria which are widely distributed all over the world. They are usually classified as pathogens, although their virulence and pathogenesis vary widely. The natural hosts of Salmonella include humans, agricultural and domestic livestock, and wild animals including birds. Humans and animals can excrete these bacteria while carrying them asymptomatically as well as during disease. It is therefore impossible to eliminate them from the environment. Following the infection of humans, the transmission of Salmonella can cause severe disease. Since water is a recognized vehicle of infection, the presence or absence of Salmonella is monitored in water where there is perceived to be a risk of infection. Salmonella can be present in all types of domestic and agricultural waste water, freshwaters, including ground and drinking waters, as well as sea water. The detection of Salmonella in water usually requires a concentration step. Since Salmonella cells can be present in low numbers and injured in the aqueous environment, their detection in water usually requires a pre-enrichment step.
SIST ISO 19250:2011SIST ISO 19250:2011



SIST ISO 19250:2011SIST ISO 19250:2011



INTERNATIONAL STANDARD ISO 19250:2010(E) © ISO 2010 – All rights reserved
1 Water quality — Detection of Salmonella spp. WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting Salmonella, and especially S. enterica subsp. enterica ser. Typhi (Salmonella ser. Typhi) and S. enterica subsp. enterica ser. Paratyphi (Salmonella ser. Paratyphi), be undertaken only in properly equipped laboratories, under the control of a skilled microbiologist, and that great care be 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 problems, 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. IMPORTANT — It is absolutely essential that tests conducted according to this International Standard be carried out by suitably trained staff. 1 Scope This International Standard specifies a method for the detection of Salmonella spp. (presumptive or confirmed) in water samples. It is possible that, for epidemiological purposes or during outbreak investigations, other media are also required. WARNING — It is possible that the method does not recover all Salmonella ser. Typhi and ser. Paratyphi. NOTE For a semi-quantitative approach, most probable number (MPN) tests can be performed using appropriate sample volumes. For these cases, the volume of the buffered peptone water is adjusted accordingly.
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 6579, Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Salmonella spp. ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture ISO 19458, Water quality — Sampling for microbiological analysis SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) 2
© ISO 2010 – All rights reserved 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 presumptive Salmonella spp. bacteria which grow in the selective enrichment medium specified, and form typical or atypical colonies on the solid selective media 3.2 confirmed Salmonella spp. bacteria which grow in the selective enrichment medium specified, and form typical and suspicious colonies on the solid selective media, and which display specfic biochemical and serological characteristics NOTE The specific biochemical and serological characteristics are determined by tests specified in this International Standard. 3.3 Salmonella detection determination of the presence or absence of Salmonella (3.4) 3.4 Salmonella spp. Salmonella microorganisms which form typical or atypical colonies on solid selective media and which display specific biochemical and serological characteristics 4 Principle 4.1 General The detection of Salmonella necessitates four successive stages (see also Annex A). Pre-enrichment is often necessary to permit detection of low numbers of Salmonella or injured Salmonella. Some Salmonella and those which are sublethally injured may require additional incubation time (4.3). Furthermore, Salmonella can be present in small numbers and are often accompanied by considerably Iarger numbers of other members of Enterobacteriaceae or of other families. Therefore, selective enrichment is necessary. 4.2 Pre-enrichment in non-selective liquid medium Buffered peptone water (B.1) is inoculated at ambient temperature with a known volume of the sample or its dilutions, then incubated at (36 ± 2) °C for (18 ± 2) h. Larger volumes can be concentrated using membrane filtration and the membrane filter is then added to buffered peptone water. NOTE For waste water it has been shown that shorter incubation times or direct inoculation of the sample in selective medium (4.3) produce better results. For a semi-quantitative approach, MPN tests can be performed using appropriate sample volumes. In these cases, adjust the volumes of the buffered peptone water accordingly. 4.3 Enrichment in selective liquid media Rappaport-Vassiliadis medium with soya (RVS broth) and Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn) are inoculated with the culture obtained in 4.2. The RVS broth is incubated at (41,5 ± 1) °C for (24 ± 3) h and the MKTTn broth at (37 ± 1) °C for (24 ± 3) h. SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) © ISO 2010 – All rights reserved
3 To detect slow-growing Salmonella spp., incubate the enrichment broth for a further (24 ± 3) h to a total of (48 ± 4) h at (41,5 ± 1,0) °C. NOTE Salmonella Typhi and Salmonella Paratyphi A are usually not important in routine water quality monitoring, but can be relevant in epidemiological investigations. MKTTn broth is used for enrichment with incubation at (36 ± 2) °C for up to (24 ± 3) h and recovers most strains of Salmonella, including some strains of Salmonella Paratyphi, but is not thought to be able to recover strains of Salmonella Paratyphi C. MKTTn broth is not used if Salmonella Typhi is suspected after the use of selenite cystine broth. 4.4 Plating out and recognition From the cultures obtained in 4.3, two selective solid media are inoculated: a) xylose lysine deoxycholate agar (XLD agar); b) any other solid selective medium complementary to XLD agar and, if applicable, appropriate for the isolation of lactose-positive Salmonella and Salmonella Typhi and Salmonella Paratyphi strains — the laboratory may choose which medium to use.
Incubate the XLD agar at (36 ± 2) °C and examine after (24 ± 3) h to check for the presence of colonies which are considered to be presumptive Salmonella. Incubate the second selective agar according to the manufacturer's recommendations. NOTE For information, brilliant green agar (BGA), bismuth sulfite agar, etc., can be used as the second plating-out medium. 4.5 Confirmation Subculture colonies of presumptive Salmonella, then plate out as described in 4.4 and confirm their identity by means of appropriate biochemical (8.5.3) and serological (8.5.4) tests.
5 Apparatus Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following. 5.1 General. Except for disposable glassware which is delivered sterile, sterilize glassware as specified in ISO 8199. Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications. 5.2 Autoclave, capable of being maintained at (121 ± 3) °C and at (115 ± 3) °C. 5.3 Water bath or incubator, capable of being maintained at (36 ± 2) °C. 5.4 Water bath or incubator, capable of being maintained at (41,5 ± 1,0) °C. 5.5 Water baths, capable of operating at (70 ± 1) °C and at 50 °C to 55 °C. 5.6 Membrane filtration apparatus, as specified in ISO 8199. 5.7 Sterile membrane filters, with a nominal pore size of 0,45 µm. The quality of membrane filters may vary from brand to brand or even from batch to batch. It is therefore advisable to check the quality on a regular basis, as specified in ISO 7704. SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) 4
© ISO 2010 – All rights reserved 5.8 pH-meter, with an accuracy of calibration of ± 0,1 pH at 20 °C to 25 °C. 5.9 Sterile forceps. 5.10 Sterile loops, approximate diameter 3 mm (10 µl volume), and inoculation needle or wire. 6 Sampling Sampling is not part of the method specified in this International Standard. Samples should be taken in accordance with ISO 19458. It is important the laboratory receive a truly representative sample which has not been damaged or changed during transport or storage. 7 Culture media and reagents NOTE For guidelines on quality assurance and performance testing, see ISO/TS 11133-1[2] and ISO/TS 11133-2[3]. 7.1 Basic materials. For uniformity of results, in the preparation of media, either use a dehydrated complete medium or use constituents of uniform quality and reagents of recognized analytical grade. Other grades of reagents may be used provided they can be shown to produce comparable results. 7.2 Water, ISO 3696[1], grade 3. 7.3 Culture media, prepared in accordance with Annex B. 7.3.1 Buffered peptone water, non-selective pre-enrichment medium buffered peptone water (BPW, B.1). 7.3.2 Rappaport-Vassiliadis broth with soya (RVS broth, B.2), selective enrichment medium. 7.3.3 Xylose lysine deoxycholate agar (XLD agar, B.3). 7.3.4 Second solid selective plating-out medium, whose choice is left to the discretion of the testing laboratory. Follow the manufacturer's instructions precisely regarding its preparation for use.
7.3.5 Nutrient agar (B.4), or other appropriate non-selective agar. 7.3.6 Triple sugar and iron agar (TSI agar, B.5).
As an alternative, iron and two sugar agar may be used. 7.3.7 Urea agar, Christensen (B.6). 7.3.8 L-Lysine decarboxylation medium (B.7). 7.3.9 Selenite cystine broth (B.8). 7.3.10 Muller-Kaufmann tetrathionate-novobiocin broth (MKTTn, B.9). 7.3.11 Filter aid (B.10). SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) © ISO 2010 – All rights reserved
5 8 Procedure See Figure A.1. 8.1 Preparation of the sample For the preparation of the sample, filtration and inoculation on isolation media, follow the instructions as specified in ISO 8199 and ISO 6887-1. Start the examination preferably immediately after taking the samples. If the samples are kept at ambient temperatures, start the examination within 12 h after sampling. Under exceptional circumstances, it is allowable for the samples to be kept at (5 ± 3) °C for up to 24 h prior to examination. The volume of the sample to be analysed depends on the type of water. Usual volumes for bathing water and drinking water are 1 000 ml to 5 000 ml. For polluted surface waters and waste water, smaller volumes are usually analysed. If sample dilutions are necessary (e.g. for waste water samples), prepare these dilutions as specified in ISO 8199. 8.2 Non-selective pre-enrichment 8.2.1 Non-selective pre-enrichment for volumes less than 10 ml Inoculate 50 ml of BPW (B.1) at room temperature with the sample or dilutions thereof and incubate at (36 ± 2) °C for (18 ± 2) h. 8.2.2 Non-selective pre-enrichment for volumes greater than 10 ml Filter a volume of water appropriate for the water being examined. Immerse the membrane filter in 50 ml of BPW (B.1). Alternatively, add the sample to the same volume of double strength BPW. Note that the latter procedure is not suitable for mineral waters with high salt content or sea water. Incubate the cultures at (36 ± 2) °C for (18 ± 2) h. 8.2.3 Recommendation for turbid or polluted water For turbid or polluted waters, sterile filter aid (B.10) can be added and the sample filtered through a sterile absorbent pad acting as a supporting base instead of using the membrane. In this case, filter an aliquot of filter aid, typically 15 ml, to form an initial layer on the absorbent pad. Mix a second aliquot, typically 15 ml, with the volume of sample and filter. For turbid or dirty waters, additional aliquots may be filtered. When filtration is complete, remove the funnel and carefully transfer the absorbent pad and filter aid to BPW (B.1). If necessary, retain a small volume of BPW to rinse the funnel so that the final volume of BPW is 100 ml. Incubate for presence or absence, or dispense as an MPN series for a semi-quantitative count. 8.3 Selective enrichment Allow the enrichment broth(s) to equilibrate to room temperature if they were stored at a lower temperature. Transfer 0,1 ml of the culture obtained in 8.2 to a tube containing 10 ml of the RVS broth (B.2). When MKTTn (B.9) is also used, transfer 1 ml of the culture obtained in 8.2 to a tube containing 10 ml of the MKTTn broth. SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) 6
© ISO 2010 – All rights reserved Incubate the inoculated RVS broth at (41,5 ± 1,0) °C for (24 ± 3) h and, if necessary (see 4.3), for (48 ± 4) h. Care should be taken that the maximum allowed incubation temperature (42,5 °C) is not exceeded. Incubate the inoculated MKTTn broth at (36 ± 2) °C for (24 ± 3) h. NOTE For RVS broth, the magnesium chloride concentration and incubation temperature have been optimized to yield good recovery without losing selectivity according to Reference [5].
8.4 Plating out 8.4.1 General Allow the XLD agar plates and the second selective plating-out medium (see ISO 6579:2002, 5.2.4.2) to equilibrate at room temperature if they were stored at a lower temperature. If necessary, dry the surface of the plates before use. 8.4.2 Plating from RVS broth Using the culture obtained in the RVS broth, inoculate, after incubation for (24 ± 3) h and, if necessary (see 4.3), for (48 ± 4) h, by means of a sterile loop (5.10), the surface of the following enrichment media so that well-isolated colonies are obtained: a) XLD agar (B.3); b) an additional selective medium (7.3.4). Invert the dishes so that the bottom is uppermost, and place them in the incubator (5.3) set at (36 ± 2) °C for (24 ± 3) h for the XLD agar. The manufacturer's instructions shall be followed for the second selective plating-out medium. 8.4.3 Plating from MKTTn broth After incubation at (36 ± 2) °C for (24 ± 3) h using the culture obtained, repeat the procedure specified in 8.4.2 with the two selective plating-out media. 8.5 Confirmation 8.5.1 General If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella may be used. Use these kits according to the manufacturer's instructions. 8.5.2 Selection of colonies for confirmation
For routine monitoring purposes, take, for confirmation, from each Petri dish of each selective medium (8.4), at least one discrete colony considered to be typical or presumptive Salmonella. If the first colony is not confirmed as Salmonella, then take a further four colonies. On XLD agar, typical Salmonella colonies usually have a black centre and a lightly transparent zone of reddish colour due to the colour change of the indicator. It is recommended that at least five colonies be identified for epidemiological studies. If on one dish there are fewer than five typical or suspect colonies, take all the typical or suspect colonies for confirmation. NOTE The recognition of Salmonella colonies 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 medium used. Shigella, Providencia and H2S-negative Salmonella spp.(e.g. Salmonella Paratyphi A) appear as pink with a darker pink centre; lactose-positive Salmonella grown on XLD are yellow with or without blackening; Enterobacteriaceae e.g. Escherichia coli, Enterobacter, Klebsiella, Citrobacter, Proteus, and Serratia appear as yellow, opaque colonies. SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) © ISO 2010 – All rights reserved
7 Streak the selected colonies on to the surface of pre-dried “non-selective” agar plates (e.g. nutrient agar, B.4) to allow the development of well-isolated colonies. Incubate the inoculated plates at (36 ± 2) °C for (24 ± 3) h. If the plating fails to produce distinct colonies, repeat in a manner which ensures that single discrete colonies are produced. Use single isolated colonies for biochemical and, if appropriate, serological confirmation. If biochemical kits are used for identification, follow the manufacturer's instructions. 8.5.3 Biochemical confirmation 8.5.3.1 General By means of an inoculating wire, inoculate the media specified in 8.5.3.2 to 8.5.3.4 with each of the cultures obtained from the colonies selected in 8.5.2.
8.5.3.2 TSI agar Streak the TSI agar (B.5) slant surface and stab the butt. Incubate at (36 ± 2) °C for (24 ± 3) h. Interpret the appearance of the medium as in Table 1. Table 1 — Interpretation of changes in medium Appearance Interpretation Butt Yellow (acid) Glucose positive (fermentation of glucose) Red or unchanged (alkaline) Glucose negative (no fermentation of carbohydrates) Black Formation of hydrogen sulfide Bubbles or cracks Gas formation from glucose Slant surface Yellow Lactose or sucrose positive (lactose or sucrose used) Red or unchanged (alkaline) Lactose and sucrose negative (neither lactose nor sucrose used) Typical Salmonella cultures show alkaline (red) slants, gas formation (bubbles) and acid (yellow) butts, with (in about 90 % of the cases) formation of hydrogen sulfide (blackening of the agar). 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. 8.5.3.3 Urea agar Streak the urea agar (B.6) slant surface. Incubate at (36 ± 2) °C for up to 24 h and examine at intervals. lf the reaction is positive, hydrolysis 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. Typical Salmonella cultures show a negative reaction, i.e. no colour production. 8.5.3.4 L-Lysine decarboxylation medium Inoculate well below the surface of the liquid L-lysine decarboxylation medium (B.7). Incubate at (36 ± 2) °C for (24 ± 3) h. Typical Salmonella cultures show a purple colour after incubation. SIST ISO 19250:2011SIST ISO 19250:2011



ISO 19250:2010(E) 8
© ISO 2010 – All rights reserved 8.5.3.5 Interpretation of the biochemical tests Salmonella generally show the reactions given in Table 2.
Table 2 — Biochemical reactions of Salmonella Testa Subclause Reaction Salmonella strains showing the reaction %b TSI glucose (acid formation) 8.5.3.2 + 100,0 TSI glucose (gas formation) 8.5.3.2 + 91,9c TSI lactose 8.5.3.2 − 99,2d TSI sucrose 8.5.3.2 − 99,5 TSI hydrogen sulfide 8.5.3.2 + 91,6e Urea hydrolysis 8.5.3.3 − 99,0 L-Lysine decarboxylation 8.5.3.4 + 94,6f a See Reference [8]. b These figures indicate only that not all strains of Salmonella show the reactions marked + or −. They may vary between geographical areas and from water source to water source. c Salmonella Typhi is anaerogenic. d The Salmonella enterica subsp. arizonae gives positive or negative lactose reactions but is always β-galactosidase positive. The Salmonella enterica subsp. diarizonae gives a negative lactose reaction, but gives a positive β-galactosidase reaction. In addition, it is possible that these strains do not produce H2S. For the study of these strains, it can be useful to carry out complimentary biochemical tests (References [9][10]). e Acid formation is sometimes difficult to recognize due to strong blackening. f Salmonella Paratyphi A is negative.
The biochemical reactions in Table 3 are typical of Salmonella spp. Table 3 — Typical biochemical reactions of Salmonella spp. to tests Test Subclause Reaction TSI lactose 8.5.3.2 − TSI glucose 8.5.3.2 + TSI sucrose 8.5.3.2 − TSI hydrogen sulfide 8.5.3.2 + Urea splitting 8.5.3.3 − L-Lysine decarboxylase 8.5.3.4 + Isolates which only vary from the typical parameters listed in Table 3 by one or two reactions can still be Salmonella and should be further investigated and sent to a recognized reference laboratory for confirmation. 8.5.4 Serological confirmation and serotyping Isolates which are typical according to the biochemical reactions listed in Table 3 are presumptive Salmonella spp. and should, if need be, be investigated further by a reference laboratory. The presence of Salmonella O-, Vi-, and H-antigens is detected by slide agglutination as specified in ISO 6579 with the appropriate sera, from pure colonies (8.5.2) and after auto-agglutinating strains have been eliminated. Isolates which give positive serological reactions are confirmed as Salmonella spp. SIST ISO 19250:2011SIST ISO 19250:2
...

NORME ISO
INTERNATIONALE 19250
Première édition
2010-07-15


Qualité de l'eau — Recherche
de Salmonella spp.
Water quality — Detection of Salmonella spp.




Numéro de référence
ISO 19250:2010(F)
©
ISO 2010

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ISO 19250:2010(F)
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ii © ISO 2010 – Tous droits réservés

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ISO 19250:2010(F)
Sommaire Page
Avant-propos .iv
Introduction.v
1 Domaine d'application .1
2 Références normatives.1
3 Termes et définitions .2
4 Principe.2
4.1 Généralités .2
4.2 Préenrichissement sur milieu non sélectif liquide .2
4.3 Enrichissement sur milieux sélectifs liquides .3
4.4 Isolement et identification .3
4.5 Confirmation .3
5 Appareillage .3
6 Échantillonnage.4
7 Milieux de culture et réactifs .4
8 Mode opératoire.5
8.1 Préparation de l'échantillon .5
8.2 Préenrichissement non sélectif .5
8.3 Enrichissement sélectif .6
8.4 Isolement.6
8.5 Confirmation .6
9 Expression des résultats.9
10 Rapport d'essai.9
Annexe A (normative) Logigramme du mode opératoire .11
Annexe B (normative) Composition et préparation des milieux de culture et réactifs .12
Annexe C (informative) Résultats de l'essai interlaboratoires.19
Bibliographie.24

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ISO 19250:2010(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée
aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du
comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,
Partie 2.
La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur
publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne
pas avoir identifié de tels droits de propriété et averti de leur existence.
L'ISO 19250 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité SC 4,
Méthodes microbiologiques.
Cette édition annule et remplace l'ISO 6340:1995, qui a fait l'objet d'une révision technique.
iv © ISO 2010 – Tous droits réservés

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ISO 19250:2010(F)
Introduction
Les Salmonella sont des bactéries qui sont largement répandues à travers le monde. Elles sont, en général,
considérées comme pathogènes bien que leur virulence et leur pouvoir pathogène varient énormément. Les
hôtes naturels des Salmonella sont la population humaine, le bétail, les animaux domestiques, ainsi que les
animaux sauvages, y compris les oiseaux. Êtres humains et animaux peuvent excréter des Salmonella tout en
étant des porteurs asymptomatiques, de même qu'en cas de maladie. Par conséquent, il est impossible de les
éliminer de l'environnement. Par infection des êtres humains, la transmission des Salmonella peut causer de
graves maladies.
Dans la mesure où l'eau est un vecteur d'infection reconnu, la présence ou l'absence de Salmonella est
contrôlée dans l'eau lorsque l'on considère qu'il existe un risque d'infection. Les Salmonella peuvent être
présentes dans les eaux usées agricoles et domestiques, les eaux douces, y compris les eaux destinées à la
consommation humaine et les eaux souterraines, ainsi que dans les eaux de mer.
La recherche de Salmonella dans l'eau requiert habituellement une étape de concentration. Dans la mesure
où les Salmonella peuvent être présentes en faible nombre et avoir subi une altération dans l'environnement
aqueux, leur recherche dans l'eau nécessite habituellement une étape de préenrichissement.

© ISO 2010 – Tous droits réservés v

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NORME INTERNATIONALE ISO 19250:2010(F)

Qualité de l'eau — Recherche de Salmonella spp.
AVERTISSEMENT — Afin de préserver la santé du personnel de laboratoire, il est essentiel d'effectuer
les essais de recherche de Salmonella, et plus particulièrement de S. enterica subsp. enterica ser.
Typhi (Salmonella ser. Typhi) et S. enterica subsp. enterica ser. Paratyphi (Salmonella ser. Paratyphi),
uniquement dans des laboratoires correctement équipés, sous la direction d'un microbiologiste
compétent, et de faire très attention à l'élimination de toutes les substances incubées.
Il convient que l'utilisateur de la présente Norme internationale connaisse bien les pratiques
courantes de laboratoire. La présente Norme internationale n'a pas pour but de traiter tous les
problèmes de sécurité qui sont, le cas échéant, liés à son utilisation. Il incombe à l'utilisateur d'établir
des pratiques appropriées en matière d'hygiène et de sécurité, et de s'assurer de la conformité à la
réglementation nationale en vigueur.
IMPORTANT — Il est absolument essentiel que les essais réalisés conformément à la présente Norme
internationale soient exécutés par un personnel ayant reçu une formation adéquate.
1 Domaine d'application
La présente Norme Internationale spécifie une méthode pour la recherche de Salmonella spp. (présomptives
ou confirmées) dans des échantillons d'eau. À des fins épidémiologiques ou au cours d'enquêtes sur des
épidémies, il peut être nécessaire d'utiliser d'autres milieux.
AVERTISSEMENT — Il est possible que cette méthode ne retrouve pas toutes les Salmonella ser.
Typhi et ser. Paratyphi.
NOTE Pour une approche semi-quantitative, la technique du nombre le plus probable (NPP) peut être mise en
œuvre en utilisant des volumes appropriés de l'échantillon. Dans ces cas, le volume d'eau peptonée tamponnée est ajusté
en conséquence.
2 Références normatives
Les documents de référence suivants sont utiles pour l'application du présent document. Pour les références
datées, seule l'édition citée s'applique. Pour les références non datées, la dernière édition du document de
référence s'applique (y compris les éventuels amendements).
ISO 6579:2002, Microbiologie des aliments — Méthode horizontale pour la recherche des Salmonella spp.
ISO 6887-1, Microbiologie des aliments — Préparation des échantillons, de la suspension mère et des
dilutions décimales en vue de l'examen microbiologique — Partie 1: Règles générales pour la préparation de
la suspension mère et des dilutions décimales
ISO 7218, Microbiologie des aliments — Exigences générales et recommandations
ISO 7704, Qualité de l'eau — Évaluation des membranes filtrantes utilisées pour des analyses
microbiologiques
ISO 8199, Qualité de l'eau — Lignes directrices générales pour le dénombrement des micro-organismes sur
milieu de culture
ISO 19458, Qualité de l'eau — Échantillonnage pour analyse microbiologique
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ISO 19250:2010(F)
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
3.1
Salmonella spp. présomptives
bactéries se développant dans le milieu d'enrichissement sélectif spécifié et formant des colonies typiques ou
atypiques sur les milieux sélectifs solides
3.2
Salmonella spp. confirmées
bactéries se développant dans le milieu d'enrichissement sélectif spécifié et formant des colonies typiques et
suspectes sur les milieux sélectifs solides et qui présentent des caractéristiques biochimiques et sérologiques
spécifiques
NOTE Les caractéristiques biochimiques et sérologiques sont déterminées par des essais spécifiés dans la présente
Norme Internationale.
3.3
recherche de Salmonella
mise en évidence de la présence ou de l'absence de Salmonella (3.4)
3.4
Salmonella spp.
Salmonella
micro-organismes formant des colonies typiques ou atypiques sur des milieux sélectifs solides et présentant
des caractéristiques biochimiques et sérologiques spécifiques
4 Principe
4.1 Généralités
La recherche de Salmonella nécessite quatre phases successives (voir également l'Annexe A).
Un préenrichissement est souvent nécessaire pour permettre la recherche de Salmonella en nombre
relativement faible ou de Salmonella ayant subi une altération. Certaines Salmonella et celles ayant subi un
dommage sublétal peuvent nécessiter une période d'incubation supplémentaire (4.3). Par ailleurs, des
Salmonella peuvent être présentes en petit nombre et sont souvent accompagnées d'autres membres de la
famille des Enterobacteriaceae ou d'autres familles, en nombre largement plus important. Un enrichissement
sélectif est donc nécessaire.
4.2 Préenrichissement sur milieu non sélectif liquide
L'eau peptonée tamponnée (B.1) est ensemencée à température ambiante avec un volume connu de
l'échantillon ou de ses dilutions, puis incubée à (36 ± 2) °C pendant (18 ± 2) h. Des volumes plus importants
peuvent être concentrés par filtration sur membrane, après quoi la membrane filtrante est transférée dans
l'eau peptonée tamponnée.
NOTE Pour les eaux usées, il a été démontré que des périodes d'incubation plus courtes ou des ensemencements
directs de l'échantillon dans le milieu sélectif (4.3) donnent de meilleurs résultats.
Pour une approche semi-quantitative, la technique du NPP peut être mise en œuvre en utilisant des volumes
appropriés de l'échantillon. Dans ces cas, ajuster les volumes d'eau peptonée tamponnée en conséquence.
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ISO 19250:2010(F)
4.3 Enrichissement sur milieux sélectifs liquides
Un milieu Rappaport-Vassiliadis au soja (bouillon RVS) et un bouillon Muller-Kauffmann
tétrathionate-novobiocine (MKTTn) sont ensemencés avec la culture obtenue en 4.2.
Le bouillon RVS est incubé à (41,5 ± 1) °C pendant (24 ± 3) h et le bouillon MKTTn à (37 ± 1) °C pendant
(24 ± 3) h.
Pour détecter les Salmonella spp. à croissance lente, incuber le bouillon d'enrichissement pendant (24 ± 3) h
supplémentaires pour une période d'incubation totale de (48 ± 4) h à (41,5 ± 1,0) °C.
NOTE Les Salmonella Typhi et les Salmonella Paratyphi A ne sont généralement pas importantes pour le contrôle de
routine de la qualité de l'eau, mais peuvent être pertinentes pour des enquêtes épidémiologiques. Le bouillon MKTTn est
utilisé pour enrichissement avec une incubation à (36 ± 2) °C pendant une période maximale de (24 ± 3) h et récupère la
plupart des souches de Salmonella, y compris certaines souches de Salmonella Paratyphi, mais il n'est pas considéré
comme apte à récupérer les souches de Salmonella Paratyphi C. Le bouillon MKTTn n'est pas utilisé lorsque la présence
de Salmonella Typhi est suspectée après l'utilisation d'un bouillon sélénite cystine.
4.4 Isolement et identification
À partir des cultures obtenues en 4.3, deux milieux sélectifs solides sont ensemencés:
a) gélose lysine xylose désoxycholate (gélose XLD);
b) tout autre milieu sélectif solide en complément de la gélose XLD et, s'il y a lieu, approprié à l'isolement de
souches de Salmonella lactose positives, de Salmonella Typhi et de Salmonella Paratyphi — le
laboratoire peut choisir le milieu à utiliser.
Incuber la gélose XLD à (36 ± 2) °C et examiner après (24 ± 3) h afin de détecter la présence de colonies
considérées comme Salmonella présomptives. Incuber le deuxième milieu de gélose sélectif conformément
aux recommandations du fabricant.
NOTE À titre informatif, il est possible d'utiliser de la gélose au vert brillant (BGA), de la gélose au sulfite de bismuth,
etc., comme deuxième milieu d'isolement.
4.5 Confirmation
Repiquer les colonies de Salmonella présomptives, puis isoler comme décrit en 4.4 et confirmer leur identité
en effectuant des essais biochimiques (8.5.3) et sérologiques (8.5.4) appropriés.
5 Appareillage
Matériel courant de laboratoire de microbiologie (voir l'ISO 7218), et en particulier, ce qui suit.
5.1 Généralités. Sauf pour la verrerie à usage unique qui est livrée stérile, stériliser la verrerie comme
spécifié dans l'ISO 8199. L'appareillage à usage unique est une solution acceptable au même titre que la
verrerie réutilisable, s'il répond à des spécifications appropriées.
5.2 Autoclave, pouvant être maintenu à (121 ± 3) °C et à (115 ± 3) °C.
5.3 Bain d'eau ou incubateur, pouvant être maintenu à (36 ± 2) °C.
5.4 Bain d'eau ou incubateur, pouvant être maintenu à (41,5 ± 1,0) °C.
5.5 Bains d'eau, pouvant fonctionner à (70 ± 1) °C et de 50 °C à 55 °C.
5.6 Appareil de filtration sur membrane, comme spécifié dans l'ISO 8199.
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ISO 19250:2010(F)
5.7 Membranes filtrantes stériles, avec un diamètre de pore nominal de 0,45 µm.
La qualité des membranes filtrantes peut varier en fonction de la marque et parfois même du lot. Il est donc
recommandé de vérifier régulièrement la qualité de ces membranes, comme spécifié dans l'ISO 7704.
5.8 pH-mètre, avec une exactitude d'étalonnage de ± 0,1 unité pH entre 20 °C et 25 °C.
5.9 Pinces stériles.
5.10 Anses stériles, diamètre d'environ 3 mm (volume de 10 µl) et aiguille ou fil d'ensemencement.
6 Échantillonnage
L'échantillonnage ne fait pas partie de la méthode spécifiée dans la présente Norme internationale. Il convient
que les échantillons soient prélevés conformément à l'ISO 19458.
Il est important que le laboratoire reçoive un échantillon réellement représentatif, non endommagé ou modifié
lors du transport ou de l'entreposage.
7 Milieux de culture et réactifs
NOTE Pour connaître les lignes directrices relatives à l'assurance qualité et aux essais de performance, voir
[2] [3]
l'ISO/TS 11133-1 et l'ISO/TS 11133-2 .
7.1 Substances de base. Dans un souci d'uniformité des résultats, utiliser soit des milieux complets
déshydratés, soit des composants de qualité uniforme et des réactifs de qualité analytique reconnue, pour la
préparation du milieu.
Des réactifs d'autres qualités peuvent être utilisés à condition qu'il ait été démontré qu'il donnent les mêmes
résultats.
[1]
7.2 Eau, ISO 3696 , qualité 3.
7.3 Milieux de culture, préparés conformément à l'Annexe B.
7.3.1 Eau peptonée tamponnée, milieu de préenrichissement non sélectif (EPT, B.1).
7.3.2 Bouillon Rappaport-Vassiliadis au soja (bouillon RVS, B.2), milieu d'enrichissement sélectif.
7.3.3 Gélose lysine xylose désoxycholate (gélose XLD, B.3).
7.3.4 Deuxième milieu d'isolement sélectif solide, dont le choix est laissé à la discrétion du laboratoire
d'essai. Suivre scrupuleusement les instructions du fabricant concernant la préparation pour utilisation.
7.3.5 Gélose nutritive (B.4), ou autre gélose non sélective appropriée.
7.3.6 Gélose ferrique aux trois sucres (gélose TSI, B.5).
De la gélose ferrique aux deux sucres peut être utilisée en variante.
7.3.7 Gélose à l'urée, Christensen (B.6).
7.3.8 Milieu de décarboxylation de la L-Iysine (B.7).
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ISO 19250:2010(F)
7.3.9 Bouillon sélénite cystine (B.8).
7.3.10 Bouillon Muller-Kauffmann tétrathionate-novobiocine (MKTTn, B.9).
7.3.11 Adjuvant de filtration (B.10).
8 Mode opératoire
Voir la Figure A.1.
8.1 Préparation de l'échantillon
Pour la préparation de l'échantillon, la filtration et l'ensemencement sur les milieux d'isolement, suivre les
instructions comme spécifié dans l'ISO 8199 et l'ISO 6887-1. Il est préférable de commencer l'examen
immédiatement après avoir prélevé les échantillons. Si les échantillons sont conservés à température
ambiante, commencer l'examen dans les 12 h suivant l'échantillonnage. Dans des cas exceptionnels, il est
permis de conserver les échantillons à (5 ± 3) °C pendant une durée maximale de 24 h avant l'examen.
Le volume de l'échantillon à analyser dépend du type d'eau. Les volumes habituels pour l'eau de baignade et
l'eau potable se situent entre 1 000 ml et 5 000 ml. Pour les eaux de surface polluées et les eaux usées,
l'analyse se fait généralement sur des volumes inférieurs.
Si des dilutions de l'échantillon sont nécessaires (par exemple pour des échantillons d'eaux usées), préparer
ces dilutions comme spécifié dans l'ISO 8199.
8.2 Préenrichissement non sélectif
8.2.1 Préenrichissement non sélectif pour des volumes inférieurs à 10 ml
Ensemencer 50 ml d'EPT (B.1) à température ambiante avec l'échantillon ou ses dilutions et incuber à
(36 ± 2) °C pendant (18 ± 2) h.
8.2.2 Préenrichissement non sélectif pour des volumes supérieurs à 10 ml
Filtrer un volume d'eau approprié pour l'eau examinée.
Immerger la membrane filtrante dans 50 ml d'EPT (B.1).
Il est également possible d'ajouter l'échantillon au même volume d'EPT de concentration double.
Il faut toutefois noter que cette procédure n'est pas adaptée aux eaux minérales à forte teneur en sel, ni aux
eaux de mer.
Incuber les cultures à (36 ± 2) °C pendant (18 ± 2) h.
8.2.3 Recommandation pour les eaux turbides ou polluées
Pour les eaux turbides ou polluées, un adjuvant de filtration stérile (B.10) peut être ajouté et l'échantillon être
filtré à travers un tampon absorbant stérile agissant comme support au lieu d'utiliser la membrane.
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ISO 19250:2010(F)
Dans ce cas, filtrer une aliquote de l'adjuvant de filtration, généralement 15 ml, pour former une première
couche sur le tampon absorbant. Mélanger une deuxième aliquote, généralement 15 ml, avec le volume de
l'échantillon et filtrer. Pour les eaux turbides ou sales, des aliquotes supplémentaires peuvent être filtrées.
Une fois la filtration terminée, enlever l'entonnoir et transférer avec précaution le tampon absorbant et
l'adjuvant de filtration dans l'EPT (B.1). Si nécessaire, conserver un petit volume d'EPT pour rincer l'entonnoir
de manière à obtenir un volume final d'EPT de 100 ml. Incuber en vue de détecter une présence ou absence
ou procéder à une répartition selon une série de NPP pour obtenir un résultat semi-quantitatif.
8.3 Enrichissement sélectif
Laisser le (les) bouillon(s) d'enrichissement s'équilibrer à température ambiante s'il(s) étai(en)t conservé(s) à
une température inférieure. Transférer 0,1 ml de la culture obtenue en 8.2 dans un tube contenant 10 ml de
bouillon RVS (B.2). Lorsqu'un bouillon MKTTn (B.9) est également utilisé, transférer 1 ml de la culture
obtenue en 8.2 dans un tube contenant 10 ml de bouillon MKTTn.
Incuber le bouillon RVS ensemencé à (41,5 ± 1,0) °C pendant (24 ± 3) h et, si nécessaire (voir 4.3), pendant
(48 ± 4) h. Il convient de s'assurer que la température d'incubation maximale tolérée (42,5 °C) n'est pas
dépassée. Incuber le bouillon MKTTn ensemencé à (36 ± 2) °C pendant (24 ± 3) h.
NOTE Pour le bouillon RVS, la concentration en chlorure de magnésium et la température d'incubation ont été
optimisées afin d'assurer un bon rendement sans perte de sélectivité conformément à la Référence [5].
8.4 Isolement
8.4.1 Généralités
Laisser les boîtes de gélose XLD et le deuxième milieu d'isolement sélectif (voir l'ISO 6579:2002, 5.2.4.2)
s'équilibrer à température ambiante s'ils étaient conservés à une température inférieure. Si nécessaire, sécher
la surface des boîtes avant utilisation.
8.4.2 Isolement à partir du bouillon RVS
À l'aide de la culture obtenue dans le bouillon RVS, ensemencer la surface des milieux d'enrichissement
suivants après incubation pendant (24 ± 3) h et, si nécessaire (voir 4.3), pendant (48 ± 4) h, à l'aide d'une
anse stérile (5.10), de manière à obtenir des colonies bien isolées:
a) gélose XLD (B.3);
b) un milieu sélectif supplémentaire (7.3.4).
Retourner les boîtes de manière que la partie inférieure se retrouve en position supérieure et les placer dans
l'incubateur (5.3) à une température de (36 ± 2) °C pendant (24 ± 3) h pour la gélose XLD. Les instructions du
fabricant doivent être suivies pour le deuxième milieu d'isolement sélectif.
8.4.3 Isolement à partir du bouillon MKTTn
Après incubation à (36 ± 2) °C pendant (24 ± 3) h à l'aide de la culture obtenue, répéter le mode opératoire
spécifié en 8.4.2 avec les deux milieux d'isolement sélectifs.
8.5 Confirmation
8.5.1 Généralités
S'ils sont identifiés comme étant fiables, des kits d'identification disponibles dans le commerce peuvent être
utilisés pour l'examen biochimique des Salmonella. Utiliser ces kits conformément aux instructions du
fabricant.
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ISO 19250:2010(F)
8.5.2 Sélection des colonies pour confirmation
En vue d'un contrôle de routine, prélever, pour la confirmation, dans chaque boîte de Petri de chaque milieu
sélectif (8.4) au moins une colonie isolée considérée comme Salmonella typique ou présomptive. Si la
première colonie n'est pas classée comme Salmonella confirmée, prélever quatre autres colonies.
Sur la gélose XLD, les colonies de Salmonella typiques ont généralement un centre noir et une zone
rougeâtre légèrement transparente en raison du changement de couleur de l'indicateur. Il est recommandé
d'identifier au moins cinq colonies en cas d'études épidémiologiques. S'il y a moins de cinq colonies typiques
ou suspectes présentes sur une boîte, prélever pour la confirmation toutes les colonies typiques ou suspectes.
NOTE L'identification des colonies de Salmonella est, dans une large mesure, une question d'expérience; leur aspect
peut varier quelque peu, non seulement entre les différents sérotypes, mais également entre les différents lots de milieux
sélectifs utilisés. Les Shigella, Providencia et les Salmonella spp. H S-négatives (comme les Salmonella Paratyphi A)
2
apparaissent en rose avec un centre rose plus foncé; les Salmonella lactose positives ayant poussé sur la gélose XLD
sont jaunes avec des noircissements possibles; les Enterobacteriaceae comme les Escherichia coli, Enterobacter,
Klebsiella, Citrobacter, Proteus et Serratia apparaissent comme des colonies jaunes et opaques.
Ensemencer en stries les colonies sélectionnées sur la surface des boîtes de gélose «non sélective»
préalablement séchées (par exemple gélose nutritive, B.4) de manière à permettre le développement de
colonies bien isolées.
Incuber les boîtes ensemencées à (36 ± 2) °C pendant (24 ± 3) h.
Si l'isolement ne produit pas de colonies séparées, le répéter de façon à s'assurer que des colonies isolées
pures sont produites. Utiliser des colonies isolées pures pour la confirmation biochimique et, si nécessaire,
pour la confirmation sérologique. Si des kits biochimiques sont utilisés pour l'identification, suivre les
instructions du fabricant.
8.5.3 Confirmation biochimique
8.5.3.1 Généralités
Au moyen d'un fil droit, ensemencer les milieux spécifiés en 8.5.3.2 à 8.5.3.4 avec chacune des cultures
obtenues à partir des colonies sélectionnées en 8.5.2.
8.5.3.2 Gélose TSI
Ensemencer en stries la surface des tranches de gélose TSI (B.5) et piquer le culot. Incuber à (36 ± 2) °C
pendant (24 ± 3) h. Interpréter l'aspect du milieu comme indiqué dans le Tableau 1.
Tableau 1 — Interprétation des changements observés dans le milieu
Aspect Interprétation
Culot
Jaune (acide) Réaction positive au glucose (fermentation du glucose)
Rouge ou inchangé (alcalin) Réaction négative au glucose (pas de fermentation de glucides)
Noir Formation de sulfure d'hydrogène
Bulles ou fissures Formation de gaz due au glucose
Surface des tranches
Jaune Réaction positive au lactose ou au saccharose (lactose ou saccharose utilisés)
Rouge ou inchangée (alcaline) Réaction négative au lactose et au saccharose (ni lactose ni saccharose utilisé)

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ISO 19250:2010(F)
Les cultures de Salmonella typiques présentent une tranche alcaline (rouge), une formation de gaz (bulles) et
des culots acides (jaunes) avec une formation (dans 90 % des cas environ) de sulfure d'hydrogène
(noircissement de la gélose).
Lorsque des Salmonella lactose positives sont isolées, la tranche TSI est jaune. Ainsi, la confirmation
préliminaire des cultures de Salmonella ne doit pas être uniquement fondée sur les résultats de l'essai réalisé
sur la gélose TSI.
8.5.3.3 Gélose à l'urée
Ensemencer en stries la surface des tranches de gélose à l'urée (B.6). Incuber à (36 ± 2) °C pendant 24 h et
examiner à
...