Water quality - Detection of Salmonella spp.

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

Qualité de l'eau - Recherche de Salmonella spp.

L'ISO 19250:2010 sp�cifie une m�thode pour la recherche de Salmonella spp. (pr�somptives ou confirm�es) dans des �chantillons d'eau. � des fins �pid�miologiques ou au cours d'enqu�tes sur des �pid�mies, il peut �tre n�cessaire d'utiliser d'autres milieux.

Kakovost vode - Določanje prisotnosti Salmonella spp.

Ta mednarodni standard opredeljuje metodo določevanja prisotnosti vrst Salmonella spp. (domnevno ali potrjeno) v vodnih vzorcih. Mogoče je, da so za epidemiološke namene ali med preiskovanjem izbruhov potrebna tudi druga sredstva.
OPOZORILO: Mogoče je, da metoda ne zajema vseh vrst Salmonella Typhy in vseh vrst Salmonella Paratyphi.
OPOMBA: Za polkvantitativni pristop se lahko izvajajo preskusi najverjetnejšega števila (MNP).

General Information

Status
Published
Public Enquiry End Date
19-May-2011
Publication Date
15-May-2011
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
03-May-2011
Due Date
08-Jul-2011
Completion Date
16-May-2011

RELATIONS

Buy Standard

Standard
ISO 19250:2010 - Water quality -- Detection of Salmonella spp.
English language
23 pages
sale 15% off
Preview
sale 15% off
Preview
Standard
SIST ISO 19250:2011
English language
31 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day
Standard
ISO 19250:2010 - Qualité de l'eau -- Recherche de Salmonella spp.
French language
24 pages
sale 15% off
Preview
sale 15% off
Preview

Standards Content (sample)

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
---------------------- Page: 1 ----------------------
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.

COPYRIGHT PROTECTED DOCUMENT
© 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. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2010 – All rights reserved
---------------------- Page: 2 ----------------------
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

© ISO 2010 – All rights reserved iii
---------------------- Page: 3 ----------------------
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.

iv © ISO 2010 – All rights reserved
---------------------- Page: 4 ----------------------
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.
© ISO 2010 – All rights reserved v
---------------------- Page: 5 ----------------------
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
© ISO 2010 – All rights reserved 1
---------------------- Page: 6 ----------------------
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.

2 © ISO 2010 – All rights reserved
---------------------- Page: 7 ----------------------
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.
© ISO 2010 – All rights reserved 3
---------------------- Page: 8 ----------------------
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).
4 © ISO 2010 – All rights reserved
---------------------- Page: 9 ----------------------
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.
© ISO 2010 – All rights reserved 5
---------------------- Page: 10 ----------------------
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;

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.

6 © ISO 2010 – All rights reserved
---------------------- Page: 11 ----------------------
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.

© ISO 2010 – All rights reserved 7
---------------------- Page: 12 ----------------------
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
Test Subclause Reaction
TSI glucose (acid formation) 8.5.3.2 100,0
TSI glucose (gas formation) 8.5.3.2 +
91,9
TSI lactose 8.5.3.2 −
99,2
TSI sucrose 8.5.3.2 99,5
TSI hydrogen sulfide 8.5.3.2 +
91,6
Urea hydrolysis 8.5.3.3 − 99,0
L-Lysine decarboxylation 8.5.3.4 +
94,6
See Reference [8].

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.
Salmonella Typhi is anaerogenic.

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

tests (References [9][10]).
Acid formation is sometimes difficult to recognize due to strong blackening.
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.

8 © ISO 2010 – All rights reserved
---------------------- Page: 13 ----------------------
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.

COPYRIGHT PROTECTED DOCUMENT

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.

+ 41 22 749 01 11 Fax
+ 41 22 749 09 47 E-mail
copyright@iso.org Web
www.iso.org Published in Switzerland
© 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
---------------------- Page: 1 ----------------------
ISO 19250:2010(F)
PDF – Exonération de responsabilité

Le présent fichier PDF peut contenir des polices de caractères intégrées. Conformément aux conditions de licence d'Adobe, ce fichier

peut être imprimé ou visualisé, mais ne doit pas être modifié à moins que l'ordinateur employé à cet effet ne bénéficie d'une licence

autorisant l'utilisation de ces polices et que celles-ci y soient installées. Lors du téléchargement de ce fichier, les parties concernées

acceptent de fait la responsabilité de ne pas enfreindre les conditions de licence d'Adobe. Le Secrétariat central de l'ISO décline toute

responsabilité en la matière.
Adobe est une marque déposée d'Adobe Systems Incorporated.

Les détails relatifs aux produits logiciels utilisés pour la création du présent fichier PDF sont disponibles dans la rubrique General Info

du fichier; les paramètres de création PDF ont été optimisés pour l'impression. Toutes les mesures ont été prises pour garantir

l'exploitation de ce fichier par les comités membres de l'ISO. Dans le cas peu probable où surviendrait un problème d'utilisation,

veuillez en informer le Secrétariat central à l'adresse donnée ci-dessous.
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2010

Droits de reproduction réservés. Sauf prescription différente, aucune partie de cette publication ne peut être reproduite ni utilisée sous

quelque forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie et les microfilms, sans l'accord écrit

de l'ISO à l'adresse ci-après ou du comité membre de l'ISO dans le pays du demandeur.

ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Publié en Suisse
ii © ISO 2010 – Tous droits réservés
---------------------- Page: 2 ----------------------
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

© ISO 2010 – Tous droits réservés iii
---------------------- Page: 3 ----------------------
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
---------------------- Page: 4 ----------------------
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
---------------------- Page: 5 ----------------------
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
© ISO 2010 – Tous droits réservés 1
---------------------- Page: 6 ----------------------
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.

2 © ISO 2010 – Tous droits réservés
---------------------- Page: 7 ----------------------
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.
© ISO 2010 – Tous droits réservés 3
---------------------- Page: 8 ----------------------
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).
4 © ISO 2010 – Tous droits réservés
---------------------- Page: 9 ----------------------
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.

© ISO 2010 – Tous droits réservés 5
---------------------- Page: 10 ----------------------
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.
6 © ISO 2010 – Tous droits réservés
---------------------- Page: 11 ----------------------
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)

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)

© ISO 2010 – Tous droits réservés 7
---------------------- Page: 12 ----------------------
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
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.