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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)

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ISO 19250:2010(E)

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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

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).

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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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