SIST EN ISO 19250:2013
(Main)Water quality - Detection of Salmonella spp. (ISO 19250:2010)
Water quality - Detection of Salmonella spp. (ISO 19250:2010)
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.
Wasserbeschaffenheit - Bestimmung von Salmonella spp. (ISO 19250:2010)
Diese Internationale Norm legt ein Verfahren zum Nachweis von Salmonella spp. (präsumtiv oder bestätigt) in Wasserproben fest. Es ist möglich, dass bei besonderen epidemiologischen Situationen oder bei Untersuchungen von Krankheitsausbrüchen auch andere Medien benötigt werden.
WARNUNG — Es ist möglich, dass das Verfahren nicht alle Salmonella ser. Typhi und ser. Paratyphi erfasst.
ANMERKUNG Für eine semi-quanitative Untersuchung können MPN-Ansätze (en: most probable number – wahrscheinlichste Zahl) unter Verwendung geeigneter Probenvolumen durchgeführt werden. Für diese Fälle wird das Volumen des gepufferten Peptonwassers entsprechend angepasst.
Qualité de l'eau - Recherche de Salmonella spp. (ISO 19250:2010)
L'ISO 19250:2010 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.
Kakovost vode - Ugotavljanje prisotnosti Salmonella spp. (ISO 19250:2010)
Ta mednarodni standard določa metodo za določanje prisotnosti Salmonella spp. (domnevne ali potrjene) v vzorcih vode. Za epidemološke namene ali med raziskovanjem izbruha so lahko zahtevani tudi drugi mediji.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 19250:2013
01-julij-2013
1DGRPHãþD
SIST ISO 6340:1998
Kakovost vode - Ugotavljanje prisotnosti Salmonella spp. (ISO 19250:2010)
Water quality - Detection of Salmonella spp. (ISO 19250:2010)
Wasserbeschaffenheit - Bestimmung von Salmonella spp. (ISO 19250:2010)
Qualité de l'eau - Recherche de Salmonella spp. (ISO 19250:2010)
Ta slovenski standard je istoveten z: EN ISO 19250:2013
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 19250:2013 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST EN ISO 19250:2013
---------------------- Page: 2 ----------------------
SIST EN ISO 19250:2013
EUROPEAN STANDARD
EN ISO 19250
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2013
ICS 07.100.20
English Version
Water quality - Detection of Salmonella spp. (ISO 19250:2010)
Qualité de l'eau - Recherche de Salmonella spp. (ISO Wasserbeschaffenheit - Bestimmung von Salmonella spp.
19250:2010) (ISO 19250:2010)
This European Standard was approved by CEN on 21 March 2013.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUR OP ÄIS CHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 19250:2013: E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 19250:2013
EN ISO 19250:2013 (E)
Contents Page
Foreword .3
2
---------------------- Page: 4 ----------------------
SIST EN ISO 19250:2013
EN ISO 19250:2013 (E)
Foreword
The text of ISO 19250:2010 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 19250:2013 by
Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by October 2013, and conflicting national standards shall be withdrawn at
the latest by October 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 19250:2010 has been approved by CEN as EN ISO 19250:2013 without any modification.
3
---------------------- Page: 5 ----------------------
SIST EN ISO 19250:2013
---------------------- Page: 6 ----------------------
SIST EN ISO 19250:2013
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: 7 ----------------------
SIST EN ISO 19250:2013
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: 8 ----------------------
SIST EN ISO 19250:2013
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: 9 ----------------------
SIST EN ISO 19250:2013
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: 10 ----------------------
SIST EN ISO 19250:2013
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: 11 ----------------------
SIST EN ISO 19250:2013
---------------------- Page: 12 ----------------------
SIST EN ISO 19250:2013
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: 13 ----------------------
SIST EN ISO 19250:2013
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: 14 ----------------------
SIST EN ISO 19250:2013
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: 15 ----------------------
SIST EN ISO 19250:2013
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: 16 ----------------------
SIST EN ISO 19250:2013
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: 17 ----------------------
SIST EN ISO 19250:2013
ISO 19250:2010(E)
Incubate the inoculated RVS broth at (41,5 ± 1,0) °C for (24 ± 3) h and, if necessary (see 4.3), for (48 ± 4) h.
Care should be taken that the maximum allowed incubation temperature (42,5 °C) is not exceeded. Incubate
the inoculated MKTTn broth at (36 ± 2) °C for (24 ± 3) h.
NOTE For RVS broth, the magnesium chloride concentration and incubation temperature have been optimized to
yield good recovery without losing selectivity according to Reference [5].
8.4 Plating out
8.4.1 General
Allow the XLD agar plates and the second selective plating-out medium (see ISO 6579:2002, 5.2.4.2) to
equilibrate at room temperature if they were stored at a lower temperature. If necessary, dry the surface of the
plates before use.
8.4.2 Plating from RVS broth
Using the culture obtained in the RVS broth, inoculate, after incubation for (24 ± 3) h and, if necessary
(see 4.3), for (48 ± 4) h, by means of a sterile loop (5.10), the surface of the following enrichment media so
that well-isolated colonies are obtained:
a) XLD agar (B.3);
b) an additional selective medium (7.3.4).
Invert the dishes so that the bottom is uppermost, and place them in the incubator (5.3) set at (36 ± 2) °C for
(24 ± 3) h for the XLD agar. The manufacturer's instructions shall be followed for the second selective
plating-out medium.
8.4.3 Plating from MKTTn broth
After incubation at (36 ± 2) °C for (24 ± 3) h using the culture obtained, repeat the procedure specified in 8.4.2
with the two selective plating-out media.
8.5 Confirmation
8.5.1 General
If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella
may be used. Use these kits according to the manufacturer's instructions.
8.5.2 Selection of colonies for confirmation
For routine monitoring purposes, take, for confirmation, from each Petri dish of each selective medium (8.4),
at least one discrete colony considered to be typical or presumptive Salmonella. If the first colony is not
confirmed as Salmonella, then take a further four colonies.
On XLD agar, typical Salmonella colonies usually have a black centre and a lightly transparent zone of
reddish colour due to the colour change of the indicator. It is recommended that at least five colonies be
identified for epidemiological studies. If on one dish there are fewer than five typical or suspect colonies, take
all the typical or suspect colonies for confirmation.
NOTE The recognition of Salmonella colonies is to a large extent a matter of experience and their appearance can
vary somewhat, not only from serovar to serovar, but also from batch to batch of the selective medium used. Shigella,
Providencia and H S-negative Salmonella spp.(e.g. Salmonella Paratyphi A) appear as pink with a darker pink centre;
2
lactose-positive Salmonella grown on XLD are yellow with or without blackening; Enterobacteriaceae e.g. Escherichia coli,
Enterobacter, Klebsiella, Citrobacter, Proteus, and Serratia appear as yellow, opaque colonies.
6 © ISO 2010 – All rights reserved
---------------------- Page: 18 ----------------------
SIST EN ISO 19250:2013
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 c
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.