1、BSI Standards PublicationBS EN ISO 19250:2013Water quality Detection of Salmonella spp.BS EN ISO 19250:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 19250:2013. It supersedes BS ISO 19250:2010, which is withdrawn.The UK participation in its preparatio
2、n was entrusted to Technical Committee EH/3, Water quality.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The
3、 British Standards Institution 2013. Published by BSI Standards Limited 2013ISBN 978 0 580 80022 1ICS 07.100.20Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30
4、 September 2010.Amendments/corrigenda issued since publicationDate Text affected31 July 2013 This corrigendum renumbers BS ISO 19250:2010 as BS EN ISO 19250:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 19250 April 2013 ICS 07.100.20 English Version Water quality - Detection of Salmon
5、ella spp. (ISO 19250:2010) Qualit de leau - Recherche de Salmonella spp. (ISO 19250:2010) Wasserbeschaffenheit - Bestimmung von Salmonella spp. (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 whic
6、h 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 Eur
7、opean 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 a
8、re 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, R
9、omania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any mean
10、s reserved worldwide for CEN national Members. Ref. No. EN ISO 19250:2013: E EN ISO 19250:2013 (E) 3 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 1
11、9250:2013 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 standar
12、ds 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 Inte
13、rnal 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, I
14、reland, 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. ii
15、BS EN ISO 19250:2013EN ISO 19250:2010(E)BS ISO 19250:2010ISO 19250:2010(E) ISO 2010 All rights reserved iiiContents Page Foreword iv Introduction.v 1 Scope1 2 Normative references1 3 Terms and definitions .2 4 Principle2 4.1 General .2 4.2 Pre-enrichment in non-selective liquid medium 2 4.3 Enrichme
16、nt in selective liquid media 2 4.4 Plating out and recognition3 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-enrichment5 8.3 Selective enrichment.5 8.4 Plating out 6 8.5 Confirmation 6 9 Expression o
17、f results9 10 Test report9 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 trial18 Bibliography23 BS ISO 19250:2010ISO 19250:2010(E) ISO 2010 All rights reserved iiiConte
18、nts Page Foreword iv Introduction .1 Scope 12 Normative references3 Terms and definition 24 Principle.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 recognition3 4.5 Confirmation 3 5 Apparatus.6 Sampling 47 Culture me
19、dia and reagents8 Procedure 58.1 Preparation of the sample 5 8.2 Non-selective pre-enrichment5 8.3 Selective enrichment.5 8.4 Plating out 6 8.5 Confirmation 6 9 Expression of results 910 Test reportAnnex A (normative) Diagram of procedure 10B Composition and preparation of culture media and reagents
20、.11C (informative) Results of the interlaboratory trial18Bibliography .23BS EN ISO 19250:2013ISO 19250:2010(E)iiiBS ISO 19250:2010ISO 19250:2010(E) ISO 2010 All rights reserved vIntroduction Salmonella species are bacteria which are widely distributed all over the world. They are usually classified
21、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
22、 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
23、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 number
24、s and injured in the aqueous environment, their detection in water usually requires a pre-enrichment step. BS EN ISO 19250:2013ISO 19250:2010(E)ivBS ISO 19250:2010ISO 19250:2010(E) ISO 2010 All rights reserved vIntroduction Salmonella specie are bacteria which are widely distributed all over the wor
25、ld. They are usually classified as pathogens, although their virulence and pathogenesis vary widely. The natural hosts of Salmonella include hum , agricultural and domestic livestock, and wild animals including birds. Humans and animals can excrete these bacteria while carrying them asymptomatically
26、 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 whe
27、re 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 cell
28、s can be present in low numbers and injured in the aqueous environment, their detection in water usually requires a pre-enrichment step. BS ISO 19250:2010INTERNATIONAL STANDARD ISO 19250:2010(E) ISO 2010 All rights reserved 1Water quality Detection of Salmonella spp. WARNING In order to safeguard th
29、e 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
30、 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 wi
31、th 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 traine
32、d 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
33、 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 fol
34、lowing 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
35、 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 dil
36、utions 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 b
37、y culture ISO 19458, Water quality Sampling for microbiological analysis BS EN ISO 19250:2013BS ISO 19250:2010ISO 19250:2010(E) 2 ISO 2010 All rights reserved3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 presumptive Salmonella spp. bacteria
38、 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 whic
39、h 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. Salm
40、onella 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
41、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 memb
42、ers 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)
43、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-quant
44、itative 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
45、 (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. BS EN ISO 19250:2013ISO 19250:2010(E)BS ISO 19250:2010ISO 19250:2010(E) ISO 2010 All rights reserved 3To detect slow-growing Salmonella spp.,
46、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
47、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 br
48、oth. 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 Salmone
49、lla 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 manufacturers 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
