1、June 2013 Translation by DIN-Sprachendienst.English price group 14No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 0
2、7.100.20!%*c“2020764www.din.deDDIN EN ISO 19250Water quality Detection of Salmonella spp. (ISO 19250:2010);English version EN ISO 19250:2013,English translation of DIN EN ISO 19250:2013-06Wasserbeschaffenheit Bestimmung von Salmonella spp. (ISO 19250:2010);Englische Fassung EN ISO 19250:2013,Englisc
3、he bersetzung von DIN EN ISO 19250:2013-06Qualit de leau Recherche de Salmonella spp. (ISO 19250:2010);Version anglaise EN ISO 19250:2013,Traduction anglaise de DIN EN ISO 19250:2013-06www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritativ
4、e.3107.13 DIN EN ISO 19250:2013-06 2 A comma is used as the decimal marker. National foreword The text of ISO 19250:2010 has been prepared by Technical Committee ISO/TC 147 “Water quality” and has been taken over as EN ISO 19250:2013 by Technical Committee CEN/TC 230 “Water analysis” (Secretariat: D
5、IN, Germany). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Working Committee NA 119-01-03-03 UA Mikrobiologie of NA 119-01-03 AA Wasseruntersuchung. Designation of the method: Detection of Salmonella spp. (K 18): Me
6、thod DIN EN ISO 19250 K 18 As a result of national implementation in Germany, the following action has been taken: The original ISO document contains three errors which have been corrected in the German version of this document and correspondingly in the English version of this DIN EN ISO Standard:
7、In Subclause 8.3, first paragraph, last sentence, the volume of the culture obtained in 8.2 which is to be transferred has been corrected from “1 ml” to 0,1 ml. In Subclause 8.5.3.3, second sentence, the incubation time has been corrected from “24 h” to (24 3) h. In Subclause B.9.3, the temperature
8、for the storage of the novobiocin solution has been corrected from “(3 2) C” to (5 3) C. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 3696 DIN ISO 3696 ISO 6579 DIN EN ISO 6579 ISO 6887-1 DIN EN ISO 6887-1 ISO 7218 DIN EN ISO 7218 IS
9、O 8199 DIN EN ISO 8199 ISO 19458 DIN EN ISO 19458 ISO/TS 11133-1 DIN ISO/TS 11133-1 ISO/TS 11133-2 DIN CEN ISO/TS 11133-2 (DIN SPEC 10126) ISO/TR 13843 DIN V ENV ISO 13843 Expert assistance and specialized laboratories will be required to perform the analyses described in this standard. Existing saf
10、ety requirements are to be observed. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to what extent additional conditions will have to be specified. This standard has been prepared by the Normenausschuss Wasserwesen (Water Practice Standard
11、s Committee) in collaboration with the Wasserchemische Gesellschaft Fachgruppe in der Gesellschaft Deutscher Chemiker (Water Chemistry Society Division of the German Chemical Society). It is part of the DIN EN ISO 19250:2013-06 3 series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammun
12、tersuchung (German standard methods for the examination of water, waste water and sludge): Detection of Salmonella spp. (K 18). Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-lea
13、f publication entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Wiley-VCH Verlag GmbH b) any other solid selective medium complementary to XLD agar and, if applicable, appropriate for the isolation of lactose-positive Salmonella and S
14、almonella 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 ma
15、nufacturers 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 appropria
16、te 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 i
17、s 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)
18、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 m. The quality of membrane filters may vary from brand to brand or even from batch to batch. It is the
19、refore advisable to check the quality on a regular basis, as specified in ISO 7704. DIN EN ISO 19250:2013-06 EN ISO 19250:2013 (E) 7 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 l volume), and inoculat
20、ion 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
21、Culture media and reagents NOTE For guidelines on quality assurance and performance testing, see ISO/TS 11133-12and ISO/TS 11133-23. 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 reagent
22、s of recognized analytical grade. Other grades of reagents may be used provided they can be shown to produce comparable results. 7.2 Water, ISO 36961, 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 w
23、ater (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 manufacture
24、rs 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 decarboxylatio
25、n 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). DIN EN ISO 19250:2013-06 EN ISO 19250:2013 (E) 8 8 Procedure See Figure A.1. 8.1 Preparation of the sample For the preparation of the sample, filtration a
26、nd 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 circumstance
27、s, 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 volume
28、s 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
29、 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 dou
30、ble 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 sam
31、ple 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 turb
32、id 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 presenc
33、e 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 (
34、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. DIN EN ISO 19250:2013-06 EN ISO 19250:2013 (E) 9 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 t
35、aken 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 accor
36、ding 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 f
37、rom 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 additi
38、onal 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 manufacturers instructions shall be followed for the second selective plating-out medium. 8.4.3 Plating from MKTTn broth After in
39、cubation 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
40、. Use these kits according to the manufacturers 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
41、 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 ident
42、ified 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
43、 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; Enterobac
44、teriaceae e.g. Escherichia coli, Enterobacter, Klebsiella, Citrobacter, Proteus, and Serratia appear as yellow, opaque colonies. DIN EN ISO 19250:2013-06 EN ISO 19250:2013 (E) 10 Streak the selected colonies on to the surface of pre-dried “non-selective” agar plates (e.g. nutrient agar, B.4) to allo
45、w 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, sero
46、logical confirmation. If biochemical kits are used for identification, follow the manufacturers 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 selec
47、ted 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 o
48、f 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
