1、November 2016 English price group 12No 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 07.100.20!%v#“2588300www.din.de
2、DIN EN ISO 14189Water quality Enumeration of Clostridium perfringens Method using membrane filtration (ISO 14189:2013);English version EN ISO 14189:2016,English translation of DIN EN ISO 14189:2016-11Wasserbeschaffenheit Zhlung von Clostridium perfringens Verfahren mittels Membranfiltration (ISO 141
3、89:2013);Englische Fassung EN ISO 14189:2016,Englische bersetzung von DIN EN ISO 14189:2016-11Qualit de leau Dnombrement de Clostridium perfringens Mthode de filtration sur membrane (ISO 14189:2013);Version anglaise EN ISO 14189:2016,Traduction anglaise de DIN EN ISO 14189:2016-11www.beuth.deDocumen
4、t comprises 21 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.11.16 DIN EN ISO 14189:2016-11 2 A comma is used as the decimal marker. National foreword The text of ISO 14189:2013 has been prepared by Technical Committee ISO/TC
5、 147 “Water quality” and has been taken over as EN ISO 14189:2016 by Technical Committee CEN/TC 230 “Water analysis” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was DIN-Normenausschuss Wasserwesen (DIN Standards Committee Water Practice), Working Committee NA
6、 119-01-03-03 UA Mikrobiologie of NA 119-01-03 AA Wasseruntersuchung. Designation of the method: Enumeration of Clostridium perfringens Method using membrane filtration (K 24): Method DIN EN ISO 14189 K 24 For the national implementation of this standard in Germany, the following should be noted: Th
7、e 4thparagraph of normative Annex A.3 “Acid phosphatase reagent” deviates from the original literature 1 according to which the buffer solution should be made up as follows: “Prepare the acetate buffer by dissolving 0,3 ml glacial acetic acid (CAS-No.: 64-19-7) and 0,4 g sodium acetate (CAS No.: 127
8、-09-3) in deionized water and make up to 100 ml. Alternatively use a commercially available product.” The acid phosphatase reagent contains a toxic and carcinogenic substance (Fast Blue B Salt). It is complicated to prepare and has a short storage life. Therefore, on recommendation of Working Commit
9、tee NA 119-01-03-03 UA Mikrobiologie, an alternative detection procedure for acid phosphatase should be taken into account. This includes anaerobic subculturing of the colonies to be tested on m-CP-agar for (21 3) h at (36 2) C and subsequent steaming with ammonium hydroxide for 20 s to 30 s. If col
10、onies that were initially dark yellow turn rose to red, acid phosphatase shall be deemed to be verified. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 3696 DIN ISO 3696 ISO 5725-2 DIN ISO 5725-2 ISO 8199 DIN EN ISO 8199 ISO 19458 DIN
11、EN ISO 19458 ISO/TR 13843 DIN V ENV ISO 13843 ISO/TS 11133-1 DIN ISO/TS 11133-1 ISO/IEC Guide 2 DIN EN 45020 DIN EN ISO 14189:2016-11 3 Expert assistance and specialized laboratories will be required to perform the analyses described in this standard. Existing safety requirements are to be observed.
12、 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 DIN-Normenausschuss Wasserwesen (DIN Standards Committee Water Practice) in collaboration wi
13、th the Wasserchemische Gesellschaft - Fachgruppe in der Gesellschaft Deutscher Chemiker (Water Chemistry Society - Division of the German Chemical Society). It is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung (German standard methods for the examination
14、 of water, waste water and sludge): Enumeration of Clostridium perfringens Method using membrane filtration (K 24). 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-leaf publicatio
15、n entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Beuth Verlag GmbH and Wiley-VCH Verlag GmbH b) all details necessary for the complete identification of the sample;c) the number of colonies presumptive C. perfringens (optional, dep
16、ending on the purpose of the investigation);d) the number of colonies confirmed as C. perfringens;e) information, if the result represents the total number of C. perfringens (vegetative cells and spores) or spores only;f) any particular occurrence(s) observed during the analysis and any operation(s)
17、 not specified in this method, which may have affected the results.11 Quality assuranceThe laboratory shall have a clearly defined quality control system to ensure that the apparatus, reagents and techniques are suitable for the test. The use of positive controls, negative controls and blanks is par
18、t of the test.Media quality control is described in ISO/TS 11133-1. For quantitative process quality control a suspension of C. perfringens is used for this purpose. Select the volume filtered to contain between 10 cfu to 80 cfu and treat the control like a sample. Compare recovery to that on a non-
19、selective agar such as blood agar. Alternatively to the suspension of C. perfringens use reference materials.NOTE Since the medium should be used as fresh as possible it is acceptable to perform media and process control for samples simultaneously in parallel with the analysis of the samples.Include
20、 a blank control in each batch of analyses. Filter 100 ml of sterile water or another appropriate diluent according to ISO 8199 and continue to treat it like a sample but without pasteurization. No colonies should be visible after incubation.Include an appropriate control for correct anaerobic condi
21、tions (e.g. anaerobic indicator strip) whenever anaerobic incubation is performed (anaerobic jar or anaerobic incubator).For the confirmation step performed by acid phosphatase test include known positive and negative control strains.At least one of the C. perfringens strains given in Table 1 shall
22、be used as a positive control for media control and the confirmation test.10EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 Bacillus subtilis WDCM 00003 shall be used as a negative control for assessing TSC medium and anaerobic conditions.Clostridium bifermentans WDCM 00079 shall be used as negative
23、control for the confirmation test.Table 1 Performance testing of Tryptose sulfite cycloserine agar (TSC agar) by comparing with a non-selective reference mediumFunction Incubation Control strainsaReference mediaMethod of controlCriteria Characteristic reac-tionsProductivity (21 3) h / (44 1) C anaer
24、obicClostridium perfrin-gens WDCM 00007; C. perfringens WDCM 00080; C. perfringens WDCM 00174TSA or blood agarQuantitative PR 0,5 Black coloniesSelectivity (21 3) h / (44 1) C anaerobicBacillus subtilis WDCM 00003 Qualitative Total inhibition (0)aRefer to the reference strain catalogue available on
25、http:/www.wfcc.info for information on culture collection strain numbers and contact details.11EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 Annex A (normative) Composition and preparation of culture media and reagentsA.1 Tryptose sulfite cycloserine agar (TSC agar), References61112A.1.1 Basal medi
26、umEnzymatic digest of casein 15 gEnzymatic digest of soya 5 gYeast extract 5 gSodium disulfite (sodium metabisulfite), anhydrous (Na2S2O5) (CAS No 7681-57-4)1 gIron(III) ammonium citrate (CAS No.: 1185-57-5) 1 gAgar 9 g to 18 gWater 1 000 mlSuspend the ingredients in the water and dissolve by heatin
27、g and stirring. Sterilize by autoclaving at (121 3) C for 15 min. Allow to cool to 45 C to 50 C. The basal medium may be stored at (5 3) C and used within 4 weeks of preparation. To prepare the complete medium the stored medium is remelted by steaming and cooled to 45 C to 50 C before adding the cyc
28、loserine solution (see A.1.2).A.1.2 D-cycloserine solutionD-cycloserine (CAS No.: 68-41-7) 4 gWater to 100 mlDissolve the D-cycloserine in the water and filter through a membrane of 0,2 m pore size. Dispense aseptically into suitable volumes, store at (-20 5) C and use within 4 weeks of preparation.
29、 Alternatively, the dispended volumes of cycloserine can be stored at (-70 10) C for a maximum of 12 months.A.1.3 Complete mediumBasal medium (A.1.1) 1 000 mlD-cycloserine solution (A.1.2) 10 mlAdd the D-cycloserine solution to the molten cooled basal medium, mix well and dispense into vented Petri
30、dishes to a depth of at least 5 mm.The final pH of the medium should correspond to 7,6 0,2 at 25 C.Use the prepared plates as fresh as possible on the same day. If storage of the prepared plates is inevitable, store the plates under anaerobic conditions at (5 3) C and use them within 7 d.12EN ISO 14
31、189:2016 (E) DIN EN ISO 14189:2016-11 Discard plates, once removed from the refrigerator, if not used. Do not return the plates to storage, as the performance of the medium deteriorates. Dry the plates well before use.A.2 Blood agarColumbia blood agar or another suitable base with 5 % blood (e.g. ho
32、rse, sheep blood).If blood agar is not available another suitable non selective nutrient rich agar may be used for subculture (e.g. Columbia agar base or tryptone soya agar).A.3 Acid phosphatase reagent1-naphthylphosphate disodium salt (CAS No.: 2183-17-7) 0,4 gFast Blue B Salt (o-Dianisidine bis(di
33、azotized) zinc double salt) (CAS No 14263-94-6)0,8 gAcetate buffer (pH 4,6 0,2) 20 mlPrepare the acetate buffer by dissolving 0,3 ml glacial acetic acid (CAS No.: 64-19-7) and 0,4 g sodium acetate (CAS No.: 127-09-3) in deionized water and make up to 1 000 ml. Alternatively use a commercially availa
34、ble product.Dissolve the ingredients in the acetate buffer and allow to stand for (60 5) min at (5 3) C to allow precipitation. Then filter the solution through a fluted filter to remove the precipitate. Store the prepared solution at (5 3) C for no longer than two weeks. If precipitation occurs aga
35、in filter again once more before use.NOTE 1 Instead of 1-naphthylphosphate disodium salt 1-naphthylphosphate monosodium salt (CAS No.: 81012-89-7) can be used.CAUTION Fast Blue B Salt is toxic and may cause cancer appropriate precautions shall be taken when weighing out, preparing and discarding the
36、 reagent.13EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 Annex B (informative) Performance dataB.1 Performance characteristics of the standard methodIn an intralaboratory trial the performance parameters like range of quantitative determination, robustness of incubation time, counting uncertainty a
37、nd efficiency, have been determined, References.210The data are summarized in Table B.1.A collaborative study within 12 laboratories from 11 countries was performed (Austria, Czech Republic, Finland, France, Germany, Hungary, Ireland, Portugal, Slovakia, The Netherlands, United Kingdom) to investiga
38、te the precision of the method. The samples (n = 108) consisted of reference material (lenticules, mixed culture, kindly provided by HPA, UK). The reference material was delivered to the participants and used to prepare the water samples. On three days within a time span of two week analyses of thre
39、e samples in triplicate each were performed according to the standard method. One plate per sample and day of analyses was used for confirmation; all colonies of these plates were tested with acid phosphatase-test. The recovery of the method was determined by using blood agar plates for cultivation.
40、 The data are presented in Tables B.1 and B.2.Table B.1 Parameters for performance characteristicsParameterIntralaboratory trialRange of quantitative determination (colonies per mem-brane 47 mm in diameter)10 to 80Robustness of incubation time 18 h to 24 h no significant difference in countsCounting
41、 uncertainty (RSD)repeatability 0,038reproducibility (intralab) 0,0348Identification (n=127)sensitivity (%) 94specificity (%) 87false positive rate 0,10false negative rate 0,09selectivity -0,15efficiency (%) 92Interlaboratory trialPrecision (RSD)arepeatability (within lab) 0,1676reproducibility with
42、in lab 0,1382reproducibility (between lab) 0,3232Recovery (%) 72aBoth, distribution variability and operational variability are included in these assessments.14EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 B.2 Statistical procedure used for the determination of the precision parameters of the colla
43、borative study Nested random effects in data analysis: Two-way ANOVAThe main principle was to fit data to a linear model (additive effect of the error, without interaction). The counts were transformed into lg scale in order to comply with the Normality pre-requisites of ANOVA. The Grubbs Test was u
44、sed to detect outlier (suspicious result for one laboratory - n6). No result was discarded.The model described in ISO Guide 354was used. Regarding the statistical pattern of the collaborative study, the original between-bottle variance was changed into a between-day variance.xkij = + k+ ki+ kijwhere
45、xkijis the measurement j of day i for the laboratory k; is the true value;kis the error due to the laboratory k;kiis the error due on ithday within the laboratory k;kijis the random error of measurement.From a practical point of view: m is the estimation of (consensus value also called assigned valu
46、e or grand mean); SL2is the variance due to the interlaboratory error (systematic error); Su2is the variance due to the “day effect” ; Sr2is the variance of the measurement error .NOTE The variance of reproducibility can be assessed as follows: sR2= sL2+ sr2.All these parameters were estimated simul
47、taneously by the method of analysis variance (ANOVA). The same number of repeated measurements for each day and the same number of days per laboratory were considered.Finally, the formula given in ISO 29201:2012, 6.2 used to convert lg scale into natural logarithms was applied to the estimated varia
48、nce. This conversion leads to an expression of the uncertainty in a relative scale (relative variance: see also ISO 29201:2012, 2.5.2):uR2= 5,3019sR2uu2= 5,3019su2ur2= 5,3019sr215EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 Table B.2 Collaborative study: Results of the determination of the precisi
49、on parametersVar (lg scale)u2uInterlaboratory error 0,0144 0,0763 27,6%“day-effect” 0,0036 0,0190 13,8%Measurement error 0,0053 0,0279 16,7%Reproducibility (between lab) 0,0197 0,1043 32,3%16EN ISO 14189:2016 (E) DIN EN ISO 14189:2016-11 Bibliography1 ISO 3696:1987, Water for analytical laboratory use Specification and test methods2 ISO/TR 13843:2000, Water quality Guidance on validati
