1、June 2014 Translation by DIN-Sprachendienst.English price group 20No 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!%2m“2157429www.din.deDDIN EN ISO 9308-2Water quality Enumeration of Escherichia coli and coliform bacteria Part 2: Most probable number method (ISO 9308-2:2012);English version EN ISO 9308-2:2014,English translation of DIN EN ISO 9308-2:2014-06Wasserbeschaffenheit Zhlung von Escherichia col
3、i und coliformen Bakterien Teil 2: Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO 9308-2:2012);Englische Fassung EN ISO 9308-2:2014,Englische bersetzung von DIN EN ISO 9308-2:2014-06Qualit de leau Dnombrement des Escherichia coli et des bactries coliformes Partie 2: Mthode du nombre l
4、e plus probable (ISO 9308-2:2012);Version anglaise EN ISO 9308-2:2014,Traduction anglaise de DIN EN ISO 9308-2:2014-06Partially supersedesDIN 38411-6:1991-06www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 54 pages05.14 DIN EN ISO 9308-2
5、:2014-06 2 A comma is used as the decimal marker. National foreword The text of ISO 9308-2:2012 has been prepared by Technical Committee ISO/TC 147 “Water quality” and has been taken over as EN ISO 9308-2:2014 by Technical Committee CEN/TC 230 “Water analysis” (Secretariat: DIN, Germany). The respon
6、sible 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. ISO 9308 consists of the following parts, under the general title Water quality Enumeratio
7、n of Escherichia coli and coliform bacteria: Part 1: Membrane filtration method for waters with low bacterial background flora Part 2: Most probable number method Part 3: Miniaturized method (Most Probable Number) for the detection and enumeration of E. coli in surface and waste water Designation of
8、 the method: Enumeration of Escherichia coli and coliform bacteria (K 6) Part 2: Most probable number method (1): Method DIN EN ISO 9308-2 K 6 1 The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 8199 DIN EN ISO 8199 ISO 19458 DIN EN ISO 1
9、9458 ISO/IEC Guide 2 DIN EN 45020 ISO/TR 13843 DIN V ENV ISO 13843 DIN EN ISO 9308-2:2014-06 3 Expert assistance and specialized laboratories will be required to perform the analyses described in this standard. Existing safety requirements are to be observed. Depending on the objective of the analys
10、is, 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 Standards Committee) in collaboration with the Wasserchemische Gesellschaft Fachgrupp
11、e 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 of water, waste water and sludge): Most probable
12、 number method (1) (K 6 1). 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 publication entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung wil
13、l continue to be published by Wiley-VCH Verlag GmbH b) all information required for the complete identification of the sample; c) the results expressed in accordance with Clause 9; d) any particular occurrence(s) observed during the course of the analysis and any operation(s) not specified in this p
14、art of ISO 9308 which may have influenced the results. 11 Quality assurance The 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 part of the te
15、st. 8DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Annex A (informative) Further microbiological information on coliform bacteria In addition to expressing -D-galactosidase, coliform bacteria are typically Gram-negative non-sporeforming, oxidase-negative, rod-shaped bacteria, which are capable of a
16、erobic and facultatively anaerobic growth in the presence of bile-salts (or other surface-active agents with similar growth-inhibiting properties), and which are usually able to ferment lactose with the production of acid and aldehyde within 48 h when incubated at a temperature of (36 2) C. In addit
17、ion to expressing -D-glucuronidase, E. coli are coliform bacteria that are able to produce indole from tryptophan within (21 3) h at (44,0 0,5) C. They give a positive result in the methyl red test and can decarboxylate l-glutamic acid but are not able to produce acetyl methyl carbinol, utilise citr
18、ate as the sole source of carbon or grow in KCN broth. Some strains of Escherichia coli which are -D-glucuronidase negative, such as Escherichia coli O157, will not be detected as E. coli. As they are -D-galactosidase positive, they will appear as coliform bacteria. 9DIN EN ISO 9308-2:2014-06EN ISO
19、9308-2:2014 (E)Annex B (normative) The Quanti-Tray5)Sealer and calculation of results B.1 General The Quanti-Tray5)Sealer is a thermal sealing unit that forms a seal between wells in the Quanti-Tray. The sealer automatically distributes liquid into the wells of the Quanti-Tray or Quanti-Tray/2000. T
20、he Quanti-Tray is used when anticipated counts are below 200 cfu/100 ml. The Quanti-Tray/2000 can be used to calculate MPN values up to 2419 cfu/100 ml. When calculating MPN, the tables supplied with the trays are the reference for all counts. A simple statistical program can also be used to calcula
21、te results. If required, the MPN can be calculated manually according to the procedures given below. B.2 Calculation of the most probable number B.2.1 Calculation of MPN for IDEXX Quanti-Tray5and Quanti-Tray/2000 B.2.1.1 Quanti-Tray (51-well) Quanti-Tray MPN was originally developed at Yale Universi
22、ty; an additional, good example of this serial dilution MPN can be found at the U.S. Food and Drug Association in the Bacteriological Analytical Manual (available on BAM Appendix 2: Most Probable Number from Serial Dilutions, October 2010). Each sample well has an approximate volume of 1,96 ml. The
23、overflow well will hold a minimum of 8,5 ml. For the calculation of the Quanti-Tray MPN (Table B.1), see Equation (B.1). NMPN= N ln N/(N X) (B.1) where NMPN is the MPN;N is the total number of wells (tubes) used in a test; X is the number of positive wells (tubes) observed in a test. B.2.1.2 Quanti-
24、Tray5)/2000 (97-well) Quanti-Tray/2000 MPN was originally derived as described by Reference 1. Small wells have a mean volume of 0,186 ml. Large wells have a mean volume of approximately 1,86 ml (ten times larger than the small wells). 5Quanti-Tray is a trademark or registered trademark of IDEXX Lab
25、oratories, Inc. or its affiliates in the United States and/or other countries. This information is given for the convenience of users of this part of ISO 9308 and does not constitute an endorsement by ISO of this product. 10DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Overflow well will hold appro
26、ximately 11 ml. For the calculation of the Quanti-Tray6)/2000 MPN (Table B.2), see Equation (B.2): mpn111eiiKKiiiiiiVd NiiVd PVdn(B.2) where diis the dilution factor at level i (e.g. 0,1 for 110 dilution); K is the number of dilution levels; niis the number of wells at level i; Nmpn is the MPN; Piis
27、 the number of positive wells at level i; Viis the volume of the wells at level i. The 95 % confidence intervals can be found at : T0 (ln Nmpn 1,96) (ln Nmpn)T1 (ln Nmpn 1,96) (ln Nmpn)where T0is the lower confidence interval; T1 is the upper confidence interval; is the standard error; and mpn2222mp
28、n mpn1lne1iiKiiiVdNiVdnNN(B.3) 6) Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. This information is given for the convenience of users of this part of ISO 9308 and does not constitute an endorsement by IS
29、O of this product. 11DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Table B.1 51-Well Quanti-Tray MPN No. of wells giving positive reaction Most probable number (MPN) per 100 ml sample 95 % Confidence limits Lower Upper 0 200,5 146,1 infiniteaA value of 1 means that there were no target bacteria det
30、ected in the test. For undiluted samples this can be reported as zero. However, if dilutions of the sample have been made, then the appropriate multiplication factor must be used. For example, if a 110 dilution has been made and no bacteria are detected then the result should be reported as 10. Tabl
31、e B.2 97-Well Quanti Tray (Quanti-Tray/2000) MPN with 95 % confidence limits Positive large wells Positive small wells MPN 95% Confidence Positive large wells Positive small wells MPN 95% Confidence Lower limit Upper limit Lower limit Upper limit 0 0 1a 0,0 3,7 25 0 33,6 22,0 48,9 0 1 1,0 0,0 3,7 25
32、 1 35,0 22,9 51,2 0 2 2,0 0,3 5,6 25 2 36,4 23,8 52,6 0 3 3,0 0,6 7,3 25 3 37,9 25,5 54,0 0 4 4,0 1,1 8,9 25 4 39,3 26,5 55,9 0 5 5,0 1,7 10,5 25 5 40,8 28,3 57,3 0 6 6,0 2,3 12,1 25 6 42,2 29,3 59,0 0 7 7,0 2,9 13,7 25 7 43,7 30,3 60,7 0 8 8,0 3,7 15,3 25 8 45,2 31,3 62,5 0 9 9,0 4,5 15,8 25 9 46,7
33、 33,3 64,2 0 10 10,0 5,2 16,9 25 10 48,2 34,4 66,0 13DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Table B.2 (continued) Positive large wells Positive small wells MPN 95% Confidence Positive large wells Positive small wells MPN 95% Confidence Lower limit Upper limit Lower limit Upper limit 0 11 11,
34、0 5,9 18,5 25 11 49,7 35,4 67,3 0 12 12,0 6,9 20,1 25 12 51,2 36,5 69,0 0 13 13,0 7,8 21,2 25 13 52,7 37,6 70,7 0 14 14,1 8,6 21,9 25 14 54,3 39,7 72,4 0 15 15,1 9,0 23,4 25 15 55,8 40,9 74,0 0 16 16,1 9,6 24,9 25 16 57,3 42,0 75,9 0 17 17,1 10,5 25,7 25 17 58,9 43,1 77,6 0 18 18,1 11,5 26,9 25 18 6
35、0,5 45,5 79,5 0 19 19,1 12,5 28,6 25 19 62,0 46,7 81,2 0 20 20,2 13,2 29,3 25 20 63,6 47,8 83,0 0 21 21,2 13,9 30,5 25 21 65,2 49,0 84,6 0 22 22,2 14,5 31,8 25 22 66,8 50,2 86,2 0 23 23,3 15,7 33,1 25 23 68,4 51,5 87,4 0 24 24,3 16,4 34,2 25 24 70,0 54,0 89,5 0 25 25,3 17,6 35,2 25 25 71,7 55,3 91,6
36、 0 26 26,4 18,3 36,5 25 26 73,3 56,6 93,9 0 27 27,4 19,5 37,7 25 27 75,0 57,8 94,6 0 28 28,4 19,7 38,6 25 28 76,6 59,1 96,1 0 29 29,5 21,0 39,9 25 29 78,3 60,4 98,6 0 30 30,5 21,7 41,2 25 30 80,0 61,7 101,0 0 31 31,5 22,5 42,3 25 31 81,7 64,6 101,6 0 32 32,6 23,9 43,4 25 32 83,3 65,9 103,6 0 33 33,6
37、 24,6 44,4 25 33 85,1 67,3 106,2 0 34 34,7 25,4 45,7 25 34 86,8 68,6 107,3 0 35 35,7 26,2 46,8 25 35 88,5 70,0 109,1 0 36 36,8 27,7 48,0 25 36 90,2 71,4 111,4 0 37 37,8 28,5 49,0 25 37 92,0 72,8 112,8 0 38 38,9 29,2 50,3 25 38 93,7 74,2 114,9 0 39 40,0 30,0 51,2 25 39 95,5 77,4 116,4 0 40 41,0 30,8
38、52,8 25 40 97,3 78,9 118,3 0 41 42,1 31,6 53,7 25 41 99,1 80,3 120,4 0 42 43,1 33,3 54,7 25 42 100,9 81,8 121,9 0 43 44,2 34,1 56,1 25 43 102,7 83,2 124,2 0 44 45,3 34,9 57,1 25 44 104,5 84,7 126,0 0 45 46,3 35,7 58,1 25 45 106,3 86,2 128,0 0 46 47,4 37,5 59,5 25 46 108,2 89,8 130,2 0 47 48,5 38,3 6
39、0,7 25 47 110,0 91,3 132,1 0 48 49,5 39,2 61,6 25 48 111,9 92,9 133,7 14DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Table B.2 (continued) Positive large wells Positive small wells MPN 95% Confidence Positive large wells Positive small wells MPN 95% Confidence Lower limit Upper limit Lower limit U
40、pper limit 1 0 1,0 0,1 5,5 26 0 35,5 23,2 52,0 1 1 2,0 0,3 5,9 26 1 36,9 24,9 53,7 1 2 3,0 0,6 7,3 26 2 38,4 25,9 55,4 1 3 4,0 1,1 8,9 26 3 39,9 26,9 56,5 1 4 5,0 1,7 10,5 26 4 41,4 27,9 58,6 1 5 6,0 2,3 12,1 26 5 42,8 29,7 60,1 1 6 7,1 3,0 13,7 26 6 44,3 30,7 61,8 1 7 8,1 3,7 15,3 26 7 45,9 31,8 63
41、,5 1 8 9,1 4,3 16,2 26 8 47,4 32,8 65,4 1 9 10,1 5,2 17,2 26 9 48,9 34,9 66,6 1 10 11,1 6,0 18,5 26 10 50,4 36,0 68,5 1 11 12,1 6,8 20,1 26 11 52,0 37,1 70,4 1 12 13,2 7,6 21,7 26 12 53,5 38,2 72,2 1 13 14,2 8,7 22,2 26 13 55,1 40,4 73,7 1 14 15,2 9,4 23,6 26 14 56,7 41,5 75,7 1 15 16,2 9,7 25,1 26
42、15 58,2 42,7 77,6 1 16 17,3 10,6 26,4 26 16 59,8 43,8 79,2 1 17 18,3 11,6 27,0 26 17 61,4 45,0 80,8 1 18 19,3 12,6 28,6 26 18 63,0 46,2 82,7 1 19 20,4 13,3 30,0 26 19 64,7 48,6 84,2 1 20 21,4 14,0 30,7 26 20 66,3 49,8 85,4 1 21 22,4 14,7 32,3 26 21 67,9 51,1 87,3 1 22 23,5 15,8 33,1 26 22 69,6 52,3
43、89,5 1 23 24,5 17,0 34,5 26 23 71,2 55,0 91,6 1 24 25,6 17,2 35,7 26 24 72,9 54,8 93,9 1 25 26,6 18,4 37,1 26 25 74,6 57,5 94,6 1 26 27,7 19,2 38,1 26 26 76,3 58,8 96,2 1 27 28,7 20,5 39,2 26 27 78,0 60,1 98,6 1 28 29,8 21,2 40,6 26 28 79,7 61,5 101,0 1 29 30,8 22,0 41,6 26 29 81,4 62,8 102,2 1 30 3
44、1,9 22,7 42,7 26 30 83,1 64,1 103,6 1 31 32,9 23,5 44,0 26 31 84,9 67,1 106,2 1 32 34,0 24,9 44,9 26 32 86,6 68,5 108,0 1 33 35,0 25,7 46,4 26 33 88,4 69,9 109,4 1 34 36,1 26,4 47,2 26 34 90,1 71,3 111,8 1 35 37,2 27,9 48,8 26 35 91,9 72,7 113,5 1 36 38,2 28,7 49,6 26 36 93,7 74,1 115,1 1 37 39,3 29
45、,5 51,0 26 37 95,5 75,6 117,5 15DIN EN ISO 9308-2:2014-06EN ISO 9308-2:2014 (E)Table B.2 (continued) Positive large wells Positive small wells MPN 95% Confidence Positive large wells Positive small wells MPN 95% Confidence Lower limit Upper limit Lower limit Upper limit 1 38 40,4 30,4 52,0 26 38 97,
46、3 78,9 119,2 1 39 41,4 31,2 53,1 26 39 99,2 80,4 121,2 1 40 42,5 32,8 54,5 26 40 101,0 81,9 123,1 1 41 43,6 33,6 55,4 26 41 102,9 83,4 125,0 1 42 44,7 34,5 56,6 26 42 104,7 84,9 127,1 1 43 45,7 35,3 58,0 26 43 106,6 86,4 128,9 1 44 46,8 36,1 58,9 26 44 108,5 90,1 130,6 1 45 47,9 37,0 60,1 26 45 110,
47、4 89,5 132,4 1 46 49,0 38,7 61,3 26 46 112,3 93,2 135,0 1 47 50,1 39,6 62,5 26 47 114,2 94,8 137,0 1 48 51,2 40,5 63,6 26 48 116,2 96,4 138,7 2 0 2,0 0,3 7,1 27 0 37,4 24,5 54,5 2 1 3,0 0,7 7,4 27 1 38,9 26,2 55,9 2 2 4,1 1,2 9,0 27 2 40,4 27,3 57,4 2 3 5,1 1,6 10,6 27 3 42,0 28,3 59,7 2 4 6,1 2,3 1
48、2,1 27 4 43,5 29,3 60,8 2 5 7,1 3,0 13,7 27 5 45,0 31,2 63,0 2 6 8,1 3,7 15,3 27 6 46,5 32,3 64,7 2 7 9,2 4,4 16,9 27 7 48,1 33,3 66,1 2 8 10,2 5,3 17,8 27 8 49,6 35,4 67,8 2 9 11,2 6,0 18,6 27 9 51,2 36,5 69,7 2 10 12,2 6,8 20,1 27 10 52,8 37,6 71,7 2 11 13,3 7,7 21,7 27 11 54,4 38,8 73,4 2 12 14,3
49、 8,5 22,9 27 12 56,0 41,0 75,0 2 13 15,4 9,4 24,0 27 13 57,6 42,2 77,2 2 14 16,4 9,8 25,1 27 14 59,2 43,3 78,6 2 15 17,4 10,4 26,8 27 15 60,8 44,5 80,6 2 16 18,5 11,4 27,5 27 16 62,4 45,7 82,3 2 17 19,5 12,4 28,8 27 17 64,1 46,9 83,7 2 18 20,6 13,5 30,4 27 18 65,7 49,4 85,3 2 19 21,6 14,1 31,2 27 19 67,4 50,7 87,3 2 20 22,7 14,8 32,6 27 20 69,1 51,9 89,5 2 21 23,7 16,0 33,8 27 21 70,8 53,2 91,6 2 22 24,8 17,2 35,0 27 22 72,5
