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1、 Reference number ISO 10705-3:2003(E) ISO 2003 INTERNATIONAL STANDARD ISO 10705-3 First edition 2003-10-01 Water quality Detection and enumeration of bacteriophages Part 3: Validation of methods for concentration of bacteriophages from water Qualit de leau Dtection et dnombrement des bactriophages P
2、artie 3: Validation des mthodes de concentration des bactriophages dans leau Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted w
3、ithout license from IHS -,-,- ISO 10705-3:2003(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes 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
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6、ral Secretariat at the address given below. ISO 2003 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 a
7、t the address below or ISOs 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 copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2003 All rights reserved Copyright International Organi
8、zation for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10705-3:2003(E) ISO 2003 All rights reserved iii Contents Page Forewordiv
9、 1 Scope1 2 Normative references .1 3 Terms and definitions.2 4 Principle.2 5 Reagents2 6 Apparatus and glassware.3 7 Sampling3 8 Preparation of sewage samples for spiking.3 9 Procedure.4 10 Calculation.5 11 Analytical quality control .6 12 Test report7 Annex A (informative) Recommended methods for
10、concentration of bacteriophages from water depending on the volume, turbidity and particle content.8 Annex B (informative) Example of a validation process of a concentration method10 Bibliography .13 Copyright International Organization for Standardization Provided by IHS under license with ISO Lice
11、nsee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10705-3:2003(E) iv ISO 2003 All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of
12、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. Inte
13、rnational 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 th
14、e 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 b
15、y 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 10705-3 was prepared by Technical Committee
16、ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 10705 consists of the following parts, under the general title Water quality Detection and enumeration of bacteriophages: Part 1: Enumeration of F-specific RNA bacteriophages Part 2: Enumeration of somatic coliphages Part 3:
17、Validation of methods for concentration of bacteriophages from water Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/
18、20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 10705-3:2003(E) ISO 2003 All rights reserved 1 Water quality Detection and enumeration of bacteriophages Part 3: Validation of methods for concentration of bacteriophages from water
19、WARNING Persons using this part of ISO 10705 should be familiar with normal laboratory practice. This part of ISO 10705 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 a
20、nd to ensure compliance with any national regulatory conditions. IMPORTANT It is imperative that personnel involved in validation of methods for concentration of bacteriophages from water have relevant experience with the methods of enumeration of bacteriophages (see ISO/TR 138431). 1 Scope This par
21、t of ISO 10705 specifies the general principles for assessing the performance of methods for the concentration of bacteriophages from water. Concentration is recommended for those water samples expected to contain 20 % of the phages spiked into the sample, heat the water and keep it at 80 C for 30 m
22、in and allow it to cool prior to use. If the number of naturally occurring bacteriophages is 2,0 NTU. For concentrating phages from 1 l of water proceed as follows: a) Add 10 ml of a magnesium chloride solution (1 mol/l) to 1 l of water sample. b) Add to the sample dropwise while stirring 3,5 ml of
23、a dipotassium hydrogen phosphate solution (1 mol/l). c) Add dropwise while stirring sodium hydroxide solution (2 mol/l) until turbidity appears (maximum final pH = 8,6). d) Slowly stir the mixture for 15 min to obtain a regular distribution of the flocs to allow the incorporation of the phage partic
24、les into the flocs. e) Allow the mixture to stand for 30 min to allow the settling of the flocs. f) Siphon off the supernatant and concentrate loose sediment by low speed centrifugation (average of 1 000 g to 1 500 g) for 15 min. g) Carefully discard the supernatant and resuspend the sediment with 3
25、0 ml of a suitable diluent. Resuspension requires vigorous shaking until flocs disappear completely. h) Assay directly or store at 4 C for a maximum of 2 d. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001
26、Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10705-3:2003(E) 10 ISO 2003 All rights reserved Annex B (informative) Example of a validation process of a concentration method B.1 Spiking material Spiking material was prepared as indi
27、cated in Clause 8. Phage counts as indicated in Clause 8 are given in Table B.1. Table B.1 Phage counts of spiking material Vial number Counts 1 Counts 2 Mean of vial J zi+ Sum of counts zi+ Vial 1 76 (z11) 77 (z12) 76,5 153 Vial 2 72 (z21) 70 (z22) 71 142 Vial 1 + Vial 2 295 (z+) Calculation of T1
28、T1 for Vial 1 (76 76,5)2/76,5 + (77 76,5)2/76,5 = 0,006 T1 for Vial 2 (72 71)2/71 + (71 72)2/71 = 0,028 T1 (Vial 1) + T1 (Vial 2) = 0,006 + 0,028 = 0,034, which is 5,99 Homogeneity within vials is then acceptable. Calculation of T2 T2 = (153 295/2)2/295/2 + (142 295/2)2/295/2 = 0,41, which is 3,84.
29、Homogeneity between vials is then acceptable. B.2 Preparation of spiked samples 125 ml, 250 ml, 500 ml and 1 000 ml volumes of spring water were spiked with 1,0 ml taken from one of the vials of the spiking material prepared as indicated in Clause 8. B.3 Concentration The above indicated water sampl
30、es and 1 000 ml of non-spiked water were concentrated using the selected method. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitt
31、ed without license from IHS -,-,- ISO 10705-3:2003(E) ISO 2003 All rights reserved 11 B.4 Evaluation of recovery Phages in the total volume of concentrate were enumerated. In parallel, two aliquots of 0,5 ml of the spiking material were assayed. Table B.2 shows the results obtained. Table B.2 Result
32、s of phage counts Phages added Phages counted in concentrated spiked volumes of Phages counted in concentrated non- spiked volume of Experiment number 2 0,5 1 ml 125 ml 250 ml 500 ml 1 000 ml 1 000 ml 1 86 87 173 96 110 140 125 0 2 74 80 154 114 104 120 127 0 3 126 115 241 78 82 100 121 0 4 85 80 16
33、5 120 126 125 90 0 5 79 82 161 105 96 114 107 0 Spiking material used in Experiment 3 does not comply with T1 and T2 when compared to each one of the other spiking samples. The remaining spiking materials comply. B.5 Data analysis Since the water used for concentration did not contain bacteriophages
34、 the recovery, , expressed as a percentage, was calculated for each one of the values obtained according to = Nc /Ns 100. The average recovery, the standard deviation and the confidence limits were calculated for each one of the volumes assayed. Results were those shown in Table B.3. Table B.3 Resul
35、ts of recovery % after concentration of Experiment number 125 ml 250 ml 500 ml 1 000 ml 1 55,5 63,6 81,0 72,0 2 74,0 67,5 77,7 82,5 3 32,8 34,2 41,6 50,1 4 72,5 76,4 75,6 54,7 5 65,0 59,6 71,0 67,1 x 60,0 60,2 69,4 65,3 s 16,9 15,8 15,9 13,1 Confidence interval 80,9; 39,1 79,9; 40,6 89,2; 49,6 81,6;
36、 49,0 No extreme values were detected applying the Dixon test. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license
37、 from IHS -,-,- ISO 10705-3:2003(E) 12 ISO 2003 All rights reserved Although spiking material used in Experiment 3 did no comply with T1 and T2 results of the experiment were this time included in the analysis of data. The arithmetic average of plus the confidence intervals were plotted (Figure B.1)
38、 and no volume effect was observed, since the confidence intervals of the of the different volumes overlap. Figure B.1 Influence of volume on recovery The 20 values were combined. The , x s and relative standard deviation ( /s x ) were calculated, giving the following values x = 63,75 s = 14,80 /s x
39、 = 0,23 which is 0,5. B.6 Analytical quality control The evaluation of recovery has been repeated several times for 1 000 ml (in this case Vmax was tested) and the arithmetic average and results are given in Table B.4. Table B.4 Results of recovery evaluation % 60,5 66,95 40,8 59,8 72,4 74,4 Except
40、for the value 40,8 %, all the values were between the confidence limits obtained during validation for 1 000 ml volumes. After calculating T1 and T2 for spiking material used in these experiments, only the spiking material that gave of 40,8 % did not comply with T1 and T2. Copyright International Or
41、ganization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10705-3:2003(E) ISO 2003 All rights reserved 13 Bibliography 1 ISO/TR
42、 13843:2000, Water quality Guidance on validation of microbiological methods 2 ISO 194583), Water quality Sampling for microbiological analysis 3 LOGAN, K.B., REES, G.E. and PRIMROSE, S.B. Rapid concentration of bacteriophages from large volumes of freshwater: evaluation of positively charged, micro
43、porous filters. Journal of Virological Methods, 1, 1980, pp. 87-97 4 SOBSEY, M.D., SCHWAB, K.J. and HANDZEL, T.R. Journal of the American Waterworks Association, 82, 1990, pp. 52-59 5 SCHULZE, E. and LENK, J. Naturwissenschaften, 70, 1983, p. S.612 3) In preparation. Copyright International Organiza
44、tion for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 10705-3:2003(E) ICS 07.100.20 Price based on 13 pages ISO 2003 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 04:00:05 MDTNo reproduction or networking permitted without license from IHS -,-,-