1、 Reference number ISO 16240:2005(E) ISO 2005INTERNATIONAL STANDARD ISO 16240 First edition 2005-04-01 Water quality Determination of the genotoxicity of water and waste water Salmonella/microsome test (Ames test) Qualit de leau Dtermination de la gnotoxicit des eaux et des eaux rsiduaires Essai de S
2、almonella/microsome (essai dAmes) ISO 16240:2005(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 compute
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5、ntral Secretariat at the address given below. ISO 2005 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
6、 at 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 2005 All rights reservedISO 16240:2005(E) ISO 2005 Al
7、l rights reserved iiiContents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Interferences 3 5 Principle . 4 6 Reagents and media . 4 7 Apparatus. 9 8 Procedure. 10 8.1 Pouring and labelling of plates 10 8.2 Test samples and their preparation 10 8.3
8、 Number of test samples . 11 8.4 Preparation 12 8.4.1 On the day prior to the test:. 12 8.4.2 On the day of testing only:. 12 8.5 Study conduct . 12 8.5.1 General. 12 8.5.2 Mutagenicity study 12 8.6 Titre determination 13 9 Colony-counting, evaluation and assessment. 13 9.1 Colony-counting 13 9.2 Ev
9、aluation 13 9.3 Assessment criteria 13 9.4 Determination of the decisive D min value. 13 10 Validity criteria 14 11 Test report 14 Annex A (normative) Nutrient broth and agar . 15 Annex B (normative) S9 fraction. 16 Annex C (normative) Verification of genotype 17 Annex D (informative) Examples of re
10、sults obtained . 18 Bibliography . 20 ISO 16240:2005(E) iv ISO 2005 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out t
11、hrough 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. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO colla
12、borates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Sta
13、ndards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements o
14、f this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16240 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. ISO 16240:2005(E) ISO 2005 All rights reserved vIntrod
15、uction It should be decided on a case-by-case basis whether, and to what extent, additional instructions may be necessary for the application of this International Standard. INTERNATIONAL STANDARD ISO 16240:2005(E) ISO 2005 All rights reserved 1Water quality Determination of the genotoxicity of wate
16、r and waste water Salmonella/microsome test (Ames test) WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility
17、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 trained staff. 1 Scope This International S
18、tandard specifies a method for the determination of the genotoxic potential of water and wastewater using the bacterial strains Salmonella typhimurium TA 100 and TA 98. This method includes sterile filtration of water and wastewater prior to the test. This International Standard is applicable only t
19、o the detection of genotoxic substances which are in the filtered aqueous phase. It is not applicable to the detection of genotoxic substances adsorbed by the retained particles. 2 Normative references The following referenced documents are indispensable for the application of this document. For dat
20、ed references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 5667-1, Water quality Sampling Part 1: Guidance on the design of sampling programmes ISO 5667-2, Water quality Sampling Part 2: Guidance on sam
21、pling techniques ISO 5667-3, Water quality Sampling Part 3: Guidance on the preservation and handling of water samples ISO 5667-14, Water quality Sampling Part 14: Guidance on quality assurance of environmental water sampling and handling ISO 5667-16, Water quality Sampling Part 16: Guidance on biot
22、esting of samples 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 number of revertants number of mutants number of visible mutant colonies per plate at the termination of the test ISO 16240:2005(E) 2 ISO 2005 All rights reserved3.2 dilution l
23、evel D denominator of the dilution coefficient (using the numerator 1) of a mixture of water or wastewater with dilution water (3.16) as integral number NOTE For undiluted water or wastewater, the dilution coefficient is by definition 1:1. The corresponding and smallest possible D value is 1. 3.3 do
24、se-response relationship reduction of the number of visible mutant colonies per plate with increasing D level 3.4 D minvalue smallest value of D at which, under the conditions of this International Standard, no positive increase in the number of visible mutant colonies per plate is detected NOTE In
25、the case of more than one D minvalue (a maximum of four are possible), the highest D value is decisive. 3.5 stock culture frozen culture for the preservation of the characteristics (e.g. genotype) of Salmonella typhimurium TA 100 and TA 98 3.6 inoculum part of a thawed stock culture used to inoculat
26、e culture medium 3.7 culture medium aqueous solution of nutrients which are required for the cultivation of the bacteria 3.8 overnight culture mixture of inoculum and culture medium, incubated for about 18 h at 37 C 1 C and gentle agitation (e.g. shaken at 100 r/min to 150 r/min) 3.9 plate solidifie
27、d mixture of water, agar and other possible constituents (e.g. inorganic salts) in Petri dishes 3.10 softagar mixture of agar, sodium chloride, histidine, biotin and water NOTE Minimal softagar contains only traces of histidine and is used for the determination of mutants. Maximal softagar contains
28、histidine in excess and is used for the determination of titres. 3.11 S9 fraction 9 000 g supernatant of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to 300 g) pretreated with an appropriate substance or substance combination for enzyme induction 3.12 cofactor solu
29、tion aqueous solution of chemicals needed for the activity of the enzymes in the S9 fraction NOTE Examples of chemicals needed are NADP, glucose-6-phosphate and inorganic salts. ISO 16240:2005(E) ISO 2005 All rights reserved 33.13 S9 mix mixture of S9 fraction and cofactor solution 3.14 titre determ
30、ination method for the determination of the number of bacteria (colony-forming units) in an overnight culture and for the determination of possible bacteriotoxic effects of the test sample 3.15 test sample sample to be used as test item after all preparative steps (e.g. sterile filtration) have been
31、 carried out 3.16 dilution water sterile water of a conductivity of u 5 S/cm used for the stepwise dilution of the test sample or used as negative control 3.17 negative control dilution water (3.16) without test sample 3.18 positive control known mutagen used to verify the sensitivity of the method
32、or the activity of the S9 mix NOTE The positive controls are dissolved in DMSO prior to use. 3.19 test mixture mixture of test sample pure or diluted with dilution water (3.16), negative or positive control, bacterial suspension, softagar and S9 mix or buffer 3.20 induction rate I difference between
33、 the mean value of mutant colonies counted on the plates treated with a dose of the test sample or with a positive control and the mean value of the corresponding plates treated with the negative control using the same strain under identical activation conditions 3.21 background growth bacterial law
34、n formed by microcolonies of non-mutated bacteria on a plate with minimal softagar due to the traces of histidine contained in this softagar 4 Interferences A strong bacteriotoxic effect of the test sample can lead to a reduction of viable bacteria and to a reduction of mutant colonies compared to t
35、he corresponding negative control counts. In an extreme case of bacteriotoxicity, the number of surviving bacteria may be reduced to such an extent (to several hundred) that the traces of histidine in the minimal softagar are sufficient to allow these bacteria to grow up to visible colonies mimickin
36、g the growth of mutant colonies. This may lead to false positive results. ISO 16240:2005(E) 4 ISO 2005 All rights reserved5 Principle The bacteria are exposed under defined conditions to various doses of the test sample and incubated for 48 h to 72 h at 37 C 1 C. Due to this exposure, genotoxic agen
37、ts contained in the test water or wastewater may be able to induce mutations in one or both marker genes (hisG46 for TA 100 and hisD3052 for TA 98) in correlation to the used doses. Such induction of mutations causes a dose-related increase in the numbers of mutant colonies. The possible mutagenic a
38、ctivity of the test sample is detected by comparing, for the used bacterial strain and the respective activation condition S9 mix (3.13); Annex B), the number of mutant colonies on plates treated with the negative control with those treated with undiluted and diluted test sample. The lowest dilution
39、 (1:N) of the test sample inducing, according to the criteria of this International Standard, no mutagenic effect under all experimental conditions (if any mutagenic effect is induced by the test sample) is the parameter relevant for the assessment of the test sample according to this International
40、Standard. Dilutions above this (1:A, AN) shall induce a mutagenic effect according to the criteria of this International Standard in at least one strain under at least one activation condition. The respective D min -value is N. If no mutagenic effect is observed under all experimental conditions, th
41、is dilution is 1:1 and the respective D min - value is 1. The test facility is qualified for the conduct of this International Standard if the Salmonella/microsome test is established in this facility according to the following criteria: several independent experiments are performed; several known m
42、utagenic and non-mutagenic reagents are tested; the mutagenic compounds are included in the positive controls of this International Standard (6.18); the results are reproducible; the results are in compliance with literature data. 6 Reagents and media As far as possible, use chemicals of reagent gra
43、de. Prepare all aqueous solutions with water of a conductivity of u 5 S/cm. If chemicals with different amounts of crystallisation water are used, recalculate the needed amounts. Always autoclave for 20 min at 121 C 2 C. Seal vessels loosely (e.g. with aluminium foil). Sealing should never be air-ti
44、ght. All compositions are given for specific final amounts. Other final amounts (N-fold) may be reached by multiplying the amounts all single components of the respective composition by N. Compositions may be subdivided under appropriate conditions into appropriate amounts. 6.1 Hydrochloric acid, c(
45、HCl) = 1 mol/l. 6.2 Sodium hydroxide solution, c(NaOH) = 1 mol/l. 6.3 Dimethyl sulfoxide (DMSO), C 2 H 6 O 4 S. ISO 16240:2005(E) ISO 2005 All rights reserved 56.4 Nutrient broth For each 1 l of water, add 3 g of beef extract, 5 g of peptone and 5 g of sodium chloride (or alternatively, 10 g of beef
46、 extract, 10 g of peptone and 5 g of sodium chloride). Warm up and stir to dissolve the compounds. Adjust the pH to 7,4 0,2 and autoclave in appropriate portions. Store under sterile conditions at 4 C 2 C for not longer than one month. For use of commercial products, see A.1. 6.5 Ampicillin solution
47、 Under sterile conditions at room temperature, dissolve 80 mg of ampicillin in 10 ml of sterile sodium hydroxide solution (0,02 mol/l). Use immediately. 6.6 Nutrient broth with ampicillin Under sterile conditions, add 3,15 ml of ampicillin solution (6.5) to 1 l of nutrient broth (6.4). Store under s
48、terile conditions at 4 C 2 C for not longer than one week. 6.7 Sodium hydrogen phosphate buffer The following solutions are needed to prepare the buffer: solution 1: 13,8 g of sodium dihydrogen phosphate monohydrate (NaH 2 PO 4 H 2 O) dissolved in 1 l water; solution 2: 14,2 g of disodium hydrogen p
49、hosphate (Na 2 HPO 4 ) dissolved in 1 l water. Stir solution 2 (e.g. with a magnetic stirrer) and add solution 1 until a pH of 7,4 is reached and remains stable. Subdivide this solution in appropriate amounts and autoclave to sterilize. Store at 4 C 2 C for not longer than one month. 6.8 Cofactor solution Dissolve the following compounds, in the amounts given, in 70 ml of sodium hydrogen phosphate buffer (6.7): 162,6 mg of magnesium chloride hexahydrate (Mg
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