CN101671735A - Improved cigarette smoke condensate Ames test - Google Patents
Improved cigarette smoke condensate Ames test Download PDFInfo
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- CN101671735A CN101671735A CN200910172253A CN200910172253A CN101671735A CN 101671735 A CN101671735 A CN 101671735A CN 200910172253 A CN200910172253 A CN 200910172253A CN 200910172253 A CN200910172253 A CN 200910172253A CN 101671735 A CN101671735 A CN 101671735A
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- cigarette smoke
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- bacterium
- positive control
- smoke condensate
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Abstract
The invention relates to an improved cigarette smoke condensate Ames test, which is characterized by comprising the following test steps: firstly, preparation of flat plates and bacteria; secondly, preparation of top agar and object to be tested; thirdly, pre-culture of the object to be tested and penetration of the flat plates; and fourthly, germ cluster counting and data statistics. The improvedcigarette smoke condensate Ames test has the advantages that two bacterial strains TA98 and TA100 sensitive to cigarette smoke are selected as the bacterial strains to be tested, and 5% S9 mixed liquor is selected as an activating system. In addition, as the cigarette smoke contains a plurality of types of materials, different from the test method of pure material, the improved cigarette smoke condensate Ames test adds a step of pre-culture of cigarette smoke condensate and bacteria, and improves the sensitivity of certain compound; the test data is more accurate; and the improved cigarette smoke condensate Ames test is more applicable to the complicated detection of cigarette smoke gene mutation.
Description
Technical field
The present invention relates to a kind of cigarette smoke mutagenicity test, particularly a kind of improved cigarette smoke condensate Ames test method.
Background technology
Chemical pollutant causes people's common concern to the potential hazard of human body.Developed over one hundred kind of short-term fast test method in the world, the genetoxic effect of detection of contamination.Year effort surplus ten such as B.N.Ames, setting up not in 1975, the perfect salmonella reverse mutation assay (also claiming Salmonella reversion test) of broken hair exhibition is widely adopted by countries in the world.This method than faster, easy, responsive, economical, and be applicable to the test mixing thing, reflect the combined effect of multiple pollutent.
Salmonella reversion test is from the most frequently used method of protokaryon level detection transgenation, it is the effective means of a kind of quick acquisition chemical substance mutagenicity and potential carinogenicity data, its principle is that Salmonella typhimurium mutant strain (histidine defect type) can not be grown on the substratum that lacks Histidine, when being tried thing and make it to grow, illustrate that its entrained sudden change is repaired or compensates, answer has promptly taken place be the sudden change of wild-type.So can detect whether tried thing be mutagen according to bacterium colony on the substratum of no Histidine generation quantity.Its concrete grammar is divided into spotting method and dull and stereotyped infiltration method, and spot test is confined to the chemical substance that can spread on agar, and most of polycyclic aromatic hydrocarbonss and the chemical substance that is insoluble in water all are not suitable for using this method.This method susceptibility is relatively poor, mainly is a kind of qualitative test, is applicable to a large amount of test-compounds of rapid screening.But flat board mixes the power of test quantitative test sample mutagenicity.This method is than the spot test sensitivity, and it is lower to obtain the required dosage of positive findings.
The concrete grammar of the dull and stereotyped infiltration method of Salmonella reversion test is with a certain amount of thing dilution different concns gradient of being tried, 0.1mL is tried thing and 0.1ml test bacterium liquid (TA97, TA98, TA100, TA102) mixing adds in the soft agar of upper strata, what need metabolism activation adds 0.5mL10%S9 (rat liver microsomes enzyme) mixed solution again, inclines to rapidly behind the mixing and paves condensation on the base plate.Do feminine gender and positive control simultaneously, every kind of dosage do 3 parallel.Sample is established 4～5 dosage usually.Plate is inverted in 37 ℃ of incubators and cultivates 48h, and each plate of same dosage returns change bacterium colony mean and beams back the ratio that becomes the bacterium colony mean certainly with each negative control ware, for causing no-load voltage ratio (MR).MR value 〉=2, and dose-response relationship is arranged, background is normal, then is judged to the mutagenesis positive
Traditional test method is selected TA97 for use, TA98, and TA100, four kinds of bacterial strains of TA102 are tested, and activation system selects the 10%S9 mixed solution for use, and experiment shows TA98 and TA100
Two kinds of bacterial strains are to the cigarette smoke sensitivity, and 10% and the basically identical as a result of 5%S9 mixed solution activation system performance.
" toxicology neasuring evaluation method of cigarette smoke mutagenicity " patent that this case applicant once applied for, its publication number CN101255456, its step is TA98, TA100 for (1) experimental strain; (2) preparation cigarette smoke agglutinator CSC; (3) preparation substratum, damping fluid and agents useful for same; (4) inoculated bacteria is in 96 orifice plates, contamination, cultivation; (5) observations and judgement.Because cigarette smoke condensate is a complicated chemical objects system, some compound needs just can play one's part to the full with microbial culture in advance, compare with the present invention, CN101255456 " toxicology neasuring evaluation method of cigarette smoke mutagenicity " has ignored this factor.
Summary of the invention
A kind of improved cigarette smoke condensate Ames test method that purpose of the present invention is specialized at existing problem in the above-mentioned prior art just, this method can improve the susceptibility of test, more is applicable to the detection of complicated cigarette smoke transgenation.
The objective of the invention is to be achieved through the following technical solutions: the improved cigarette smoke condensate Ames test of the present invention comprises following testing sequence:
1. the preparation of flat board and bacterium
Behind 1.5% nutrient agar autoclaving, add phosphoric acid salt storing solution and 40% glucose solution, pour the 90mm plate after fully shaking up into, each chemicals dosage carries out parallel with three flat boards, after preparing enough flat boards, put into biochemical incubator, 37 ℃ of overnight incubation have pollution-free to remove moisture and inspection; Increase bacterium work subsequently, get 2 sterilization angle flasks, add nutrient broth 10mL, scrape the TA98 that takes a morsel, TA100 bacterial classification, be seeded in the meat soup; 37 ℃ of shaking culture 10h make bacterium reach 1-2 * 10
9Individual/mL;
2. top-agar and tried thing and prepare
With adding 0.5mmoL/L Histidine-biotin solution behind the agar-agar soln high pressure, be sub-packed in test tube with 2mL then, and in 45 ℃ of water-baths, be incubated; Configuration is tried thing-cigarette smoke condensate and positive control then, and positive control is respectively, and during no activation system: the TA98 positive control is 40 μ g/mL nitrofluorenes, and the TA100 positive control is 10 μ g/mL; When activation system was arranged: TA98, TA100 positive control were 20 μ g/mL anthranylamines; Negative control is a solvent control;
3. tried pre-cultivation of thing and dull and stereotyped the infiltration
Draw 0.1mL and tried thing and 0.1mL bacterium liquid mixing, under 37 ℃ of conditions, cultivate 30min in advance; Need activatory to add the 0.5mL5%S9 mixed solution in addition; Afterwards, get top layer substratum one pipe (2mL) that melts and in 45 ℃ of water-baths, be incubated, add 0.2mL and tried thing bacterium liquid mixed solution, activatory adds 0.7mL and is tried thing, bacterium liquid and S9 mixed solution, mixing is poured on the bottom platform substratum rapidly, and pivotal plate is evenly distributed on the bottom top layer substratum, square curing is cultivated 48h for 37 ℃;
4. counting bacterium colony and data statistics
Count each flat-plate bacterial colony number, and carry out result's statistics, and then the mutagenicity of evaluation of flue gas condensation product.
Characteristics of the present invention are: experimental subjects is selected for use and use bacterial strain, activation system to select 5%S9 mixed solution (because 10% and the basically identical as a result that shows of 5%S9 activation system) for use to the TA98 of cigarette smoke sensitivity and two kinds of bacterial strains of TA100 as test.And because cigarette smoke contains multiple material, be different from the test method of pure substance, the present invention has increased cigarette smoke condensate and the pre-incubated step of bacterium, has improved the susceptibility of some compound, testing data is more accurate, more is applicable to the detection of complicated cigarette smoke transgenation.
Embodiment
The present invention is further described below in conjunction with example:
A certain homemade fire-cured tobacco type brand cigarette is carried out Salmonella reversion test.
At first preparation is dull and stereotyped, behind 1.5% nutrient agar autoclaving, adds phosphoric acid salt storing solution and 40% glucose solution, pours the 90mm plate into after fully shaking up, and 37 ℃ of overnight incubation have pollution-free to remove moisture and inspection; Increase bacterium work subsequently, get 2 sterilization angle flasks, add nutrient broth 10mL, scrape the TA98 that takes a morsel, TA100 bacterial classification, be seeded in the meat soup.37 ℃ of shaking culture 10h make bacterium reach 1-2 * 10
9Individual/mL.
The preparation top-agar with adding 0.5mmoL/L Histidine-biotin solution behind the agar-agar soln high pressure, is sub-packed in test tube with 2mL then, and is incubated in 45 ℃ of water-baths; Preparation is tried thing (cigarette smoke condensate) and positive control then.The concentration of cigarette smoke condensate is respectively: 25,50,100,125,250,500 μ g/mL; Positive control is respectively, and during no activation system: the TA98 positive control is 40 μ g/mL nitrofluorenes, and the TA100 positive control is 10 μ g/mL; When activation system was arranged: TA98, TA100 positive control were 20 μ g/mL anthranylamines.Negative control is a solvent control.
Then, draw 0.1mL and tried thing and 0.1mL bacterium liquid mixing, under 37 ℃ of conditions, cultivate 30min in advance; Need activatory to add the 0.5mL5%S9 mixed solution in addition.Afterwards, get top layer substratum one pipe (2mL) that melts and in 45 ℃ of water-baths, be incubated, add 0.2mL and tried thing bacterium liquid mixed solution, activatory adds 0.7mL and is tried thing, bacterium liquid and S9 mixed solution, mixing is poured on the bottom platform substratum rapidly, and pivotal plate is evenly distributed on the bottom top layer substratum, square curing is cultivated 48h for 37 ℃; Count each dull and stereotyped sudden change colony number that returns.
Claims (1)
1, a kind of improved cigarette smoke condensate Ames test is characterized in that: comprise following testing sequence:
1). the preparation of flat board and bacterium
Behind 1.5% nutrient agar autoclaving, add phosphoric acid salt storing solution and 40% glucose solution, pour the 90mm plate after fully shaking up into, each chemicals dosage carries out parallel with three flat boards, after preparing enough flat boards, put into biochemical incubator, 37 ℃ of overnight incubation have pollution-free to remove moisture and inspection; Increase bacterium work subsequently, get 2 sterilization angle flasks, add nutrient broth 10mL, scrape the TA98 that takes a morsel, TA100 bacterial classification, be seeded in the meat soup; 37 ℃ of shaking culture 10h make bacterium reach 1-2 * 10
9Individual/mL;
2). top-agar and tried thing and prepare
With adding 0.5mmoL/L Histidine-biotin solution behind the agar-agar soln high pressure, be sub-packed in test tube with 2mL then, and in 45 ℃ of water-baths, be incubated; Configuration is tried thing-cigarette smoke condensate and positive control then, and positive control is respectively, and during no activation system: the TA98 positive control is 40 μ g/mL nitrofluorenes, and the TA100 positive control is 10 μ g/mL; When activation system was arranged: TA98, TA100 positive control were 20 μ g/mL anthranylamines; Negative control is a solvent control;
3). tried pre-cultivation of thing and dull and stereotyped the infiltration
Draw 0.1mL and tried thing and 0.1mL bacterium liquid mixing, under 37 ℃ of conditions, cultivate 30min in advance; Need activatory to add the 0.5mL5%S9 mixed solution in addition; Afterwards, get top layer substratum one pipe (2mL) that melts and in 45 ℃ of water-baths, be incubated, add 0.2mL and tried thing bacterium liquid mixed solution, activatory adds 0.7mL and is tried thing, bacterium liquid and S9 mixed solution, mixing is poured on the bottom platform substratum rapidly, and pivotal plate is evenly distributed on the bottom top layer substratum, square curing is cultivated 48h for 37 ℃;
4). counting bacterium colony and data statistics
Count each flat-plate bacterial colony number, and carry out result's statistics, and then the mutagenicity of evaluation of flue gas condensation product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910172253A CN101671735A (en) | 2009-09-24 | 2009-09-24 | Improved cigarette smoke condensate Ames test |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910172253A CN101671735A (en) | 2009-09-24 | 2009-09-24 | Improved cigarette smoke condensate Ames test |
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| CN101671735A true CN101671735A (en) | 2010-03-17 |
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| CN200910172253A Pending CN101671735A (en) | 2009-09-24 | 2009-09-24 | Improved cigarette smoke condensate Ames test |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851658A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Method for detecting mutagenicity of total particulate matter of cigarette smoke |
| CN104212871A (en) * | 2014-09-23 | 2014-12-17 | 云南中烟工业有限责任公司 | Positive control setting method of cigarette filter tip water extract Ames test |
| CN104762365A (en) * | 2015-04-22 | 2015-07-08 | 云南中烟工业有限责任公司 | Detection method for bacterium reverse mutation of buccal tobacco product |
| CN104962604A (en) * | 2015-07-31 | 2015-10-07 | 云南中烟工业有限责任公司 | Method for detecting bacterial reverse mutability of total particulate matters of cigarette smoke |
| CN106281992A (en) * | 2015-05-18 | 2017-01-04 | 北京慧荣和科技有限公司 | Full-automatic Ames test instrument |
| CN106997216A (en) * | 2017-05-19 | 2017-08-01 | 云南中烟工业有限责任公司 | A kind of constant pressure storehouse of cigarette smoking machine and its method for suction experiment |
-
2009
- 2009-09-24 CN CN200910172253A patent/CN101671735A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851658A (en) * | 2010-04-23 | 2010-10-06 | 云南烟草科学研究院 | Method for detecting mutagenicity of total particulate matter of cigarette smoke |
| CN104212871A (en) * | 2014-09-23 | 2014-12-17 | 云南中烟工业有限责任公司 | Positive control setting method of cigarette filter tip water extract Ames test |
| CN104762365A (en) * | 2015-04-22 | 2015-07-08 | 云南中烟工业有限责任公司 | Detection method for bacterium reverse mutation of buccal tobacco product |
| CN104762365B (en) * | 2015-04-22 | 2017-09-22 | 云南中烟工业有限责任公司 | Suck the detection method of tobacco product reverse mutation |
| CN106281992A (en) * | 2015-05-18 | 2017-01-04 | 北京慧荣和科技有限公司 | Full-automatic Ames test instrument |
| CN106281992B (en) * | 2015-05-18 | 2024-02-09 | 北京慧荣和科技有限公司 | Full-automatic Ames experiment instrument |
| CN104962604A (en) * | 2015-07-31 | 2015-10-07 | 云南中烟工业有限责任公司 | Method for detecting bacterial reverse mutability of total particulate matters of cigarette smoke |
| CN104962604B (en) * | 2015-07-31 | 2018-01-30 | 云南中烟工业有限责任公司 | Detection method for cigarette smoke condensates reverse mutation |
| CN106997216A (en) * | 2017-05-19 | 2017-08-01 | 云南中烟工业有限责任公司 | A kind of constant pressure storehouse of cigarette smoking machine and its method for suction experiment |
| CN106997216B (en) * | 2017-05-19 | 2023-06-09 | 云南中烟工业有限责任公司 | Constant-pressure bin for cigarette smoking machine and smoking experiment method thereof |
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Application publication date: 20100317 |