CN101255456A - Toxicology neasuring evaluation method of cigarette smoke mutagenicity - Google Patents
Toxicology neasuring evaluation method of cigarette smoke mutagenicity Download PDFInfo
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- CN101255456A CN101255456A CNA2008100494519A CN200810049451A CN101255456A CN 101255456 A CN101255456 A CN 101255456A CN A2008100494519 A CNA2008100494519 A CN A2008100494519A CN 200810049451 A CN200810049451 A CN 200810049451A CN 101255456 A CN101255456 A CN 101255456A
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Abstract
The invention relates to a toxicology evaluation method of measuring for cigarette smoke mutagenicity, characterized in: the method comprises the steps of: (1) selecting experimental bacterial strain; (2) preparing the needed material for the experiment; (3) preparing cigarette smoke condensate CSC; (4) inoculating and cultivating cells; and (5) observing the result and judging. Compared with traditional AMES experiment method, the method is easy to operate, highly sensitive, and more reliable.
Description
Technical field
The invention belongs to the toxicology neasuring evaluation method in cigarette smoke toxicologic study field, particularly a kind of cigarette smoke mutagenicity.
Background technology
In recent years, in the world a large amount of research work have been carried out in the hazardness evaluation of tobacco product and cigarette additive.
1999 FDA (FDA (Food and Drug Adminstration)) require IOM (Institute for Medical Research of American Academy of Sciences) to formulate a cover scientific methods, be used to assess the security and the effect of life-time service medicament production or tobacco substitute product (being used for reducing rather than eliminating tobacco harm).According to present result of study, the biomarker of experiencing tobacco product is verified and uses.But the early stage index of biomarker almost is not verified being used to predict on the disease progression in later stage, and present existing knowledge still is not enough to support to carry out formal " low harm " cigarette product assessment.The in vitro toxicity test is not the direct mensuration of harm, but can guarantee that tobacco product can not increase potential harm, and toxicology data can be supported the evaluation of " low harm " cigarette product to a certain extent.
CORESTA (international tobacco scientific research cooperation center) has also set up relevant working group's (smoke of tobacco in vitro toxicity test job group), its main task is in vitro toxicity test philosophy and the correct according to international endorsement, with character and the unique property that adapts to smoke of tobacco, determine main method.According to result of study, for the bacterial mutagenesis analysis, working group recommends the mutagenesis of em Salmonellas to analyze (Ames Test, Salmonella reversion test).
From the international and domestic flue gas toxicologic study work of carrying out, Salmonella reversion test has become the toxicological evaluation method of the universally recognized cigarette smoke mutagenicity of international tobacco circle.
The mutant of Salmonella typhimurium (Salmonella typhimurium) (being the histidine defect type) bacterial strain can not be grown on the substratum of no Histidine, in that have can normal growth on the substratum of Histidine.But as when in the substratum of no Histidine, having mutagen to exist, then but the reverse mutation of mutant salmonella type is wild-type (phenotype), thereby on no Histidine is cultivated, also can grow, so can form quantity, check whether tried thing is mutagen according to bacterium colony.Different histidine defect type Salmonella typhimuriums can detect different mutation types.TA97, TA98, TA100 and four kinds of bacterial strains of TA102 are adopted in general AMES experiment, and (wherein TA97 and TA98 can detect various frame shift type mutagenic compound, TA100 can detect the mutagenic compound that cause base pair replacement, TA102 can detect some mutagenic compound that other test strain can not detect or seldom detect), need two kinds of bacterial strains (a kind of and TA100 among TA97, the TA98) at least.Some mutagen needs just can make the mutant salmonella type produce reverse mutation behind the metabolism activation, and the metabolism activation system can use the S-9 mixed solution of polychlorobiphenyl (PCB) inductive rat liver homogenate preparation.
Based on the ultimate principle of above-mentioned Salmonella reversion test, the invention provides a kind of toxicology novel method that is used for the evaluating cigarette flue gas mutagenicity---micro fluctuation laboratory method (Microtitre fluctuation test).
Traditional Salmonella reversion test method such as flat board mix method, some examination method etc., tests loaded down with trivial detailsly, adopt semi-solid nutrient solution, and required reagent is many, makes the ware complexity, and needs artificial counting plate colony number, and workload is very big.
Summary of the invention
Purpose of the present invention just is being based on the situation of above-mentioned prior art and the toxicology neasuring evaluation method of a kind of cigarette smoke mutagenicity of providing, this method is applied to the evaluating cigarette flue gas mutagenicity with Salmonella reversion test, it is more convenient that the Salmonella reversion test of evaluating cigarette flue gas mutagenicity is operated, and experiment susceptibility is higher, the result is more reliable.
The objective of the invention is to be achieved through the following technical solutions: the toxicology neasuring evaluation method of cigarette smoke mutagenicity of the present invention may further comprise the steps:
(1) selects experimental strain, adopt two strains to meet the Salmonella typhimurium saltant TA98 and the TAI00 of Salmonella reversion test standard;
(2) preparing experiment material therefor comprises the minimum medium, phosphate buffered saline buffer, activation system S-9 mixed solution, the Vogel-Bonner damping fluid stock solution that are used for cell cultures;
(3) preparation cigarette smoke agglutinator CSC passes through organic phase (ethyl acetate) respectively with flue gas and collects with inorganic (basic culture solution) mutually, after the organic solvent volatilization, mixes with inorganic nutrient solution, and with cell culture fluid concentration is adjusted into 1 cigarette/ml;
(4) inoculation, cultivation cell, TA98 and TA100 are inoculated in the nutrient broth medium, in 37 ℃ of vibrations (100 times/min) cultivate 10h, what every hole added that 50 μ l contain 500 bacteriums in 96 orifice plates is tried the bacterium mixture, and add each concentration and tried thing, respectively tried thing and adopted 0.0025 respectively, 0.005,0.01 and 0.02 cigarette/ml concentration is tested, 48 holes of every kind of bacterium inoculation of every kind of concentration, hatch the substratum 150 μ L that bring Selection In behind the 16h for 37 ℃, observe after continuing to cultivate 72h, to become yellow by purple positive for culture in the hole in;
(5) the positive hole count of each concentration of every kind of CSC of every kind of bacterium is counted in observations and judgement, is X-coordinate with concentration, the logarithm of positive hole count is the ordinate zou mapping, adopt straight-line regression fit equation respectively, calculate 50% sudden change dosage, as the index of weighing CSC mutagenesis ability.
In the present invention, the described bacterium mixture that tried comprises 1 * Vogel-Bonner salt buffer, 16g/L glucose, 0.002g/L Histidine, 0.0003g/L vitamin H, 0.024mol/L phosphate buffered saline buffer, 0.0066mol/L Repone K, 0.0016mol/L magnesium chloride, 0.001mol/L G-6-P, 0.0008mol/L coenzyme II, 2% liver S-9 liquid.Described selection substratum comprises 1 * Vogel-Bonner salt buffer, 8g/L glucose, 0.0067g/L purpurum bromocresolis.
Being prepared as follows of the various experiment materials that the present invention is used:
(1) minimum medium: extractum carnis 5.0g, tryptone (or mixed protein peptone) 10.0g, sodium-chlor 5.0g adds distilled water 1000ml, and heating for dissolving is transferred pH to 7.4, autoclaving after the packing ,-20 ℃ of preservations standby (preservation period is no more than half a year)
(2) the 0.2mol phosphate buffered saline buffer (PBS, pH7.4): take by weighing 63.03g Na
2HPO
412H
20,3.74g NaH
2PO
42H
20, be dissolved in the 1000ml deionized water, transfer pH to 7.4, autoclaving or filtration sterilization.
(3) 10%S-9 mixed solution (activation system): every 10ml contains aseptic 0.2mol/L phosphate buffered saline buffer (pH6.4) 6.0ml, 1.65mol/L Klorvess Liquid 0.2ml, 0.4mol/L magnesium chloride solution 0.2ml, 0.05mol/L G-6-P salts solution 1.0ml, 0.025mol/L coenzyme-II solution 1.6ml and rat liver homogenate liquid (S-9) 1.0ml, mixing (preparation of prison time spent is put in the ice bath stand-by).Wherein the preparation method of S-9 is as follows: healthy male adult (5-6 age in week) SD or Wistar rat, and about body weight 150g.Polychlorobiphenyl (Aroclor 1254 or homemade PCB-pentachloro-) is dissolved in the Semen Maydis oil, concentration 200g/L, press 500mg/kg BW abdominal injection (aseptic technique) 1 time, sacrificed by decapitation animal behind the 5d, get liver, the active oxyphorase of inhibition microsomal enzyme that deenergizes (is removed) for several times with fresh ice-cold 0.15mol/L Klorvess Liquid flushing liver in the back of weighing.Every gram liver (weight in wet base) adds 0.1mo l/L Klorvess Liquid 3ml, shreds liver with the sterilization scissors in the ice bath, glass homogenizer (be lower than 4000r/min, 1-2min) or tissue homogenizer (20000r/min makes liver homogenate in 1min).Low temperature (0-4 ℃) high speed centrifugation (9000g 10min) is got supernatant liquor (S-9 component) and is sub-packed in aseptic freeze pipe or the small jar, about every bottle of 2ml, with liquid nitrogen or dry ice quick-frozen postposition-80 ℃ cryopreservation (preservation period is no more than 1 year).
(4) nutrient broth agar substratum: agar powder 1.5g adds in the 100ml nutrient broth medium, and heating and melting is transferred the sterilization of pH (6.4) back.Identify as the Viola crystallina sensitization test of genotype identification, anti-penbritin and tsiklomitsin test, sensitivity to ultraviolet light test and bacterium vigor.
(5) bottom substratum: under the aseptic condition, (80 ℃) while hot, add aseptic Vogel-Bonner stock solution 2ml and 40% glucose solution 5ml successively in the 1.5% agar powder solution (distilled water) of every 100ml sterilization, abundant mixing, pour plate immediately into, every ware (diameter 90mm) 25ml, 37 ℃ of overnight incubation have pollution-free to remove moisture and inspection.
(6) Vogel-Bonner damping fluid stock solution (50 times): microcosmic salt (NaNH
4HPO
44H
2O) 16.5g, citric acid (C
6H
8O
7H
2O) 10.0g, dipotassium hydrogen phosphate (K
2HPO
4) 50.0g, sal epsom (MgSO
47H
2O) 1.0g adds and steams pomegranate water to 100ml, and mattress goes out after the dissolving.(annotate: treat again sal epsom slowly to be put into after other reagent dissolve fully and wherein continue dissolving, otherwise easily separate out precipitation.)
(7) top layer substratum: add 10ml 0.5mol/L Histidine (molecular weight 155)-vitamin H (molecular weight 244) solution in the 0.6% agar powder solution (0.5% sodium chloride solution) that every 100ml melts.Mixing, the sterilization of packing (100ml triangular flask) back.Time spent is melted the packing small test tube, and every pipe 2ml is incubated in 45 ℃ of water-baths.
(8) Histidine vitamin H flat board (Histidine need be tested usefulness): contain bottom substratum 914ml, phosphoric acid salt stock solution 20ml, the 40% glucose solution 50ml of sterilization, 0.4% Histidine aqueous solution 10ml and 0.5mmol/LD-biotin solution 6ml among every 1000ml.
(9) dilution of bacteria: after increasing bacterium culture counting, be diluted to 1 * 10 with trying inoculum
6Individual/ml.
(10) tried inoculum: contain glucose 16mg/ml in the Vogel-Bonner damping fluid (1 times), Histidine 2 μ g/ml vitamin Hs 0.3 μ g/ml, S-9 mixture 50 μ L.
(11) select substratum: contain glucose 16mg/ml in the Vogel-Bonner damping fluid (1 times), purpurum bromocresolis (BCP) 6.67 μ g/ml
Method provided by the invention has more convenient operation, higher, the more reliable advantage of result of susceptibility with respect to traditional AMES experimental technique.
Description of drawings
Accompanying drawing is a schema of the present invention.
Embodiment
The present invention is further described below in conjunction with dissimilar cigarette (example):
Example one
Domestic a certain Virginian-type cigarette is estimated.
Cigarette smoke passes through organic phase (ethyl acetate) respectively and collects with inorganic (basic culture solution) mutually.After the organic solvent volatilization, mix with nutrient solution, and concentration is adjusted into 1 cigarette/ml with cell culture fluid.-80 ℃ of preservations.
TA98 and TA100 are inoculated in the 5mL nutrient broth medium after identifying, (100 times/min) cultivate 10h ,-4 ℃ store for future use in 37 ℃ of vibrations.
Culturing bacterium adopts spectrophotometer to measure OD value and calculating concentration under the 600nm wavelength.What every hole added that 50 μ l contain 500 bacteriums in 96 orifice plates is tried bacterium mixture (1 * Vogel-Bonner salt buffer, 16g/L glucose, 0.002g/L Histidine, 0.0003g/L vitamin H, 0.024mol/L phosphate buffered saline buffer, 0.0066mol/L Repone K, 0.0016mol/L magnesium chloride, 0.001mol/L G-6-P, 0.0008mol/L is coenzyme II, and 2% liver S-9 liquid and respective concentration are tried thing).Respectively tried thing and adopted 0.0025 respectively, 0.005,0.01 and 0.02 cigarette/ml concentration is tested, 48 holes of every kind of bacterium inoculation of every kind of concentration, hatch the substratum 150 μ L (1 * Vogel-Bonner salt buffer, 8g/L glucose, 0.0067g/L purpurum bromocresolis) that bring Selection In behind the 16h for 37 ℃, observe after continuing to cultivate 72h, culture is become yellow positive in the hole by purple.
Counting the positive hole count of each concentration of every kind of CSC of every kind of bacterium, is X-coordinate with concentration, and the logarithm of positive hole count is the ordinate zou mapping, adopts straight-line regression fit equation respectively, calculates 50% sudden change dosage.
Bacterium half mutagenesis dosage calculation result is: 0.036cigs/ml.
Example two
Domestic a certain blended type cigarette is estimated.
Cigarette smoke passes through organic phase (ethyl acetate) respectively and collects with inorganic (basic culture solution) mutually.After the organic solvent volatilization, mix with nutrient solution, and concentration is adjusted into 1 cigarette/ml with cell culture fluid.-80 ℃ of preservations.
TA98 and TA100 are inoculated in the 5mL nutrient broth medium after identifying, (100 times/min) cultivate 10h ,-4 ℃ store for future use in 37 ℃ of vibrations.
Culturing bacterium adopts spectrophotometer to measure OD value and calculating concentration under the 600nm wavelength.What every hole added that 50 μ l contain 500 bacteriums in 96 orifice plates is tried bacterium mixture (1 * Vogel-Bonner salt buffer, 16g/L glucose, 0.002g/L Histidine, 0.0003g/L vitamin H, 0.024mol/L phosphate buffered saline buffer, 0.0066mol/L Repone K, 0.0016mol/L magnesium chloride, 0.001mol/L G-6-P, 0.0008mol/L is coenzyme II, and 2% liver S-9 liquid and respective concentration are tried thing).Respectively tried thing and adopted 0.0025 respectively, 0.005,0.01 and 0.02 cigarette/ml concentration is tested, 48 holes of every kind of bacterium inoculation of every kind of concentration, hatch the substratum 150 μ L (1 * Vogel-Bonner salt buffer, 8g/L glucose, 0.0067g/L purpurum bromocresolis) that bring Selection In behind the 16h for 37 ℃, observe after continuing to cultivate 72h, culture is become yellow positive in the hole by purple.
Counting the positive hole count of each concentration of every kind of CSC of every kind of bacterium, is X-coordinate with concentration, and the logarithm of positive hole count is the ordinate zou mapping, adopts straight-line regression fit equation respectively, calculates 50% sudden change dosage.
Bacterium half mutagenesis dosage calculation result is: 0.034cigs/ml.
Example three
External a certain cigarette is estimated.
Cigarette smoke passes through organic phase (ethyl acetate) respectively and collects with inorganic (basic culture solution) mutually.After the organic solvent volatilization, mix with nutrient solution, and concentration is adjusted into 1 cigarette/ml with cell culture fluid.-80 ℃ of preservations.
TA98 and TA100 are inoculated in the 5mL nutrient broth medium after identifying, (100 times/min) cultivate 10h ,-4 ℃ store for future use in 37 ℃ of vibrations.
Culturing bacterium adopts spectrophotometer to measure OD value and calculating concentration under the 600nm wavelength.What every hole added that 50 μ l contain 500 bacteriums in 96 orifice plates is tried bacterium mixture (1 * Vogel-Bonner salt buffer, 16g/L glucose, 0.002g/L Histidine, 0.0003g/L vitamin H, 0.024mol/L phosphate buffered saline buffer, 0.0066mol/L Repone K, 0.0016mol/L magnesium chloride, 0.001mol/L G-6-P, 0.0008mol/L is coenzyme II, and 2% liver S-9 liquid and respective concentration are tried thing).Respectively tried thing and adopted 0.0025 respectively, 0.005,0.01 and 0.02 cigarette/ml concentration is tested, 48 holes of every kind of bacterium inoculation of every kind of concentration, hatch the substratum 150 μ L (1 * Vogel-Bonner salt buffer, 8g/L glucose, 0.0067g/L purpurum bromocresolis) that bring Selection In behind the 16h for 37 ℃, observe after continuing to cultivate 72h, culture is become yellow positive in the hole by purple.
Counting the positive hole count of each concentration of every kind of CSC of every kind of bacterium, is X-coordinate with concentration, and the logarithm of positive hole count is the ordinate zou mapping, adopts straight-line regression fit equation respectively, calculates 50% sudden change dosage.
Bacterium half mutagenesis dosage calculation result is: 0.042cigs/ml.
Claims (3)
1, a kind of toxicology neasuring evaluation method of cigarette smoke mutagenicity, it is characterized in that: it may further comprise the steps:
(1) selects experimental strain, adopt two strains to meet the Salmonella typhimurium saltant TA98 and the TAI00 of Salmonella reversion test standard;
(2) preparing experiment material therefor comprises the minimum medium, phosphate buffered saline buffer, activation system S-9 mixed solution, the Vogel-Bonner damping fluid stock solution that are used for cell cultures;
(3) preparation cigarette smoke agglutinator CSC passes through organic phase (ethyl acetate) respectively with flue gas and collects with inorganic (basic culture solution) mutually, after the organic solvent volatilization, mixes with inorganic nutrient solution, and with cell culture fluid concentration is adjusted into 1 cigarette/ml;
(4) inoculation, cultivation cell, TA98 and TA100 are inoculated in the nutrient broth medium, in 37 ℃ of vibrations (100 times/min) cultivate 10h, what every hole added that 50 μ l contain 500 bacteriums in 96 orifice plates is tried the bacterium mixture, and add each concentration and tried thing, respectively tried thing and adopted 0.0025 respectively, 0.005,0.01 and 0.02 cigarette/ml concentration is tested, 48 holes of every kind of bacterium inoculation of every kind of concentration, hatch the substratum 150 μ L that bring Selection In behind the 16h for 37 ℃, observe after continuing to cultivate 72h, to become yellow by purple positive for culture in the hole in;
(5) the positive hole count of each concentration of every kind of CSC of every kind of bacterium is counted in observations and judgement, is X-coordinate with concentration, the logarithm of positive hole count is the ordinate zou mapping, adopt straight-line regression fit equation respectively, calculate 50% sudden change dosage, as the index of weighing CSC mutagenesis ability.
2, the toxicology neasuring evaluation method of cigarette smoke mutagenicity according to claim 1, it is characterized in that: the described bacterium mixture that tried comprises 1 * Vogel-Bonner salt buffer, 16g/L glucose, 0.002g/L Histidine, 0.0003g/L vitamin H, 0.024mol/L phosphate buffered saline buffer, 0.0066mol/L Repone K, 0.0016mol/L magnesium chloride, 0.001mol/L G-6-P, 0.0008mol/L coenzyme II, 2% liver S-9 liquid.
3, the toxicology neasuring evaluation method of cigarette smoke mutagenicity according to claim 1 is characterized in that: described selection substratum comprises 1 * Vogel-Bonner salt buffer, 8g/L glucose, 0.0067g/L purpurum bromocresolis.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101825528A (en) * | 2010-04-23 | 2010-09-08 | 云南烟草科学研究院 | Three-stage trapping method of cigarette mainstream smoke for smoke toxicity detection |
| CN102220405A (en) * | 2011-04-22 | 2011-10-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for detecting lung injuries caused by smoke gas of tobacco and application thereof |
| CN102321565A (en) * | 2011-09-06 | 2012-01-18 | 广东省微生物研究所 | Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof |
| CN101693915B (en) * | 2009-07-27 | 2012-03-14 | 浙江中烟工业有限责任公司 | Method for determining and evaluating cytotoxicity caused by cigarette smoke |
| CN102586385A (en) * | 2012-01-20 | 2012-07-18 | 云南烟草科学研究院 | Gas-liquid contact type cigarette bacterial reverse mutation assay method under full smoke gas exposure |
| CN103018431A (en) * | 2012-12-05 | 2013-04-03 | 陕西中烟工业有限责任公司 | Toxicology biological assay method for cigarette smoke condensate |
| 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 |
| CN110656154A (en) * | 2019-11-28 | 2020-01-07 | 江西中烟工业有限责任公司 | Mutation-causing detection method for urine after rat smoke inhalation exposure |
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2008
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| CN101693915B (en) * | 2009-07-27 | 2012-03-14 | 浙江中烟工业有限责任公司 | Method for determining and evaluating cytotoxicity caused by cigarette smoke |
| CN101825528A (en) * | 2010-04-23 | 2010-09-08 | 云南烟草科学研究院 | Three-stage trapping method of cigarette mainstream smoke for smoke toxicity detection |
| CN102220405A (en) * | 2011-04-22 | 2011-10-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for detecting lung injuries caused by smoke gas of tobacco and application thereof |
| CN102321565A (en) * | 2011-09-06 | 2012-01-18 | 广东省微生物研究所 | Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof |
| CN102321565B (en) * | 2011-09-06 | 2013-04-24 | 广东省微生物研究所 | Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof |
| CN102586385A (en) * | 2012-01-20 | 2012-07-18 | 云南烟草科学研究院 | Gas-liquid contact type cigarette bacterial reverse mutation assay method under full smoke gas exposure |
| CN103018431A (en) * | 2012-12-05 | 2013-04-03 | 陕西中烟工业有限责任公司 | Toxicology biological assay method for cigarette smoke condensate |
| 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 |
| CN110656154A (en) * | 2019-11-28 | 2020-01-07 | 江西中烟工业有限责任公司 | Mutation-causing detection method for urine after rat smoke inhalation exposure |
| CN110656154B (en) * | 2019-11-28 | 2022-11-25 | 江西中烟工业有限责任公司 | Mutation-causing detection method for urine after rat smoke inhalation exposure |
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