CN104830946A - Technology for detecting TK6 cell chromosome damage genetic toxicity caused by environmental water and application of technology - Google Patents
Technology for detecting TK6 cell chromosome damage genetic toxicity caused by environmental water and application of technology Download PDFInfo
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- CN104830946A CN104830946A CN201510242537.3A CN201510242537A CN104830946A CN 104830946 A CN104830946 A CN 104830946A CN 201510242537 A CN201510242537 A CN 201510242537A CN 104830946 A CN104830946 A CN 104830946A
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Abstract
The invention provides a technology for detecting TK6 cell chromosome damage genetic toxicity caused by environmental water and an application of the technology and belongs to the fields of environmental science and health risk assessment. The technology for detecting the TK6 cell chromosome damage genetic toxicity caused by the environmental water comprises a pre-treatment process on an environmental water sample, a cell culture and contamination method, a micronucleus slice preparation process and a result analysis method. The provided micronucleus experiment method can be directly applied to monitoring of the genetic toxicity of the environmental water, and important genetic toxicity effect information is provided for water pollution health risk management.
Description
Technical field
The present invention relates to a kind of environment water and cause TK6 cell chromosome damage genetoxic high-throughput method for quick, belong to environmental science and health risk assessment field, be detect the genotoxic experimental technique of environmental water sample, utilize TK6 lymphocyte micronuclei experiment testing environment water body to cause detection technique and the application thereof of eukaryotic cell chromosome damage genetoxic size specifically.
Background technology
Environment water complex inheritance Toxicity Monitoring has vital role in Environmental Health comprehensive monitoring, can specify the harm of environmental pollution to population health.The detection of physical and chemical index is often paid close attention in environmental monitoring, pollutant load as concrete in composite target and heavy metals etc. such as COD, then judge environmental situation, but contaminated environment can produce great damaging effect to the health of exposed population group and indefinite actually.Even if each pollutant load meets national standard, but may there is composite toxicity effect in it under common exposure condition, is therefore necessary to measure the toxicity of environmental sample to organism.Genetoxic refers to that the chemical factors in environment acts on organism, its genetic material is sustained damage and the toxic action caused.The damage of genetic material is often closely related with the generation of the various diseases such as cancer, and therefore the genetoxic of testing environment sample can predict its toxicity size to organism.
Existing genetoxic detection method needs to be further improved to be applicable to the monitoring of comprehensive water-body genetoxic.Genotoxic detection method is divided into multiple according to the difference of mechanism, wherein can reflect the damaging action of tested material to the genetic material such as karyomit(e), DNA to detect the experiment that chromosome damage is endpoint detection.Detection method comprises single cell gel electrophoresis technique, chromosomal aberration test and micronucleus test etc.Wherein single cell gel electrophoresis technique is the experimental technique that reaction tested material causes DNA break effect, and laboratory operating procedures is comparatively complicated, is not suitable for the monitoring of extensive sample; Chromosome Aberration Test is the basic skills detecting chemical substance genome quantity and structure, although experimental implementation is comparatively simple, is only suitable for the observation of hyperploid in principle, therefore applies limited; Micronucleus test is the experimental technique detecting chromosome damage, can detect rhexis and chromosome doubling.Micronucleus derives from akinetic chromosome fragment (namely lacking kinetochore) in mitotic division process, or the whole chromosome (spindle body is impaired) at the two poles of the earth cannot be migrated at anaphase of cell division, in the kytoplasm that anaphase of cell division still stays daughter cell, become micronucleus.Micronucleus test operation is comparatively simple, and result accuracy rate is high, compares and is applicable to the monitoring of extensive sample genetoxic.But traditional micronucleus test is subject cell mainly with human peripheral lymphocyte, require to gather peripheral blood that is healthy, grownup, collection crowd requires that the age is lower than 35 one full year of life, interference without smoking, the factor such as to drink, the source of subject cell is required comparatively strict, otherwise may false positive results be caused; Need after blood specimen collection in addition to carry out immediately to cultivate, the process such as contamination, collection in worksite blood sample is all needed before each test, the required sample size of Micronuclei of Human Peripheral Lymphocytes experiment is larger in addition, and there are differences between each blood sample quality gathered, this makes the widespread use of micronucleus test in environmental monitoring be restricted.
The micronucleus test being subject vector with TK6 people lymphocytoblast can meet environment water genetoxic monitoring requirements.For making micronucleus test meet the demand, stable, sensitive, a representative clone is needed to carry out alternative human peripheral and carry out micronucleus test.TK6 people lymphocytoblast is the stable clone built, its source is human lymphoblastoid, stable can carry out passage cultivation, TK6 cell is existing in micronucleus test to be applied comparatively widely, it is stable, highly sensitive that result shows its result, but need to be set up for the specific experiment scheme that environment water genetoxic detects and perfect.
Summary of the invention
For solving the application limitations of existing micronucleus test in environmental sample genetoxic, the present invention has carried out improvement to a certain degree to micronucleus test, human peripheral lymphocyte is replaced by using TK6 people's lymphoblastoid cell line, simplify laboratory operating procedures, reduce the consumption of tested water body, achieve high-throughput rapid detection; Use stable clone to ensure result is more stable, accurately simultaneously, difference when avoiding end user's peripheral blood lymphocyte between different cell derived.The present invention with TK6 people lymphocytoblast for subject cell, cause chromosome damage genetoxic size for testing environment water body example, content comprises the pre-treatment of environment water body example, TK6 cell cultures, cell contamination, micronucleus film-making, the experimentation such as Micronuclei and interpretation of result.
1 water sample pre-treatment
(1) first filter through 0.45 μm of glass fiber filter after environment water body example collection, the enrichment of HLB solid-phase extraction column and acetone wash-out, merging, and the concrete pre-treatment step such as drying;
(2) experiment reagent preparation;
(3) recovery of bacterium and preculture, poisoning dosage set, contaminate after microbial culture and end-result measure;
(4) calculate beta galactose enzymic activity (U), inductivity (R) by the absorption photometric value of sample under 600nm and 420nm, and then judge whether it has genetoxic.
3, method as claimed in claim 2, wherein step (1) is specially:
(1) filter membrane is activated: use the glass fiber filter of 0.45 μm after activation to filter water sample, remove the impurity that in water sample, particle diameter is larger.Activation method is: use retort furnace that the glass fiber filter (GF/A, 70mm, Whatman) of 0.45 μm is activated 3h at 450 DEG C of-500 DEG C of temperature, be placed on more than equilibrium at room temperature 24h in moisture eliminator, use front 1h ultrapure water reactivate after activation;
(2) HLB solid-phase extraction column activation: use the organism etc. in HLB solid-phase extraction column (Waters Oasis, 6mL) enrichment water body, detect for genetoxic.Every root HLB solid-phase extraction column first soaks 5min with 6mL methylene dichloride, then solution is made slowly to flow out, after liquid level is parallel with upper strata filter plate, close upper lock gate, add 6mL methyl alcohol, close upper lock gate again after dichloromethane solution is flowed out completely, add 6mL ultrapure water afterwards and make it slow outflow, in most rear pillar, leave a small amount of ultrapure water;
(3) filter: after treating sediment deposition, then filter.The glass fiber filter of 0.45 μm is put in core filtration unit correspondence position, core filtration unit connects vacuum pump (1500MA) and filters, the Brown Glass Brown glass bottles and jars only preservation that the water sample rinse after filtration is clean;
(4) water sample crosses post: HLB solid-phase extraction column is placed in solid-phase extracting instrument top joint place, solid-phase extraction column top is connected with large discharge sampling thief, is positioned over by the sampling head of sampling thief in the vial of the water sample after filtration is housed; Solid-phase extracting instrument is connected with filter flask and vacuum pump, opens vacuum pump and the careful valve opening solid-phase extracting instrument, regulate the flow of every root pillar, flow rate control at 6 ~ 8mL/min, every root HLB post enrichment 2L water sample.Drain moisture in part post with vacuum pump after enrichment completes, with Nitrogen evaporator, pillar is dried up, can-20 DEG C of preservations if do not use immediately;
(5) sample elution: in stink cupboard, carries out wash-out with 10mL acetone to HLB post, and acetone is naturally flowed down in without external force situation, and elutriant 50mL Brown Glass Brown glass bottles and jars only is collected, and residual acetone rubber suction bulb is blown down.Acetone can be more excessive a little, ensures that sample eluent reaches 10mL;
(6) same sample is merged: merged by the elutriant of same sample, first carrying out nitrogen to acetone eluant blows concentrated, identical water sample is merged to same sample pipe after residue about 200 μ L, by other each effective a small amount of acetone rinsing 3-4 time, be merged in same sample pipe equally, blow to complete drying with Nitrogen evaporator;
(7) the ratio DMSO constant volume sample of 10 μ L concentrated solutions is settled to according to 1L water sample ,-20 DEG C of preservations.
2 cell cultures
(1) cell recovery: by frozen TK6 cell quick-thawing in 37 DEG C of water-baths, the centrifugal 5min of 125xg, discards frozen storing liquid, cell adds the RPMI1640 substratum of 10mL containing 10% foetal calf serum, at 37 DEG C, 5%CO
224h is cultivated in incubator.
(2) passage: carry out cell counting, when cell number reaches 10
6need go down to posterity in time during cells/mL, make cell concn remain at 2 × 10
5~ 1 × 10
6between cells/mL.Within general 2 ~ 3 days, once need go down to posterity, answer strict implement aseptic technique in cell cultures and succeeding generations, prevent cell contaminated.
3 micronucleus test steps
(1) plating cells: by cell to be gone down to posterity by 2 × 10
5cells/mL is taped against in 24 orifice plates, uses the substratum bed board of 10% serum; Every hole adds final concentration is simultaneously that the cytochalasin-B of 6 μ g/mL is to form dikaryocyte;
(2) cell contamination: add certain volume tested material, draws through experiment: the concentration of contamination of groundwater sample is advisable with 0.2-0.5L water sample extract concentrated solution, and surface water concentration of contamination is advisable with 0.02-0.05L extract concentrated solution; Every hole cumulative volume is 1mL, cultivates 24h;
(3) counting before experiment, gets 1 × 10
6individual cell, centrifugal collecting cell (125xg, 5min), abandoning supernatant, adds 1mLPBS damping fluid;
(4) centrifugal abandoning supernatant, continues to add 1mL PBS buffer solution cell;
(5) centrifugal abandoning supernatant, adds the stationary liquid (methyl alcohol: Glacial acetic acid=4: 1 (volume ratio)) of 1mL precooling, dispels mixing;
(6) centrifugally discard stationary liquid, add 500 μ L stationary liquids, dispel mixing;
(7) drip sheet: taken out from frozen water by slide glass, drip sheet (10cm height), spontaneous drying;
(8) dye: with Giemsa dye liquor PBS (pH6.8) according to 1: 9 volume ratio dilute, dye 13min in glass dye vat:
(9) wash-out: rinse slide glass gently and be about 15s under tap water, notices that current can not be too large and be not directly flushed on cell, prevents cell to blow off;
(10) micronucleus is observed: use inverted microscope, with 40 × object lens observation of cell or collection image;
(11) micronucleus permillage (MNC ‰) is counted, and pollution index (P1).
The judging criterion of 4 dikaryocytes and micronucleus
(1) dikaryocyte judges need meet following judging criterion simultaneously:
1 cell has two equal-sized main cores; In 1 cell, contained micronucleus number is no more than 6; Slight nuclear substance can be had between two main cores to be connected; Cytolemma will keep complete; Contact or light overlap can be had between two main cores.
(2) micronucleus judges need meet following judging criterion simultaneously:
Diameter is the 1/16-1/3 of main core; There is no the refraction of light; Nuclear substance is not had to be connected with between main core; The overlapping of border can be had with main core, but respective nuclear membrane can be seen clearly.
The calculating of 5 micronuclear rateses
Dikaryocyte number/1000 dikaryocyte of micronucleus permillage=containing micronucleus
The micronuclear rates of pollution index (PI)=experimental group micronuclear rates-blank group micronuclear rates/negative control group
6 interpretations of result
When the genetoxic of water sample is larger, its micronuclear rates is also higher.Finally, whether there is significant difference between the micronucleus permillage (negative control group micronucleus permillage-blank group micronucleus permillage) of statistical study sample micronucleus permillage (sample micronucleus permillage-blank group micronucleus permillage) and negative control group.
The ratio of micronucleus permillage is also known as pollution index (PI value), can evaluate the size of water sample to cytogenetic toxicity effect, the pollution level of reaction water sample.Generally, can think when PI value is between 0 ~ 1.5 and substantially not pollute; It is slight pollution between 1.5 ~ 2; It is intermediate pollution between 2 ~ 3.5; More than 3.5 is serious pollution.
Accompanying drawing explanation
Fig. 1 is schema of the present invention.
Embodiment
The present invention is the implementation method of environment water body example being carried out to the detection of micronucleus test genetoxic, the method has been applied in Basin of Huaihe River Environmental Health comprehensive monitoring, have detected the genetoxic of part surface water, underground water, to have carried out exploring to its practical application and perfect.Embody rule situation is as follows:
12 groundwater sample are acquired respectively in 3 villages of some areas, F city, 1 central water supply sample, and 3 surface waters, according to step shown in summary of the invention, the testing sequences such as pre-treatment, cell cultures, Micronuclei test and micronuclear rates counting are carried out to the sample gathered.Concrete sampling point information is in table 1.
Table 1 water sampling information
1 water sample pre-treatment
Each sample collecting water sample at least 10L, after the glass fiber filter filtration of activated 0.45 μm, uses HLB solid-phase extraction column (activation) enrichment wherein organism, every root HLB post enrichment 2L water sample.Dry up HLB post after enrichment completes and preserve.Use acetone wash-out enriched substance afterwards, dry up with nitrogen after merging same sample, with 10 μ L DMSO constant volumes before experiment.
2 micronucleus tests
(1) cell cultures: recovery TK6 cell, this cell is suspension cell, cell counting before going down to posterity, and keeps cell concn 2 × 10
5~ 1 × 10
6between;
(2) cell contamination: the cell that growth selection is in good condition, centrifugal segregation substratum, adjusts cell concn 2 × 10 after cell counting
5cells/mL,
The toxicity of sample may cause necrocytosis too greatly, cannot detect micronucleus, therefore first adds the testing sample of different concns gradient before experiment, selects the poisoning dosage be applicable to.Different concns gradient is set: in 0.1 ~ 1.0 μ L concentration range, arrange 10 concentration gradients carry out preliminary experiment, incubation time 24h, by microscope observing cell growth conditions, determine best poisoning dosage.TK6 cell is comparatively responsive, can remarkable cell growth inhibiting when poisoning dosage is higher, and experiment finds that underground water poisoning dosage is comparatively suitable under 0.2 ~ 0.5L water sample concentration, and the poisoning dosage of surface water is comparatively suitable under 0.02 ~ 0.05L water sample concentration.
The poisoning dosage of this experiment is: underground water 0.3L, surface water 0.03L, and contamination adds the cytochalasin-B that final concentration is 6 μ g/mL simultaneously, and arranges blank, positive control (1.0 μ g/mL MMC) simultaneously.Last cumulative volume is 1mL.
(3) micronucleus film-making: pass through centrifugal collecting cell after contamination time terminates, the stationary liquid adding precooling after washing twice with cold PBS pre-fixes cell, recentrifuge collecting cell, carry out cell after adding 500 μ L stationary liquids and drip sheet, at room temperature dye with the Giemsa dye liquor prepared after seasoning, cleaning and drying after namely by microscope observing cell core.
3 experimental results
Directly observe under the microscope, or shooting image, count the quantity of micronucleus in 1000 dikaryocytes, the micronuclear rates of calculation sample, and calculate its pollution index.Experiment concrete outcome is as shown in table 2.
Table 2 underground water and surface water micronucleus test result
Note: negative control micronuclear rates is 6 ‰; PI=(sample micronuclear rates-blank group micronuclear rates)/negative control group micronuclear rates
From micronucleus test result can, the micronuclear rates of surface water is higher, and genetoxic is comparatively large, and pollution index is shown as serious pollution water body; The micronuclear rates of central water supply sample is less, and pollution index is lower than 1.5, pollution-free; Have 4 sample micronuclear rateses higher in groundwater sample, pollution index is shown as slight pollution, should arouse attention.
Result display survey region surface water genetoxic is large, and local resident may cause Health hazard by edible aquatic living things, use polluted-water after irrigating; Its genetoxic of different groundwater sample of the same area differs greatly in addition, and part groundwater sample demonstrates higher genetoxic, comparatively large as its potential health hazard of resident living tap water, is not suitable for continuing to drink.
The practical application of 4 micronucleus tests
Environment water body example genetoxic detects can understand the impact of water body environment on around exposed population group's health, can enriched environment health comprehensive monitoring syllabus and content.Correlative study result contributes to understanding Polluted area environmental health problems, and the Health hazard that exposed region crowd can be helped to evade environmental pollution cause, has actual application value and meaning.
The genetoxic size of TK6 people lymphocytoblast testing environment water body is adopted in the present invention, simplifying by using stable clone with peripheral blood lymphocyte is the environment genetoxic detection method of subject vector, simplify experiment flow, decrease water body example usage quantity simultaneously, be conducive to the application of micronucleus test in environment water complex inheritance Toxicity Monitoring.
Claims (7)
1. an environment water genetoxic high-throughput method for quick, described method is TK6 cell micronucleus experimental technique.
2. detection method as claimed in claim 1, described TK6 cell micronucleus experimental technique is:
(1) first filter through 0.45 μm of glass fiber filter after environment water body example collection, the enrichment of HLB solid-phase extraction column and acetone wash-out, merging, and the concrete pre-treatment step such as drying;
(3) recovery of bacterium and preculture, poisoning dosage set, contaminate after microbial culture and end-result measure;
(4) calculate beta galactose enzymic activity (U), inductivity (R) by the absorption photometric value of sample under 600nm and 420nm, and then judge whether it has genetoxic.
3. method as claimed in claim 2, wherein step (1) is specially:
(1) filter membrane is activated: use the glass fiber filter of 0.45 μm after activation to filter water sample, remove the impurity that in water sample, particle diameter is larger; Activation method is: use retort furnace that the glass fiber filter (GF/A, 70mm, Whatman) of 0.45 μm is activated 3h at 450 DEG C of-500 DEG C of temperature, be placed on more than equilibrium at room temperature 24h in moisture eliminator, use front 1h ultrapure water reactivate after activation;
(2) HLB solid-phase extraction column activation: use the organism etc. in HLB solid-phase extraction column (Waters Oasis, 6mL) enrichment water body, detect for genetoxic; Every root HLB solid-phase extraction column first soaks 5min with 6mL methylene dichloride, then solution is made slowly to flow out, after liquid level is parallel with upper strata filter plate, close upper lock gate, add 6mL methyl alcohol, close upper lock gate again after dichloromethane solution is flowed out completely, add 6mL ultrapure water afterwards and make it slow outflow, in most rear pillar, leave a small amount of ultrapure water;
(3) filter: after treating sediment deposition, then filter; The glass fiber filter of 0.45 μm is put in core filtration unit correspondence position, core filtration unit connects vacuum pump (1500MA) and filters, the Brown Glass Brown glass bottles and jars only preservation that the water sample rinse after filtration is clean;
(4) water sample crosses post: HLB solid-phase extraction column is placed in solid-phase extracting instrument top joint place, solid-phase extraction column top is connected with large discharge sampling thief, is positioned over by the sampling head of sampling thief in the vial of the water sample after filtration is housed; Solid-phase extracting instrument is connected with filter flask and vacuum pump, opens vacuum pump and the careful valve opening solid-phase extracting instrument, regulate the flow of every root pillar, flow rate control at 6 ~ 8mL/min, every root HLB post enrichment 2L water sample.Drain moisture in part post with vacuum pump after enrichment completes, with Nitrogen evaporator, pillar is dried up, can-20 DEG C of preservations if do not use immediately;
(5) sample elution: in stink cupboard, carries out wash-out with 10mL acetone to HLB post, and acetone is naturally flowed down in without external force situation, and elutriant 50mL Brown Glass Brown glass bottles and jars only is collected, and residual acetone rubber suction bulb is blown down.Acetone can be more excessive a little, ensures that sample eluent reaches 10mL;
(6) same sample is merged: merged by the elutriant of same sample, first carrying out nitrogen to acetone eluant blows concentrated, identical water sample is merged to same sample pipe after residue about 200 μ L, by other each effective a small amount of acetone rinsing 3-4 time, be merged in same sample pipe equally, blow to complete drying with Nitrogen evaporator;
(7) the ratio DMSO constant volume sample of 10 μ L concentrated solutions is settled to according to 1L water sample ,-20 DEG C of preservations.
4. detection method as claimed in claim 2, wherein step (2) is:
(1) cell recovery: by frozen TK6 cell quick-thawing in 37 DEG C of water-baths, the centrifugal 5min of 125xg, discards frozen storing liquid, cell adds RPMI 1640 substratum of 10mL containing 10% foetal calf serum, at 37 DEG C, 5%CO
224h is cultivated in incubator;
(2) passage: carry out cell counting, when cell number reaches 10
6need go down to posterity in time during cells/mL, make cell concn remain at 2 × 10
5~ 1 × 10
6between cells/mL.
5. detection method as claimed in claim 2, wherein step (3) is:
(1) plating cells: by cell to be gone down to posterity by 2 × 10
5cells/mL is taped against in 24 orifice plates, uses the substratum bed board of 10% serum; Every hole adds final concentration is simultaneously that the cytochalasin-B of 6 μ g/mL is to form dikaryocyte;
(2) cell contamination: add certain volume tested material, draws through experiment: the concentration of contamination of groundwater sample is advisable with 0.2-0.5L water sample extract concentrated solution, and surface water concentration of contamination is advisable with 0.02-0.05L extract concentrated solution; Every hole cumulative volume is 1mL, cultivates 24h;
(3) counting before experiment, gets 1 × 10
6individual cell, centrifugal collecting cell (125xg, 5min), abandoning supernatant, adds 1mLPBS damping fluid;
(4) centrifugal abandoning supernatant, continues to add 1mL PBS buffer solution cell;
(5) centrifugal abandoning supernatant, adds the stationary liquid (methyl alcohol: Glacial acetic acid=4: 1 (volume ratio)) of 1mL precooling, dispels mixing;
(6) centrifugally discard stationary liquid, add 500 μ L stationary liquids, dispel mixing;
(7) drip sheet: taken out from frozen water by slide glass, drip sheet (10cm height), spontaneous drying;
(8) dye: with Giemsa dye liquor PBS (pH6.8) according to 1: 9 volume ratio dilute, dye 13min in glass dye vat;
(9) wash-out: rinse slide glass gently and be about 15s under tap water, notices that current can not be too large and be not directly flushed on cell, prevents cell to blow off;
(10) micronucleus is observed: use inverted microscope, with 40 × object lens observation of cell or collection image;
(11) micronucleus permillage (MNC ‰) is counted, and pollution index (PI).
6. detection method as claimed in claim 2, wherein step (4) is:
(1) dikaryocyte judges need meet following judging criterion simultaneously:
1 cell has two equal-sized main cores; In 1 cell, contained micronucleus number is no more than 6; Slight nuclear substance can be had between two main cores to be connected; Cytolemma will keep complete; Contact or light overlap can be had between two main cores;
(2) micronucleus judges need meet following judging criterion simultaneously:
Diameter is the 1/16-1/3 of main core; There is no the refraction of light; Nuclear substance is not had to be connected with between main core; The overlapping of border can be had with main core, but respective nuclear membrane can be seen clearly;
Wherein, the calculating of micronuclear rates:
Dikaryocyte number/1000 dikaryocyte of micronucleus permillage=containing micronucleus;
The micronuclear rates of pollution index (PI)=experimental group micronuclear rates-blank group micronuclear rates/negative control group.
7. the application of detection method in testing environment water body genetoxic that one of claim 1-6 is described.
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| CN112014189A (en) * | 2020-09-17 | 2020-12-01 | 北京市化工职业病防治院(北京市职业病防治研究院) | Method for preparing slide for micronucleus and chromosome test |
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| CN103276042A (en) * | 2013-05-27 | 2013-09-04 | 中国科学院生态环境研究中心 | Method for detecting genetic toxicity of organic pollutants in water |
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| CN104140997A (en) * | 2013-05-07 | 2014-11-12 | 中国科学院生态环境研究中心 | In-vitro micronucleus detection method for genetic toxicity of water outlet of drinking water disinfection process |
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| CN110261213A (en) * | 2019-06-11 | 2019-09-20 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | A kind of method of medical instrument limit extraction |
| CN110261213B (en) * | 2019-06-11 | 2022-04-19 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | Limit leaching method for medical instruments |
| CN112014189A (en) * | 2020-09-17 | 2020-12-01 | 北京市化工职业病防治院(北京市职业病防治研究院) | Method for preparing slide for micronucleus and chromosome test |
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