CN101570785A - Method for detecting potential inherent toxicity of organic pollutants in water body - Google Patents
Method for detecting potential inherent toxicity of organic pollutants in water body Download PDFInfo
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Abstract
The invention discloses a method for detecting potential inherent toxicity of organic pollutants in a water body, which comprises the following steps: using resin to perform enrichment of water samples first; and applying a comet assay of Euglena gracilis to research the inherent toxicity of the organic pollutants in the water body. The key technique lies in the innovation of glue manufacturing process, namely adopting fractioning at a first layer, then adopting the conventional 'sandwich' glue manufacturing method, and manufacturing the glue by using a normal melting point glue with a higher melting point at a third layer, thus the defect that a rubber plate in the conventional method is easy to damage is overcome. By performing optimization research on cracking time and electrophoresis conditions of algae comets, a perfect image result is obtained. At present, the method for detecting the potential inherent toxicity of the organic pollutants in the water body through algae single cell gel electrophoresis (SCGE) is not reported, and algae are applied to the SCGE to widen the research objects of the SCGE.
Description
Technical field
The invention belongs to Environmental Health genetic toxicology field, be specifically related to the potential genotoxic method of organic pollutant in a kind of detection water body.
Background technology
Chinese scholars has successively been found to exist in tap water and the source water since the mutagenicity organic pollutant, and relevant epidemiology survey result shows that also Organic pollutants have certain relevant with human some cancer in the drinking-water.Therefore, harmful organic pollutant in the water is investigated and extremely urgent to its genotoxic research, even had the people to propose source water whether to contain the genetoxic material, should be the leading indicator of evaluating water quality.Organic genetoxic is an important process of environmental monitoring in a kind of effective testing method evaluation water so set up.
Biological detection to various environmental factor genetoxic effects has been occupied crucial status in toxicologic study, they have brought into play positive effect in environmental pollution monitoring and environment protection.A large amount of genetic analysis methods is used to discern sexual cell mutagenic compound, somatocyte mutagenic compound, potential carcinogen, and the various hereditary changes relevant with human health, and related method is above 200 kinds.The indicator terminal scope related according to the genetoxic effect detection method can be divided into three major types to them.The first kind detects transgenation; Second class detects chromosome aberration, comprises the abnormal change of chromosome structure and/or number; The 3rd class is measured formation, sister chromatid exchange, somatocyte reorganization and the DNA bond rupture etc. of the exciting of the sign of dna damage such as dna damage reparation, dna adduct.The detection of transgenation mainly contains forward and reverse two classes.Forward mutation changes wild type gene, makes the related gene inactivation and shows detectable phenotypic variation.On the contrary, reverse mutation is by sudden change the gene function of inactivation in the former muton to be recovered, thus performance wild-type phenotype.Chromosome aberration generally comprises the change of chromosome structure and number, and alteration of chromosome structure comprises rhexis, inversion, transposition and repeats etc.Detect chromosome aberration and adopt Cytogenetic techniques usually.The system that is used to detect chromosomal structural abnormality generally is in higher organism, the especially mammalian body and vitro system.In recent years, because development of molecular biology and development of technology make and have produced many new genotoxicity testing method in the genetic toxicology field.Although detection in Gene Mutation amount " gold standard " is direct dna sequencing, and automated DNA order-checking ability also has considerable progress, but order-checking remain one more loaded down with trivial details, expense is high, need the work of expensive device, is not suitable on a large scale dna mutation in the examination genome.
DNA is biological intravital genetic material, and its stable normal breeding and growth for life entity is vital.Yet various physics and chemistry and biotic factor in the environment---all may cause dna damage as ionizing rays, various oxygenant, germ etc.Single cell gel electrophoresis (single cell gel electrophoresis assay, be called for short SCGE, have another name called Using Comet Assay, comet assay) being one is widely used in the unicellular dna single chain of eukaryote, two strands, base point of instability and repairs the quantification detection means that postpones the site, this method is easy, quick, sensitive, and every 109Da can detect characteristics such as 0.1 fracture and obtain widespread use.Compare with chromosome aberration, micronucleus, the SCE of classics, SCGE can be used for the detection of viable cell DNA, also can be used for the analysis of dead cell DNA, makes SCGE not only can study biological effect under the low dosage, also can be used for studying the biological effect under the high dosage; SCGE can provide the information of DNA repair ability again simultaneously, and this makes SCGE be highly suitable for estimating the genetoxic that is tried thing.
Single cell gel electrophoresis technique is to be initiated in 1984 by Ostling, is improved respectively by Singh and Olive again thereafter, forms present alkalescence and neutral cracking two class methods.Although this technology principle, method are simple, its operating process is very difficult.In making the process of glue, generally on the sanding slide glass, drip 2～3 layers of gels, at every turn with cover glass lid pressure,, very easily damage offset plate taking off when getting cover glass.In cracking, electrophoresis and washing process thereafter, gel soaks for a long time, washes, though extremely light vibration, the levitating of also offset plate may being got excited, the damage of offset plate is often to the failure of an experiment.
The SCGE technology is suitable for all eukaryotic cells theoretically, the sample cell requirement is few, cell can be cultured cells system or somatic tissue's isolated cells, and present exhausted cell has root cells and blade and the yeast cell etc. of human lymphocyte, epidermic cell, hemocyte, inoblast, testicular cell, tumour cell, animal liver cell, hemocyte, plant.Do not appear in the newspapers yet rarely seen report as yet abroad but the Using Comet Assay of relevant algae is domestic.Algae is the important component part of ecotope, and algae is used for SCGE, expands the research object of SCGE.
Summary of the invention
Technical problem to be solved by this invention provides the potential genotoxic method of organic pollutant in a kind of detection water body, just provides a kind of potential genotoxic method of organic pollutant in little algae dna damage detection water body of utilizing specifically.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The potential genotoxic method of organic pollutant comprises the steps: in a kind of detection water body
(1) pre-treatment of water sample: gather water sample, after leaving standstill 24h, filter and remove suspended particle, by the enrichment of resin absorption post, get rid of moisture with nitrogen again, use methyl alcohol, acetone and methylene dichloride wash-out successively, elutriant concentrates, dries up under 50 ℃, use dimethyl sulfoxide (DMSO) (DMSO) dissolving at last, the water sample enriched substance, it is standby to keep in Dark Place under-18 ℃;
(2) cell preparation: microalgae cell is inoculated in the water sample enriched substance that step (1) obtains contaminates, the microalgae cell of not contamination is compared; The above-mentioned normal microalgae cell that dyed the microalgae cell of poison or not contamination respectively through centrifugal removal liquid portion, with PBS suspension cell again, is obtained cell suspension, and centrifugal purification is standby;
(3) shop glue:
(3a) (solvent is PBS with 1%g/mL, 1%g/mL adds 1g NMA among the 100mL PBS, identical to give a definition) normal fusing point agarose (NMA) 60～120 μ L on the frosted slide glass, feel secure, scrape off again, can make the first layer glue be more prone to be fixed on the slide glass like this;
(3b) preparation the first layer glue: the normal fusing point agarose (NMA) of 60～120 μ L 1%g/mL (solvent is PBS) is dropped on the frosted slide glass that step (3a) obtains, covered rapidly, 4 ℃ solidify 5～10min;
(3c) preparation second layer glue: add the low melting-point agarose (LMA) of 0.7%g/mL (solvent is PBS) in the cell that after step (2) is handled, obtains, resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5～1 * 10
6Individual cell/mL gets 50～120 μ L and is added on the first layer glue, covered, and 4 ℃ solidify 30～40min;
(3d) the 3rd layer of glue of preparation: the normal fusing point agarose (NMA) of Dropwise 5 0～120 μ L 1%g/mL (solvent is PBS) on second layer glue, covered, 4 ℃ solidify 5～10min;
(4) cracking: the slide glass that step (3d) is prepared removes cover glass, immerses 4 ℃ alkaline bleach liquor cleavage liquid (2.5mol/L NaOH, 1.0mmol/L Na
2-EDTA, 0.01% (w/v) SDS, pH=10, with the TritonX-100 of preceding adding 1% (v/v), 10% (v/v) dimethyl sulfoxide (DMSO)), cracking 10min～2h;
(5) electrophoresis: take out the slide glass after the cracking, put into electrophoresis chamber, in groove, slowly inject alkaline electrophoresis liquid (pH=13.0), begin electrophoresis after leaving standstill 20min, electric current 200～300mA, voltage 18～25V, electrophoresis time are 20min;
(6) neutralization: with pH7.5,0.4mmol/L Tris-HCL damping fluid submergence slide glass, the each 15min of rinsing, in and 3 times;
(7) dyeing: bromination second pyridine (EB) the 30 μ L of Dropwise 50 μ g/mL on the glue, add a cover cover glass, microscopy in the 24h; Perhaps, under 4 ℃, the condition of moist, black out, preserve the slide glass that step (6) obtains, dye again when needing to observe, microscopy in the 24h;
(8) dna damage degree analyzing: excite observation down at green glow with fluorescent microscope, take pictures, after obtaining the comet image, measure the parameter of DNA migration with the CASP software analysis: tail momentum (TM) and Olive tail momentum (OTM), estimate the potential genetoxic of organic pollutants in water body with it.The experiment establish at least 3 parallel, get its mean value.Statistics is carried out ANOVA analysis with Origin 7.5 softwares, and experimental group and control group are carried out significant t-test, with p＜0.05 as the significance foundation.CASP software is adopted in the comet image analysis, and in evaluating, (olive tailmoment OTM) has reflected dna content and tail of a comet shape facility in the tail of a comet to Olive tail square simultaneously, is the common counter of quantification dna damage degree.Provide tail momentum (TM) parameter as the reference index in addition, so that comprehensive reaction dna degree of impairment.Every slide glass is analyzed 50 cells at least.
In the step (1), described resin is XAD-2 or XAD-8 resin, preferred XAD-2 resin.
In step (2) and the step (3), described microalgae cell is very thin Euglena cell.
Preferably, in the step (3a), on the frosted slide glass, feel secure with the normal fusing point agarose 100 μ L of 1%g/mL.
Preferably, in the step (3b), with the normal fusing point agarose drop of 100 μ L 1%g/mL on the prefabricated frosted slide glass that obtains of step (3a).
Preferably, in the step (3c), get 60 μ L cell suspensions and be added drop-wise on the first layer glue.
Preferably, in the step (3d), on second layer glue, drip the normal fusing point agarose of 100 μ L 1%g/mL.
Preferably, in the step (4), the cracking time is 20min.
Preferably, in the step (5), electrophoretic current is 200mA, and voltage is 20V, and electrophoresis time is 20min.
Beneficial effect: the present invention compared with prior art has following advantage:
(1) since in the water organism be trace organic substance and of a great variety, form complicatedly, concentration, polarity difference are very big, general concentration is at g/L, the minimum ng/L level that reaches, treatment step such as therefore must at first carry out enrichment before to water sample analysis, concentrate.Being used for the organic method of enrichment water at present mainly contains: liquid-liquid extraction method, solid phase extraction and solid-phase microextraction method.With regard to the surface water, because the organic content of its water body own is very low, the wherein organic requirement of the often inaccessible enrichment of liquid-liquid extraction method, this is because liquid-liquid extraction method is unfavorable for a large amount of water samples.The solid-phase microextraction method has the few and timesaving advantage of amount of samples, but used extracting head is relatively more expensive, is not possessing broad spectrum (selectivity is stronger) aspect the organism enrichment, and its application has certain limitation.Solid phase extraction has two kinds of methods of commercialization solid phase extraction column and macroporous adsorbent resin enrichment.The C18 pillar is adopted in the organic compound enrichment in the EPA of environmental protection general administration of the United States Federal 525 methods regulation surface water, and the domestic porous resin that then adopts mostly comes organic compound in the enrichment surface water.The author finds by test, utilizes the C18 pillar can't realize the enrichment of organic compound in the large volume water sample, and the enrichment of small volume water sample then can't detect very little owing to the organism enrichment.Organism has the advantage that sample size is big, the enrichment multiple is high and extract the organism broad spectrum in the employing macroporous adsorbent resin solid phase extraction extraction water.Utilize XAD-2 or XAD-8 resin to realize the enrichment of organic compound in the water body, can make organism enrichment multiple reach 10 by concentrating
5-10
6, be specially adapted to the low water sample of organic concentration in the water.
(2) carry out the water sample enrichment with resin earlier, use the Using Comet Assay of very thin Euglena, the genetoxic of the organic pollutant in the water is studied, set up a kind of effective testing method and estimate organic genetoxic in the water.Water is the important substance basis of Sustainable development, and the protection water surrounding promotes continuous development of society economy exactly, therefore.Carry out organic pollutants in water body to the genetoxic evaluation, the coordinated development of this management to water surrounding, guarantee people ' s health, protection population resource and social economy and environment all is significant.
(3) adopted special glue method; it is the first layer frictioning; adopt the method for traditional " sandwich " glue then; use the higher normal fusing point glue NMA glue of fusing point instead at the 3rd layer; the fusing point height of NMA is not easy to come unstuck; can finely protect colloid, therefore solve the easily impaired shortcoming of traditional method offset plate substantially.
(4) algae is the important component part of ecotope, and algae is used for SCGE, expands the research object of SCGE.The SCGE technology is suitable for all eukaryotic cells theoretically, the sample cell requirement is few, cell can be cultured cells system or somatic tissue's isolated cells, and present exhausted cell has root cells and blade and the yeast cell etc. of human lymphocyte, epidermic cell, hemocyte, inoblast, testicular cell, tumour cell, animal liver cell, hemocyte, plant.Do not appear in the newspapers yet rarely seen report as yet abroad but the Using Comet Assay of relevant algae is domestic.
Description of drawings
Fig. 1 is the synoptic diagram of film-making of the present invention.Wherein, 1 is 1%NMA, and 2 is 0.7%LMA﹠amp; Algae, 3 is 1%NMA, 4 are the frosted slide glass.
Fig. 2 for organic extraction to the undamaged comet image of very thin Euglena DNA (intac nucleus---do not have the tail of a comet).
Fig. 3 for organic extraction to the comet image of very thin Euglena dna damage (nucleus of damaged---have the very long tail of a comet)
Fig. 4 a and Fig. 4 b are that different concns concentrates the influence of Taihu Lake water sample to very thin Euglena dna damage, wherein a is the Olive tail momentum effects of different concns water sample to very thin Euglena dna damage, b is the tail momentum effects of different concns water sample to very thin Euglena dna damage, and * compares p＜0.05 with blank.
Embodiment:
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
The pre-treatment of 1 water sample:
In June, 2008, gather water sample 20L by State Bueau of Environmental Protection's method for normalizing at Taihu Lake Mei Liangwan waterhead area (Wuxi).After leaving standstill 24h, filter and remove suspended particle, by the enrichment of XAD-2 resin (Sigma) adsorption column.Flow velocity is 30～40mL/min, gets rid of moisture with nitrogen again, uses methyl alcohol, acetone and methylene dichloride wash-out successively, and elutriant concentrates, dries up under 50 ℃, uses dimethyl sulfoxide (DMSO) (DMSO) dissolving at last, is settled to 2.0mL, and it is standby to keep in Dark Place under-18 ℃.
2 very thin Euglenas are cultivated:
Very thin Euglena is provided by Inst. of Hydrobiology, Chinese Academy of Sciences typical case culture collection council fresh water algae algae kind storehouse (FACHB), adopts Checcucci (1976) culture medium culturing.Mei Liang gulf water with Taihu Lake is contaminated as poisonous substance by XAD-2 resin (Sigma) adsorption column enriched substance, establishes three concentration treatment group, is equivalent to 1,5 and 10 times of former water sample concentration respectively, and establishes blank (CK) and solvent control group (DMSO).3 of each concentration are parallel.Inoculum density is 0.5 * 10
5About individual cell/mL, culture temperature is 80-90 μ mol/ (m for (25 ± 1) ℃, light intensity
2S), light dark period is 12h: 12h, cultured continuously 6d.Regularly shake and move at random the position of each bottle every day.
3 cell preparation:
Select well-grown normal or dyed the frustule 0.5～1mL of poison, 2000rpm, the centrifugal removal liquid portion of 5min.With PBS suspension cell again, obtain cell suspension, centrifugal purification is standby.
4 concentrate the degree of injury detection of water sample to DNA:
4.1 shop glue:
At first the NMA 100 μ L with 1%g/mL feel secure on the frosted slide glass, scrape off again.Again 100 μ L1%g/mLNMA are dropped in ready-formed frosted slide glass, add a cover cover glass rapidly, 4 ℃ solidify 10min.The LMA that in the cell of preparation, adds 0.7%g/mL then, resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5～1 * 10
6Individual cell/mL gets 60 μ L and is added on the first layer glue, adds a cover cover glass, and 4 ℃ solidify 30min.Drip 100 μ L 1%g/mL NMA on the LMA layer of final set, covered, 4 ℃ solidify 10min.
4.2 cracking:
The slide glass for preparing is removed cover glass, immerse 4 ℃ alkaline bleach liquor cleavage liquid, cracking 20min.
4.3 electrophoresis:
Take out the slide glass after the cracking, put into electrophoresis chamber, in groove, slowly inject alkaline electrophoresis liquid, begin electrophoresis after leaving standstill 20min, electric current 200～300mA, voltage 18～25V, electrophoresis time are 20min.
4.4 neutralization:
With the each 15min of 0.4mmol/L Tris-HCL damping fluid (pH=7.5) submergence slide glass rinsing, in and 3 times.
4.5 dyeing:
Bromination second pyridine (EB) the 30 μ L of Dropwise 50 μ g/ml add a cover cover glass, microscopic examination on the glue.Perhaps, preserve film under 4 ℃, the condition of moist, black out, dye again during observation, microscopy in the 24h.
4.6DNA degree of injury analysis:
(BX41 Olympus) excites observation down at green glow, takes pictures with cold CCD under the automatic exposure condition with accompanying software with fluorescent microscope.After obtaining the comet image, measure the parameter of DNA migration with the CASP software analysis: tail momentum (TM), Olive tail momentum (OTM), estimate the degree of injury of DNA with it.Statistics is carried out ANOVA analysis with Origin 7.5 softwares, and experimental group and control group are carried out significant t-test, with p＜0.05 as the significance foundation.CASP software is adopted in the comet image analysis, and in evaluating, (olive tail moment OTM) has reflected dna content and tail of a comet shape facility in the tail of a comet to Olive tail square simultaneously, is the common counter of quantification dna damage degree.Provide tail momentum (TM) parameter as the reference index in addition, so that comprehensive reaction dna degree of impairment.Every slide glass is analyzed 50 cells at least.
Solution formula is as follows described in the embodiment:
1 no Ca
2+, Mg
2+Phosphate buffered saline buffer (PBS)
Liquid 1:NaH
2PO
42H
2O 0.15M (23.4g/L) adds 2.34g in the 100mL deionized water.
Get 96mL.
Liquid 2:Na
2HPO
412H
2O 0.15M (53.72g/L) adds 26.86g in the 500mL deionized water.
Get 404mL.
Liquid 3:NaCl 0.145M (8.5g/L) adds 4.25g in the 500mL deionized water.
Get 500mL.
2Checcucci (1976) nutrient solution:
CH
3COONa * 3H
2O 1.0g, (NH4)
2HPO
41.0g, KH
2PO
41.0g, MgSO
4* 7H
2O 0.2g, FeSO
4* nH
2O 0.003g, CaCl
20.02g, Na
2EDTAT 0.0637g; Trace element mother liquor 1ml; VITAMIN: VB1100 μ g, VB121 μ g; Be dissolved in the 1000ml deionized water.
Trace element mother liquor: FeC
6H
5O
75mg, Na
2SiO
35mg, ZnSO
425 μ g, Na
2MoO
420 μ g, CuSO
45H
2O 10 μ g, CoCl
210 μ g, MnCl
2200 μ g, Na
2(EDTA) 5mg, Na
2MoO
45 μ g.
Nutrient solution (not adding trace element and VITAMIN) is prepared the back place a night, 121 ℃ of high-temperature sterilizations are 20 minutes then, add the VITAMIN and the micro-mother liquor of filtration sterilization after the cooling.
3 normal fusing point agarose gels, the NMA of 1%g/mL, PBS are solvent, matching while using;
Low melting-point agarose glue, the LMA of 0.7%g/mL, PBS are solvent, matching while using.
4 alkaline bleach liquor cleavage liquid
Storing solution: weighing 1.44gNaOH, 1.3401gNa
2-EDTA, 0.012gSDS is settled to 120ml with deionized water.
Use liquid: in storing solution, add 1% (v/v) TritonX-100 and 10% (v/v) DMSO, and in 4 ℃ of refrigerations down.
5 alkaline electrophoretic buffers
Weighing 43.2gNaOH and 1.302gNa
2-EDTA is dissolved in the 3500ml deionized water.
6 neutral buffered liquid
Weighing 48.5gTris is dissolved in 1000ml and goes dried up.HCl with 10mol/L regulates pH=7.5.Room temperature preservation.
7 ethidium bromide staining liquid
The weighing ethidium bromide is dissolved in dried up, is mixed with storing solution.Lucifuge is stored.Time spent is with 10 times of storing solution dilutions.Matching while using.
Claims (9)
1, the potential genotoxic method of organic pollutant in a kind of detection water body is characterized in that this method comprises the steps:
(1) pre-treatment of water sample: gather water sample, after leaving standstill 24h, filter and remove suspended particle, by the enrichment of resin absorption post, get rid of moisture with nitrogen again, use methyl alcohol, acetone and methylene dichloride wash-out successively, elutriant concentrates, dries up under 50 ℃, use dmso solution at last, the water sample enriched substance, it is standby to keep in Dark Place under-18 ℃;
(2) cell preparation: microalgae cell is inoculated in the water sample enriched substance that step (1) obtains contaminates, the microalgae cell of not contamination is compared; The above-mentioned normal microalgae cell that dyed the microalgae cell of poison or not contamination respectively through centrifugal removal liquid portion, with PBS suspension cell again, is obtained cell suspension, and centrifugal purification is standby;
(3) shop glue:
(3a) normal fusing point agarose 60～120 μ L with 1%g/mL feel secure on the frosted slide glass, scrape off again;
(3b) preparation the first layer glue: with the normal fusing point agarose drop of 60～120 μ L 1%g/mL on the slide glass that step (3a) obtains, covered rapidly, 4 ℃ solidify 5～10min;
(3c) preparation second layer glue: add the low melting-point agarose of 0.7%g/mL in the cell that after step (2) is handled, obtains, resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5～1 * 10
6Individual cell/mL gets 50～120 μ L and is added on the first layer glue, covered, and 4 ℃ solidify 30～40min;
(3d) the 3rd layer of glue of preparation: the normal fusing point agarose of Dropwise 5 0～120 μ L 1%g/mL on second layer glue, covered, 4 ℃ solidify 5～10min;
(4) cracking: the slide glass that step (3d) is prepared removes cover glass, immerses 4 ℃ alkaline bleach liquor cleavage liquid, cracking 10min～2h;
(5) electrophoresis: take out the slide glass after the cracking, put into electrophoresis chamber, in groove, slowly inject alkaline electrophoresis liquid, begin electrophoresis after leaving standstill 20min, electric current 200～300mA, voltage 18～25V, electrophoresis time are 20min;
(6) neutralization: with pH7.5,0.4mmol/L Tris-HCL damping fluid submergence wave carrier piece, the each 15min of rinsing, in and 3 times;
(7) dyeing: the bromination second pyridine 30 μ L of Dropwise 50 μ g/mL on the glue, add a cover cover glass, microscopy in the 24h;
(8) dna damage degree analyzing: excite down at green glow with fluorescent microscope and to observe, take pictures, obtain the comet image after, measure the parameter of DNA migration with the CASP software analysis: tail momentum and Olive tail momentum, estimate the potential genetoxic of organic pollutants in water body.
2, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (1), and described resin is XAD-2 or XAD-8 resin.
3, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in step (2) and the step (3), described microalgae cell is very thin Euglena cell.
4, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (3a), feels secure on the frosted slide glass with the normal fusing point agarose 100 μ L of 1%g/mL.
5, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (3b), with the normal fusing point agarose drop of 100 μ L 1%g/mL on the prefabricated slide glass that obtains of step (3a).
6, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (3c), gets 60 μ L cell suspensions and is added drop-wise on the first layer glue.
7, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (3d), drips the normal fusing point agarose of 100 μ L 1%g/mL on second layer glue.
8, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (4), the cracking time is 20min.
9, the potential genotoxic method of organic pollutant in the detection water body according to claim 1 is characterized in that in the step (5), electrophoretic current is 200mA, and voltage is 20V, and electrophoresis time is 20min.
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