CN103076444A - Method for identifying plasticizer in distilled spirit by caenorhabditis elegans - Google Patents
Method for identifying plasticizer in distilled spirit by caenorhabditis elegans Download PDFInfo
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Abstract
The invention discloses a method for identifying a plasticizer in a stilled spirit by caenorhabditis elegans, and relates to a method for indentifying the plasticizer. The method solves the problems in a conventional plasticizer detection method that the operation is complex, the dependence degree for a device is large, and the cost is high. The method comprises the steps as follows: step one, preparing a cholesterol ethanol solution; step two, preparing a phosphate buffer solution; step three, preparing culture media of elegans and step four, coating the culture media of the elegans with a colon bacillus OP50 bacterium solution uniformly, culturing the culture media for 8-12h at the temperature of 37 DEG C, putting in 8-12 sexually mature caenorhabditis elegans, culturing for 12h, and then observing the egg-laying quantity of the elegans. If the egg-laying quantity of the elegans is lower than a normal level, the fact that a distilled spirit to be detected contains the plasticizer is indicated. The detection method has low requirements for experimental conditions, and does not require any special high-precision analytical instruments or detection devices, the experiment can be operated conveniently, the cost is low, and the method can be mastered easily.
Description
Technical field
The present invention relates to a kind of method of identifying plasticiser.
Background technology
Plasticiser (plasticizer) has another name called plastifier, is the phthalic ester homolog.Rise in May, 2011 and successively detect 6 kinds of phthalate plasticiser compositions such as DEHP, DINP, DNOP, DBP, DMP, DEP in the Taiwan Food, detect DIDP in the medicine, June 1, the Ministry of Public Health promptly issued bulletin, with phthalic ester (also being phthalate ester) class material, list the non-edible material from soybeans of the illegal interpolation of possibility in the food and the food additives list of easily abusing in.And " drunkard " wine plasticiser that the end of the year in 2012 was exposed exceeds standard 2.6 times, causes that people are about plasticiser and to the particularly panic and query of high-grade wine food security of liquor.
Plasticiser is a kind of Environmental Hormone, can cause immunity and fecundity to descend to humans and animals.At present the tolerable 60 kilograms of adult's daily intake scopes in various countries are the 1.2-8.4 milligram, are that the mouse of taking " plasticiser " is found in investigation of materials with white mouse, and the offspring under absurd fantastic is take female as main, and can affect its normal ovulation; Even birth is lower male, its reproductive organs compared with normal little by 2/3rds, and sperm quantity also subtracts greatly, shows that " plasticiser " has genotoxicity.Some researchs show that also plasticiser can be larger to child's potential hazard, shows certain development toxicity.
Adjacent benzene class plasticiser consumption is huge, usable range is wide, contaminated area and the number that affects are just all luxuriant to be considerable, and phthalate can be described as a most widely class synthetic environment pollutant of distribution on global at present, can detect in air, water, soil and dust.Phthalate has inrichment at the biosome escheat, and the significantly residue of this compounds is arranged in the aquatic organism, and final people expose wherein by approach such as food and air.Detection about plasticiser there is no unified standard.Have complicated operation in conventional chemical detection and the instrumental analysis, the instrument dependency degree is large, the deficiency that cost is high.
Summary of the invention
The objective of the invention is provides a kind of method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor in order to solve complicated operation in the conventional plasticiser detection method, the instrument dependency degree is large, cost is high technical matters.
Utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, with 15～20g agar powder, 2～3g peptone, 0.10～0.12g CaCl
2, 0.11～0.13g MgSO
4Join in the beaker of 1000mL with 2～3g NaCl, add 1ml cholesterol ethanolic solution and 25mL phosphate buffer, be settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 15～25min, then be cooled to 50～60 ℃, under gnotobasis, add 0.5～5ml liquor to be measured, then under gnotobasis, pour into potpourri in the 6cm double dish, be dry 8h～12h under 20 ℃ the gnotobasis in temperature, namely get nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 1 * 10
9Individual/mL～2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 0.8～1.2mL, cultivate 8～12h in 37 ℃, access 8～12 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs, the Caenorhabditis elegans laying is lower than normal level (being normal spawning quantity more than 21), proves that then liquor to be checked contains plasticiser.
The present invention identifies in liquor whether contain plasticiser by plasticiser to the genotoxicity of nematode by measuring the nematode spawning rate.An amount of alcohol is added into micro-liquor in the nematode culture medium the not impact of breeding of nematode, if the nematode spawning rate of growing on this nutrient culture media is lower than normal level, also has plasticiser just can determine this type of liquor.
The present invention compares with traditional method of utilizing plasticiser in the analytical instrument evaluation liquor, and susceptibility is high, utilizes nematode breeding index, can intuitively determine efficiently whether contain plasticiser in the liquor.This detection method is low to requirement for experiment condition, does not need special high-precision analytical instrument and checkout equipment, and convenient experimental operation is with low cost, is easy to grasp.
Description of drawings
Fig. 1 is that experiment one is to the laying of testing every Caenorhabditis elegans in four, a represents to test the laying of every Caenorhabditis elegans among the figure, b represents to test the laying of every Caenorhabditis elegans in two, c represents to test the laying of every Caenorhabditis elegans in three, d represents to test the laying of every Caenorhabditis elegans in four, e represents to test the laying of every Caenorhabditis elegans in five, f represents to test the laying of every Caenorhabditis elegans in six, g represents to test the laying of every Caenorhabditis elegans in seven, h represents to test the laying of every Caenorhabditis elegans in eight, and i represents to test the laying of every Caenorhabditis elegans in nine.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the combination in any between each embodiment.
Embodiment one: present embodiment utilizes Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, with 15～20g agar powder, 2～3g peptone, 0.10～0.12g CaCl
2, 0.11～0.13g MgSO
4Join in the beaker of 1000mL with 2～3g NaCl, add 1ml cholesterol ethanolic solution and 25mL phosphate buffer, be settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 15～25min, then be cooled to 50～60 ℃, under gnotobasis, add 0.5～5ml liquor to be measured, then under gnotobasis, pour into potpourri in the 6cm double dish, be dry 8h～12h under 20 ℃ the gnotobasis in temperature, namely get nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 1 * 10
9Individual/mL～2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 0.8～1.2mL, in 37 ℃ of cultivation 8～12h, access 8～12 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs, the Caenorhabditis elegans laying is lower than normal level, proves that then liquor to be checked contains plasticiser.
Embodiment two: what present embodiment and embodiment one were different is with 16～19g agar powder, 2g peptone, 0.10g CaCl in the step 2
2, 0.11g MgSO
4Join in the beaker of 1000mL with 2g NaCl.Other is identical with embodiment one.
Embodiment three: what present embodiment and embodiment one were different is with 15g agar powder, 2g peptone, 0.10g CaCl in the step 2
2, 0.11g MgSO
4Join in the beaker of 1000mL with 2g NaCl.Other is identical with embodiment one.
Embodiment four: what present embodiment and embodiment one were different is with 20g agar powder, 3g peptone, 0.12g CaCl in the step 2
2, 0.13g MgSO
4Join in the beaker of 1000mL with 3g NaCl.Other is identical with embodiment one.
Embodiment five: what present embodiment and embodiment one were different is to be cooled to 52～58 ℃ in the step 4.Other is identical with embodiment one.
Embodiment six: what present embodiment and embodiment one were different is to be cooled to 55 ℃ in the step 4.Other is identical with embodiment one.
Embodiment seven: present embodiment and embodiment one are different is to be dry 10h under 20 ℃ the gnotobasis in temperature in the step 4.Other is identical with embodiment one.
Embodiment eight: present embodiment and embodiment one are different is that evenly to be painted into concentration in the nematode culture medium in the step 5 be 1 * 10
9Individual/mL～2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL.Other is identical with embodiment one.
Embodiment nine: what present embodiment and embodiment one were different is in cultivating 10h in 37 ℃ in the step 5.Other is identical with embodiment one.
Embodiment ten: what present embodiment and embodiment one were different is 10 sexually matured Caenorhabditis elegans of access in the step 5.Other is identical with embodiment one.
Adopt following experimental verification effect of the present invention:
Experiment one: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g GaGl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, cool the temperature to 55 ℃, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Do not add any other material in this experimental procedure four, in this experiment behind the 24h Caenorhabditis elegans spawning rate be the nematode breeding level of growing in the normal nutrient culture media.
Experiment two: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g GaGl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, cool the temperature to 55 ℃, under the superclean bench gnotobasis, add the 5ml absolute ethyl alcohol, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Add in this experimental procedure four and do not contain the absolute alcohol that comprises any impurity of plasticiser, in this experiment behind the 24h Caenorhabditis elegans spawning rate be in normal level.
Experiment three: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 15g agar powder, 2g peptone, 0.10g CaCl
2, 0.11g MgSO
4, 2g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 15～25min, cool the temperature to 50～60 ℃, under the superclean bench gnotobasis, add the 0.5ml absolute ethyl alcohol, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 8h～12h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 1 * 10
9Individual/mL/mL Escherichia coli OP50 bacterium liquid 0.8mL, in 37 ℃ of cultivation 8h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Add in this experimental procedure four and do not contain the absolute alcohol that comprises any impurity of plasticiser, in this experiment behind the 24h Caenorhabditis elegans spawning rate be in normal level.
Experiment four: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 5mL ethanol mass concentration is 53% liquor under the superclean bench gnotobasis, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Add the ethanol mass concentration do not contain plasticiser in this experimental procedure four and be 53% liquor, in this experiment behind the 24h nematode spawning rate be in normal level.
Experiment five: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water; Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 5mL ethanol mass concentration is 42% liquor under the superclean bench gnotobasis, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Add in this experimental procedure four and do not contain 42% liquor that plasticiser ethanol mass concentration is, in this experiment behind the 24h nematode spawning rate be in normal level.
Experiment six: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water; Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 1mL plasticiser (phthalic ester) content under the superclean bench gnotobasis is that 0.1mg/L, mass concentration are 42% ethanol, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Adding plasticiser concentration in this experimental procedure four is that 0.1mg/L, ethanol mass concentration are 42% liquor, in this experiment behind the 24h nematode spawning rate significantly be lower than normal level.
Experiment seven: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 1mL plasticiser (phthalic ester) content is that the mass concentration of 0.2mg/L is 42% ethanol under the superclean bench gnotobasis, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Adding plasticiser concentration in this experimental procedure four is that 0.2mg/L, ethanol mass concentration are 42% liquor, in this experiment behind the 24h nematode spawning rate significantly be lower than normal level.
Experiment eight: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 1mL plasticiser (phthalic ester) content under the superclean bench gnotobasis is that 0.3mg/L, ethanol mass concentration are 42% ethanol, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Adding plasticiser concentration in this experimental procedure four is 42% liquor of 0.3mg/L, ethanol, in this experiment behind the 24h nematode spawning rate significantly be lower than normal level.
Experiment nine: utilize Caenorhabditis elegans to identify that the method for plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, take by weighing 18g agar powder, 2.5g peptone, 0.11g CaCl
2, 0.12g MgSO
4, 2.5g NaCl is in the 1000mL beaker, adds 1ml cholesterol ethanolic solution and 25mL phosphate buffer, is settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 20min, treat that temperature is down to 55 ℃, adding 5mL plasticiser (phthalic ester) content under the superclean bench gnotobasis is that 0.1mg/L, ethanol mass concentration are 42% ethanol, under the superclean bench gnotobasis, potpourri is poured in the 6cm double dish, be dry 10h under 20 ℃ the gnotobasis in temperature at last, namely obtain nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL, in 37 ℃ of cultivation 10h, access 10 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs.
Adding plasticiser concentration in this experimental procedure four is that 0.1mg/L, ethanol mass concentration are 42% liquor, in this experiment behind the 24h nematode spawning rate significantly be lower than normal level.
Claims (10)
1. utilize Caenorhabditis elegans to identify the method for plasticiser in the liquor, it is characterized in that the method for utilizing Caenorhabditis elegans to identify plasticiser in the liquor is as follows:
One, take by weighing the 1.0g cholesterol, be dissolved in the absolute ethyl alcohol, being mixed with concentration is the cholesterol ethanolic solution of 10mg/ml;
Two, preparation phosphate buffer: take by weighing 13.6g KH
2PO
4, 1.8g KOH, adding distil water is settled to 100mL, regulating the pH value is 6.0;
Three, with 15～20g agar powder, 2～3g peptone, 0.10～0.12g CaCl
2, 0.11～0.13g MgSO
4Join in the beaker of 1000mL with 2～3g NaCl, add 1ml cholesterol ethanolic solution and 25mL phosphate buffer, be settled to 1000ml with distilled water;
Four, beaker is sealed, put into high-pressure steam sterilizing pan in 120 ℃ of sterilization 15～25min, then be cooled to 50～60 ℃, under gnotobasis, add 0.5～5ml liquor to be measured, then under gnotobasis, pour into potpourri in the 6cm double dish, be dry 8h～12h under 20 ℃ the gnotobasis in temperature, namely get nematode culture medium;
Five, evenly being painted into concentration in the nematode culture medium is 1 * 10
9Individual/mL～2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 0.8～1.2mL, in 37 ℃ of cultivation 8～12h, access 8～12 sexually matured Caenorhabditis elegans, cultivate 24h and observe the quantity of laying eggs, the Caenorhabditis elegans laying is lower than normal level, proves that then liquor to be checked contains plasticiser.
2. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that in the step 2 16～19g agar powder, 2g peptone, 0.10g CaCl
2, 0.11g MgSO
4Join in the beaker of 1000mL with 2g NaCl.
3. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that in the step 2 15g agar powder, 2g peptone, 0.10g CaCl
2, 0.11g MgSO
4Join in the beaker of 1000mL with 2g NaCl.
4. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that in the step 2 20g agar powder, 3g peptone, 0.12g CaCl
2, 0.13g MgSO
4Join in the beaker of 1000mL with 3g NaCl.
5. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that being cooled in the step 4 52～58 ℃.
6. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that being cooled in the step 4 55 ℃.
7. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that in the step 4 that in temperature be dry 10h under 20 ℃ the gnotobasis.
8. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that in the step 5 that evenly being painted into concentration in nematode culture medium is 1 * 10
9Individual/mL～2 * 10
9Individual/mL Escherichia coli OP50 bacterium liquid 1mL.
9. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that cultivating 10h in 37 ℃ in the step 5.
10. the described method of utilizing Caenorhabditis elegans to identify plasticiser in the liquor according to claim 1 is characterized in that 10 sexually matured Caenorhabditis elegans of access in the step 5.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN103875606A (en) * | 2014-03-13 | 2014-06-25 | 中国科学院东北地理与农业生态研究所 | caenorhabditis elegans stress response behavior observation platform base and manufacturing method thereof |
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| CN105738606A (en) * | 2014-12-09 | 2016-07-06 | 兰州红虫生物工程有限责任公司 | Kit for screening drugs, and use method thereof |
| CN105911290A (en) * | 2016-04-15 | 2016-08-31 | 中国科学院东北地理与农业生态研究所 | Method for identifying pesticide D-butyl ester with application of nematode Hsp12.2 protein expression as marker |
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| CN109342709A (en) * | 2018-11-21 | 2019-02-15 | 蚌埠医学院 | A method of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity |
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