CN105021772A - Method for detecting copper by pharyngeal motion of Caenorhabditis elegans - Google Patents

Method for detecting copper by pharyngeal motion of Caenorhabditis elegans Download PDF

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Publication number
CN105021772A
CN105021772A CN201510400626.6A CN201510400626A CN105021772A CN 105021772 A CN105021772 A CN 105021772A CN 201510400626 A CN201510400626 A CN 201510400626A CN 105021772 A CN105021772 A CN 105021772A
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mixed liquor
copper
concentration
caenorhabditis elegans
sterilizing
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CN201510400626.6A
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Chinese (zh)
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王云彪
陈展彦
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Northeast Institute of Geography and Agroecology of CAS
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Northeast Institute of Geography and Agroecology of CAS
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Priority to CN201510400626.6A priority Critical patent/CN105021772A/en
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Abstract

A method for detecting copper by pharyngeal motion of Caenorhabditis elegans relates to a method for detection of copper. The invention aims to solve technical problems of complex operation, high dependency on instruments and high appraisal cost of existing copper detection methods. The method for detecting copper by pharyngeal motion of Caenorhabditis elegans comprises the following steps: 1, preparation of mixed liquor and sterilization; and 2, addition of Caenorhabditis elegans and observation. According to the detection method provided by the invention, pharyngeal motion frequency of Caenorhabditis elegans is used as an index. In comparison with a traditional copper identification method by an analytical instrument, the method provided by the invention can be used to intuitively and efficiently discriminate copper. In addition, the detection method has low requirements on experiment conditions, requires no special high precision analytical instrument or detection equipment, is convenient to operate in experiments, is low-cost and is easy to train and grasp. The method provided by the invention is applied in detecting a liquid with the concentration of copper in an aqueous solution being greater than or equal to 0.013 micron mol/L.

Description

A kind of method utilizing nematode to swallow dynamic detection copper
Technical field
The present invention relates to a kind of method detecting copper.
Background technology
Caenorhabditis elegans (Caenorhabditis elegans, is abbreviated as C.elegans) lets out pipe guiding principle animal for Nemathelminthes, is widely used at scientific research field as a kind of typical module biology.Caenorhabditis elegans entire body is transparent, is easy to observe form and behavior, and life cycle is short, and cultivation is convenient and cheap, is easy to preserve and be convenient to test operation.The experimental study of Caenorhabditis elegans has formed a series of International standardization and standardized operation.Bulb is the important feed of nematode and locomotive organ, and nematode pharynx dynamic frequency is the important behavioral indicator characterizing its health, is also the conventional behavior end points of nematode toxicology.
Copper is as representative heavy metal, and along with the increase in range of application and market, the ecologic environment that copper three waste discharge causes and human health problems constantly occur, evaluates and the Ecological Environment Risk that controls copper causes people to pay close attention to day by day.There is complicated operation in conventional copper detection method, instrument dependency degree is large, causes appraisal cost high.
Summary of the invention
There is complicated operation to solve existing copper detection method in the present invention, instrument dependency degree is large, causes the technical matters that appraisal cost is high, and provide a kind of method utilizing nematode to swallow dynamic detection copper.
A kind of method utilizing nematode to swallow dynamic detection copper of the present invention is carried out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL ~ 0.12 × 10 -3g/mL, MgSO 4concentration be 0.11 × 10 -3g/mL ~ 0.13 × 10 -3the concentration of g/mL, NaCl is 2 × 10 -3g/mL ~ 3 × 10 -3the concentration of g/mL, KCl is 2 × 10 -3g/mL ~ 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after during cultivation 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved, if the pharynx dynamic frequency of Caenorhabditis elegans is less than 100 times/minute, show containing copper in liquid to be checked, if the pharynx dynamic frequency of Caenorhabditis elegans is more than or equal to 100 times/minute, show in liquid to be checked not containing copper.
Advantage of the present invention: detection method of the present invention make use of the pharynx dynamic frequency of Caenorhabditis elegans as index, identifies compared with the method for copper with traditional analytical instrument that utilizes, intuitively can screen copper efficiently; In addition, detection method of the present invention is low to requirement for experiment condition, does not need special high-precision analytical instrument and checkout equipment, convenient experimental operation, with low cost, is easy to training and grasps.
The present invention is applied to the liquid that the concentration detecting copper in aqueous solution is more than or equal to 0.013 μm of ol/L.
Embodiment
Embodiment one: present embodiment is that a kind of method utilizing nematode to swallow dynamic detection copper is carried out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL ~ 0.12 × 10 -3g/mL, MgSO 4concentration be 0.11 × 10 -3g/mL ~ 0.13 × 10 -3the concentration of g/mL, NaCl is 2 × 10 -3g/mL ~ 3 × 10 -3the concentration of g/mL, KCl is 2 × 10 -3g/mL ~ 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after during cultivation 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved, if the pharynx dynamic frequency of Caenorhabditis elegans is less than 100 times/minute, show containing copper in liquid to be checked, if the pharynx dynamic frequency of Caenorhabditis elegans is more than or equal to 100 times/minute, show in liquid to be checked not containing copper.
Embodiment two: the difference of present embodiment and embodiment one is: by CaCl in step one 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 20min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing.Other are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one or two is: add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains in step 2 successively and enter 10 Caenorhabditis elegans in each hole of 24 hole double dish, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.Other are identical with embodiment one or two.
Embodiment four: the difference of present embodiment and embodiment one to three is: the testing liquid described in step 2 is the liquid that the concentration of copper is more than or equal to 0.013 μm of ol/L.Other are identical with embodiment one to three.
Embodiment five: the difference of present embodiment and embodiment one to four is: CaCl in the mixed liquor described in step one 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL.Other are identical with embodiment one to four.
By following verification experimental verification beneficial effect of the present invention:
Test one: this test is contrast test:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.
Testing liquid described in step 2 is deionized water.
The pharynx that naked-eye observation obtains Caenorhabditis elegans is dynamic higher than 200 times/minute.
Test two: this test is a kind of method utilizing nematode to swallow dynamic detection copper, specifically carries out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.
The solution of to be copper ion concentration the be 0.013 μm of ol/L of the testing liquid described in step 2.
Naked-eye observation obtains that the pharynx of Caenorhabditis elegans is dynamic is less than 100 times/minute.
Test three: this test is a kind of method utilizing nematode to swallow dynamic detection copper, specifically carries out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.
The solution of to be copper ion concentration the be 0.13 μm of ol/L of the testing liquid described in step 2.
Naked-eye observation obtains that the pharynx of Caenorhabditis elegans is dynamic is less than 100 times/minute.
Test four: this test is a kind of method utilizing nematode to swallow dynamic detection copper, specifically carries out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.
The solution of to be copper ion concentration the be 1.3 μm of ol/L of the testing liquid described in step 2.
Naked-eye observation obtains that the pharynx of Caenorhabditis elegans is dynamic is less than 100 times/minute.

Claims (5)

1. utilize nematode to swallow the dynamic method detecting copper, it is characterized in that utilizing nematode to swallow the dynamic method detecting copper carries out according to the following steps:
One, mixed liquor is prepared and sterilizing: by CaCl 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 15min ~ 25min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing; CaCl in described mixed liquor 2concentration be 0.1 × 10 -3g/mL ~ 0.12 × 10 -3g/mL, MgSO 4concentration be 0.11 × 10 -3g/mL ~ 0.13 × 10 -3the concentration of g/mL, NaCl is 2 × 10 -3g/mL ~ 3 × 10 -3the concentration of g/mL, KCl is 2 × 10 -3g/mL ~ 3 × 10 -3g/mL;
Two, add nematode and observe: in each hole of 24 hole double dish, add mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains successively and enter 8 ~ 12 Caenorhabditis elegans, after during cultivation 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved, if the pharynx dynamic frequency of Caenorhabditis elegans is less than 100 times/minute, show containing copper in liquid to be checked, if the pharynx dynamic frequency of Caenorhabditis elegans is more than or equal to 100 times/minute, show in liquid to be checked not containing copper.
2. a kind of method utilizing nematode to swallow dynamic detection copper according to claim 1, is characterized in that CaCl in step one 2, MgSO 4, NaCl and KCl be dissolved in distilled water, obtain mixed liquor, mixed liquor being put into high-pressure steam sterilizing pan is sterilizing 20min under the condition of 120 DEG C in temperature, and after taking out, cooling, obtains the mixed liquor after sterilizing.
3. a kind of method utilizing nematode to swallow dynamic detection copper according to claim 1, it is characterized in that adding successively in each hole of 24 hole double dish in step 2 mixed liquor, the 10 μ L testing liquids after the sterilizing that 1mL step one obtains and enter 10 Caenorhabditis elegans, after when cultivating 24h, the pharynx of naked-eye observation Caenorhabditis elegans is moved.
4. according to claim 1ly a kind ofly utilize nematode to swallow the dynamic method detecting copper, the testing liquid that it is characterized in that described in step 2 is the liquid that the concentration of copper is more than or equal to 0.013 μm of ol/L.
5. a kind of method utilizing nematode to swallow dynamic detection copper according to claim 1, is characterized in that CaCl in the mixed liquor described in step one 2concentration be 0.1 × 10 -3g/mL, MgSO 4concentration be 0.12 × 10 -3the concentration of g/mL, NaCl is 3 × 10 -3the concentration of g/mL, KCl is 3 × 10 -3g/mL.
CN201510400626.6A 2015-07-09 2015-07-09 Method for detecting copper by pharyngeal motion of Caenorhabditis elegans Pending CN105021772A (en)

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Cited By (1)

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CN105911238A (en) * 2016-04-13 2016-08-31 中国科学院东北地理与农业生态研究所 Method for detecting titanium dioxide by using Caenorhabditis elegans mutant RB2612

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Publication number Priority date Publication date Assignee Title
CN105911238A (en) * 2016-04-13 2016-08-31 中国科学院东北地理与农业生态研究所 Method for detecting titanium dioxide by using Caenorhabditis elegans mutant RB2612

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Application publication date: 20151104