CN103875606A - caenorhabditis elegans stress response behavior observation platform base and manufacturing method thereof - Google Patents
caenorhabditis elegans stress response behavior observation platform base and manufacturing method thereof Download PDFInfo
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- CN103875606A CN103875606A CN201410091287.3A CN201410091287A CN103875606A CN 103875606 A CN103875606 A CN 103875606A CN 201410091287 A CN201410091287 A CN 201410091287A CN 103875606 A CN103875606 A CN 103875606A
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Abstract
The invention relates to a caenorhabditis elegans stress response behavior observation platform base and a manufacturing method thereof. The problem that an existing elegans toxicity test cultivating medium is inaccurate in counting, large in death judging error, long in observation time and inaccurate in behavior observation is solved. The caenorhabditis elegans stress response behavior observation platform base comprises MgSO4, CaCl2, agarose, KCl, Alamar blue and distilled water according to mass percent. The method comprises the steps that weighed agarose and KCl are poured into distilled water for even stirring, and after heating boiling, cooling is carried out; MgSO4, CaCl2 and Alamar blue are added, and the pH value is adjusted through a KOH solution of 5 mol/L; the materials are poured into a 24-hole culture plate, and the liquid level height is 0.1 cm-0.2 cm; and the materials stand on a horizontal table until the materials are solidified, and the caenorhabditis elegans stress response behavior observation platform base is obtained. The caenorhabditis elegans stress response behavior observation platform base is mainly used for an existing elegans toxicity test.
Description
Technical field
The present invention relates to a kind of C. Elegans Automatic Screening stress response behavior observation platform substrate and preparation method thereof.
Background technology
C. Elegans Automatic Screening Caenorhabditiselegans belongs to Nemathelminthes (Nematoda), can experience more delicately surrounding environment change, its cycle history of life is short, can produce indicative function to environmental change at short notice, because the feature of easily cultivating, preserve is widely used as model organism, in the experimental sciences such as toxicology, carry out the behavior observational study in the fields such as environmental science, life science, and be used as the susceptibility indicator organism of ecotope health assessment.
The C. Elegans Automatic Screening carrier that carries out toxicology test behavior observation is generally agar medium or tested liquid.C. Elegans Automatic Screening health is transparent, and body is long generally in 1mm left and right, visible under the microscope.Classical toxicologic index comprises lethality, behavior variation etc., and these indexs all require test nematode to carry out dead judgement, counting and behavior observation etc.The wherein judgement of nematode death, need to be by instrument, if touch the reactionless death that is defined as, nematode on the point of dying often touches reactionlessly to this, makes dead borer population virtual height; And polypide after touching is impaired, cannot carry out follow-up physiological and biochemical analysis.
In traditional medium, the culture matrix colors such as nematode and agar, water liquid are close, unfavorable counting and observation; C. Elegans Automatic Screening usually has situation about piercing in agaropectin, occurs losing worm phenomenon, and this has increased the difficulty of counting and behavior judgement; Under microscope, in observation water liquid, nematode often has fluctuation water wave, occurs under-enumeration, this can affect to nematodal accounting accurately, particularly polypide rocks with water, and cannot judge that whether nematode dead.Generally a death of coercing lower 10 insects conventional in toxicological test is identified, at least need 5min, insect cannot be found owing to piercing in agaropectin, makes sum often lower than 10.And it is carried out to behavior measure, especially may be up to 0.5h.Therefore traditional medium is mistaken C. Elegans Automatic Screening observation effort in toxicological test, and efficiency is extremely low, also makes the accuracy and precision of result of the test decline to some extent.
Summary of the invention
The present invention be to solve that inaccurate, the dead error in judgement of counting is large at the bottom of existing nematode toxicological test medium, observation time long, not problem accurately of behavior observation, and provide a kind of C. Elegans Automatic Screening stress response behavior observation platform substrate and preparation method thereof.
A kind of C. Elegans Automatic Screening stress response behavior observation platform of the present invention substrate is by mass percentage by 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water composition of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA; Described MgSO
4and CaCl
2mass ratio be 1:1.
The preparation method of above-mentioned a kind of C. Elegans Automatic Screening stress response behavior observation platform substrate carries out according to the following steps:
One, take by mass percentage 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA;
Two, agarose step 1 being taken and KCl put in the distilled water that step 1 takes, and stir, and are heated to be cooled to 40 DEG C～45 DEG C after boiling, obtain mixed solution A;
Three, the MgSO that adds step 1 to take in the mixed solution A obtaining to step 2
4, CaCl
2with ALMA indigo plant, the KOH solution that is 5mol/L by concentration after stirring and dissolving is adjusted to 6.8～7.2 by pH value, obtains mixed solution B;
Four, mixed solution B step 3 being obtained pours in 24 porocyte culture plates, and liquid level is 0.1cm～0.2cm;
Five, the 24 porocyte culture plates that mixed solution B is housed are at room temperature placed on horizontal stand and are solidified to it, obtain the substrate of C. Elegans Automatic Screening stress response behavior observation platform;
Advantage of the present invention: 1,, at the bottom of observation platform of the present invention has the black-and-blue medium by the blue colour developing of ALMA, compared with at the bottom of the approximate medium of nematode body colour, nematode is apparent, observes more convenient with existing agar, water liquid etc.
2, the invention enables living nematode in the higher substrate of hardness, to show as standing upright and violent Tail swaying behavior, and dead nematode is without standing and swinging, this makes nematodal accounting and dead judgement directly perceived visible, there will not be and lose worm, leakage worm phenomenon, observation time shortens in 1min, efficiency obviously improves, and the accuracy and precision of result of the test also obtains lifting; Due to polypide touchless hurt, just can carry out further physiological and biochemical analysis to nematode, make to utilize C. Elegans Automatic Screening to carry out that ecological risk is evaluated in biological monitoring fast and Environmental Health becomes possibility.
3, the present invention is compared with agar, water liquid culture medium, use nematode stress respondent behavior observation platform of the present invention to carry out the test of acute toxicology stress response, survival and the behavior etc. of nematode do not have difference, i.e. observation platform substrate of the present invention is the survival on nematode and behavior generation impact not.
Embodiment
Embodiment one: a kind of C. Elegans Automatic Screening stress response behavior observation platform substrate of present embodiment is by mass percentage by 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water composition of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA; Described MgSO
4and CaCl
2mass ratio be 1:1.
At the bottom of the observation platform of present embodiment has the black-and-blue medium by the blue colour developing of ALMA, compared with at the bottom of the approximate medium of nematode body colour, nematode is apparent, observes more convenient with existing agar, water liquid etc.
Present embodiment makes living nematode in the higher substrate of hardness, show as standing upright and violent Tail swaying behavior, and dead nematode is without standing and swinging, this makes nematodal accounting and dead judgement directly perceived visible, there will not be and lose worm, leakage worm phenomenon, observation time shortens in 1min, efficiency obviously improves, and the accuracy and precision of result of the test also obtains lifting; Due to polypide touchless hurt, just can carry out further physiological and biochemical analysis to nematode, make to utilize C. Elegans Automatic Screening to carry out that ecological risk is evaluated in biological monitoring fast and Environmental Health becomes possibility.
Present embodiment is compared with agar, water liquid culture medium, use nematode stress respondent behavior observation platform of the present invention to carry out the test of acute toxicology stress response, survival and the behavior etc. of nematode do not have difference, i.e. the observation platform substrate of present embodiment is the survival on nematode and behavior generation impact not.
Embodiment two: present embodiment is different from embodiment one: by mass percentage by 18%～22% MgSO
4, 18%～22% CaCl
2, blue and 30%～65% the distilled water composition of 5%～10% agarose, 1.5%～2.5% KCl, 1%～2% ALMA.Other are identical with embodiment one.
Embodiment three: present embodiment is different from embodiment one or two: by mass percentage by 16% MgSO
4, 16% CaCl
2, blue and 59% the distilled water composition of 6% agarose, 2% KCl, 1% ALMA.Other are identical with embodiment one or two.
Embodiment four: present embodiment is different from embodiment one to three: by mass percentage by 22% MgSO
4, 22% CaCl
2, blue and 41% the distilled water composition of 11% agarose, 2.5% KCl, 1.5% ALMA.Other are identical with embodiment one to three.
Embodiment five: present embodiment is different from one of embodiment one to four: by mass percentage by 25% MgSO
4, 25% CaCl
2, blue and 31% the distilled water composition of 14% agarose, 3% KCl, 2% ALMA.Other are identical with one of embodiment one to four.
Embodiment six: the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform substrate of present embodiment carries out according to the following steps:
One, take by mass percentage 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA;
Two, agarose step 1 being taken and KCl put in the distilled water that step 1 takes, and stir, and are heated to be cooled to 40 DEG C～45 DEG C after boiling, obtain mixed solution A;
Three, the MgSO that adds step 1 to take in the mixed solution A obtaining to step 2
4, CaCl
2with ALMA indigo plant, the KOH solution that is 5mol/L by concentration after stirring and dissolving is adjusted to 6.8～7.2 by pH value, obtains mixed solution B;
Four, mixed solution B step 3 being obtained pours in 24 porocyte culture plates, and liquid level is 0.1cm～0.2cm;
Five, the 24 porocyte culture plates that mixed solution B is housed are at room temperature placed on horizontal stand and are solidified to it, obtain the substrate of C. Elegans Automatic Screening stress response behavior observation platform.
Embodiment seven: present embodiment is different from one of embodiment one to six: step 2 is heated to be cooled to 42 DEG C after boiling.Other are identical with one of embodiment one to six.
Embodiment eight: present embodiment is different from one of embodiment one to seven: the KOH solution that is 5mol/L by concentration after step 3 stirring and dissolving is adjusted to 7.0 by pH value.Other are identical with one of embodiment one to seven.
Embodiment nine: present embodiment is different from one of embodiment one to eight: described in step 4, the bore dia of 24 porocyte culture plates is 11.0mm, hole height is 0.5cm.Other are identical with one of embodiment one to eight.
Embodiment ten: present embodiment is different from one of embodiment one to nine: step 4 liquid level is 0.15cm.Other are identical with one of embodiment one to nine.
Adopt following verification experimental verification effect of the present invention:
Test one: one, take by mass percentage the MgSO of 25g
4, 25g CaCl
2, the ALMA indigo plant of KCl, 2g of agarose, 3g of 15g and the distilled water of 30g;
Two, agarose step 1 being taken and KCl put in the distilled water that step 1 takes, and stir, and are heated to be cooled to 45 DEG C after boiling, obtain mixed solution A;
Three, the MgSO that adds step 1 to take in the mixed solution A obtaining to step 2
4, CaCl
2with ALMA indigo plant, the KOH solution that is 5mol/L by concentration after stirring and dissolving is adjusted to 7.0 by pH value, obtains mixed solution B;
Four, mixed solution B step 3 being obtained pours in 24 porocyte culture plates, and liquid level is 0.15cm;
Five, the 24 porocyte culture plates that mixed solution B is housed are at room temperature placed on horizontal stand and are solidified to it, obtain the substrate of C. Elegans Automatic Screening stress response behavior observation platform.
Utilize C. Elegans Automatic Screening stress response behavior observation platform of the present invention substrate to carry out C. Elegans Automatic Screening acute toxinology experiment, because contrast at the bottom of nematode body colour and medium fast black base is obvious, and living nematode shows as standing and swing, what make that the judgement of dead nematode becomes is directly perceived and simple, and counting efficiency obviously improves.Observation time shortens to 30～50s, without losing worm, leaking worm phenomenon, has improved the accuracy and precision of testing result.
Claims (10)
1. a C. Elegans Automatic Screening stress response behavior observation platform substrate, is characterized in that the substrate of C. Elegans Automatic Screening stress response behavior observation platform is by mass percentage by 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water composition of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA; Described MgSO
4and CaCl
2mass ratio be 1:1.
2. a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 1 substrate, is characterized in that by mass percentage by 18%～22% MgSO
4, 18%～22% CaCl
2, blue and 30%～65% the distilled water composition of 5%～10% agarose, 1.5%～2.5% KCl, 1%～2% ALMA.
3. a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 1 substrate, is characterized in that by mass percentage by 16% MgSO
4, 16% CaCl
2, blue and 59% the distilled water composition of 6% agarose, 2% KCl, 1% ALMA.
4. a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 1 substrate, is characterized in that by mass percentage by 22% MgSO
4, 22% CaCl
2, blue and 41% the distilled water composition of 11% agarose, 2.5% KCl, 1.5% ALMA.
5. a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 1 substrate, is characterized in that by mass percentage by 25% MgSO
4, 25% CaCl
2, blue and 31% the distilled water composition of 14% agarose, 3% KCl, 2% ALMA.
6. the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform as claimed in claim 1 substrate, is characterized in that the preparation method of C. Elegans Automatic Screening stress response behavior observation platform substrate carries out according to the following steps:
One, take by mass percentage 15%～25% MgSO
4, 15%～25% CaCl
2, blue and 30%～65% the distilled water of 3%～15% agarose, 1%～3% KCl, 1%～2% ALMA;
Two, agarose step 1 being taken and KCl put in the distilled water that step 1 takes, and stir, and are heated to be cooled to 40 DEG C～45 DEG C after boiling, obtain mixed solution A;
Three, the MgSO that adds step 1 to take in the mixed solution A obtaining to step 2
4, CaCl
2with ALMA indigo plant, the KOH solution that is 5mol/L by concentration after stirring and dissolving is adjusted to 6.8～7.2 by pH value, obtains mixed solution B;
Four, mixed solution B step 3 being obtained pours in 24 porocyte culture plates, and liquid level is 0.1cm～0.2cm;
Five, the 24 porocyte culture plates that mixed solution B is housed are at room temperature placed on horizontal stand and are solidified to it, obtain the substrate of C. Elegans Automatic Screening stress response behavior observation platform.
7. the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 6 substrate, is characterized in that step 2 is heated to be cooled to 42 DEG C after boiling.
8. the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 6 substrate, is characterized in that after step 3 stirring and dissolving that the KOH solution that is 5mol/L by concentration is adjusted to 7.0 by pH value.
9. the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 6 substrate, is characterized in that the bore dia of 24 porocyte culture plates is 11.0mm described in step 4, and hole height is 0.5cm.
10. the preparation method of a kind of C. Elegans Automatic Screening stress response behavior observation platform according to claim 6 substrate, is characterized in that step 4 liquid level is 0.15cm.
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