CN103875606B - A kind of preparation method of C. Elegans Automatic Screening stress response measuring behavior platform base - Google Patents
A kind of preparation method of C. Elegans Automatic Screening stress response measuring behavior platform base Download PDFInfo
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- CN103875606B CN103875606B CN201410091287.3A CN201410091287A CN103875606B CN 103875606 B CN103875606 B CN 103875606B CN 201410091287 A CN201410091287 A CN 201410091287A CN 103875606 B CN103875606 B CN 103875606B
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- automatic screening
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- measuring behavior
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Abstract
A preparation method for C. Elegans Automatic Screening stress response measuring behavior platform base, the present invention relates to a kind of preparation method of C. Elegans Automatic Screening stress response measuring behavior platform base.The present invention be to solve to count at the bottom of existing nematode toxicological test medium that large, the observation time of inaccurate, dead error in judgement is long, measuring behavior not problem accurately.C. Elegans Automatic Screening stress response measuring behavior platform base is by mass percentage by MgSO
4, CaCl
2, the blue and distilled water composition of agarose, KCl, ALMA.Method: the agarose taken and KCl are poured in distilled water and stirs, rear cooling is boiled in heating; Add MgSO
4and CaCl
2, and ALMA is blue, with the KOH solution adjust pH of 5mol/L.Pour 24 well culture plates into, liquid level is 0.1 ~ 0.2cm; Being statically placed in solidifying on horizontal stand, namely obtaining C. Elegans Automatic Screening stress response measuring behavior platform base.The present invention is mainly used in nematode toxicological test.
Description
Technical field
The present invention relates to a kind of preparation method of C. Elegans Automatic Screening stress response measuring behavior platform base.
Background technology
C. Elegans Automatic Screening Caenorhabditiselegans belongs to Nemathelminthes (Nematoda), surrounding environment change can be experienced more delicately, its cycle history of life is short, indicative function can be produced at short notice to environmental change, because the feature of easily cultivate, preserving is widely used as model organism, in the experimental sciences such as toxicology, carry out the measuring behavior research in the field such as environmental science, life science, and be used as the susceptibility indicator organism of ecotope health assessment.
The C. Elegans Automatic Screening carrier carrying out toxicology test measuring behavior is generally agar medium or tested liquid.C. Elegans Automatic Screening health is transparent, and body is long general at about 1mm, visible under the microscope.Classical toxicologic index comprises lethality, Behavioral change etc., and these indexs all require to carry out death determination, counting and measuring behavior etc. to test nematode.Wherein the judgement of nematode death, needs by instrument, if touch reactionless, is defined as death, and nematode on the point of dying often touches reactionless 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, nematode is close with the culture matrix color such as agar, water liquid, unfavorable counting and observation; C. Elegans Automatic Screening usually has situation about piercing in agaropectin, occurs losing worm phenomenon, which increases the difficulty of counting and behavior judgement; Under microscope, in observation water liquid, nematode often has fluctuation water wave, occurs under-enumeration, and it is accurate that this can affect nematodal accounting, and particularly polypide rocks with water, and cannot judge that whether nematode is dead.Generally one conventional in toxicological test is coerced to the death identification of lower 10 insects, at least need 5min, insect cannot find owing to piercing in agaropectin, makes sum often lower than 10.And behavior measure is carried out to it, especially may up to 0.5h.Therefore conventional medium makes C. Elegans Automatic Screening observation effort in toxicological test mistake, 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 to count at the bottom of existing nematode toxicological test medium that large, the observation time of inaccurate, dead error in judgement is long, measuring behavior not problem accurately, and provide a kind of C. Elegans Automatic Screening stress response measuring behavior platform base and preparation method thereof.
A kind of C. Elegans Automatic Screening stress response measuring behavior platform base of the present invention by mass percentage by 15% ~ 25% MgSO
4, 15% ~ 25% CaCl
2, 3% ~ 15% agarose, 1% ~ 3% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water composition; Described MgSO
4and CaCl
2mass ratio be 1:1.
The preparation method of above-mentioned a kind of C. Elegans Automatic Screening stress response measuring behavior platform base carries out according to the following steps:
One, the MgSO of 15% ~ 25% is taken by mass percentage
4, 15% ~ 25% CaCl
2, 3% ~ 15% agarose, 1% ~ 3% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water;
Two, agarose step one taken and KCl put in the distilled water that step one takes, and stir, and be cooled to 40 DEG C ~ 45 DEG C, obtain mixed solution A after being heated to boiling;
Three, the MgSO that step one takes is added in the mixed solution A obtained to step 2
4, CaCl
2blue with ALMA, by the KOH solution that concentration is 5mol/L, pH value is adjusted to 6.8 ~ 7.2 after stirring and dissolving, obtains mixed solution B;
Four, mixed solution B step 3 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 at room temperature are placed on horizontal stand solidify to it, namely obtain C. Elegans Automatic Screening stress response measuring behavior platform base;
Advantage of the present invention: 1, observation platform of the present invention has by the bottom of the black-and-blue medium of ALMA indigo plant colour developing, and compared with at the bottom of the medium that existing agar, water liquid etc. are approximate with nematode body colour, nematode is apparent, observes more convenient.
2, the invention enables living nematode in the substrate that hardness is higher, show as standing upright and violent Tail swaying behavior, and dead nematode is without standing and swinging, this make to nematodal accounting and death determination intuitively visible, there will not be and lose worm, leakage worm phenomenon, within observation time shortens to 1min, efficiency significantly 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 biological monitoring fast and evaluated ecological risk and Environmental Health becomes possibility.
3, the present invention is compared with agar, water liquid culture medium, nematode stress respondent behavior observation platform of the present invention is used to carry out the test of acute toxicology stress response, the survival of nematode and behavior etc. do not have difference, and namely observation platform substrate of the present invention does not have an impact to the survival of nematode and behavior.
Embodiment
Embodiment one: a kind of C. Elegans Automatic Screening stress response measuring behavior platform base of present embodiment by mass percentage by 15% ~ 25% MgSO
4, 15% ~ 25% CaCl
2, 3% ~ 15% agarose, 1% ~ 3% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water composition; Described MgSO
4and CaCl
2mass ratio be 1:1.
The observation platform of present embodiment has by the bottom of the black-and-blue medium of ALMA indigo plant colour developing, and compared with at the bottom of the medium that existing agar, water liquid etc. are approximate with nematode body colour, nematode is apparent, observes more convenient.
Present embodiment makes living nematode in the substrate that hardness is higher, show as standing upright and violent Tail swaying behavior, and dead nematode is without standing and swinging, this make to nematodal accounting and death determination intuitively visible, there will not be and lose worm, leakage worm phenomenon, within observation time shortens to 1min, efficiency significantly 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 biological monitoring fast and evaluated ecological risk and Environmental Health becomes possibility.
Present embodiment is compared with agar, water liquid culture medium, nematode stress respondent behavior observation platform of the present invention is used to carry out the test of acute toxicology stress response, the survival of nematode and behavior etc. do not have difference, and namely the observation platform substrate of present embodiment does not have an impact to the survival of nematode and behavior.
Embodiment two: present embodiment and embodiment one unlike: by mass percentage by 18% ~ 22% MgSO
4, 18% ~ 22% CaCl
2, 5% ~ 10% agarose, 1.5% ~ 2.5% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water composition.Other are identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two unlike: by mass percentage by 16% MgSO
4, 16% CaCl
2, 6% agarose, 2% KCl, 1% ALMA blue and 59% distilled water composition.Other are identical with embodiment one or two.
Embodiment four: present embodiment and embodiment one to three unlike: by mass percentage by 22% MgSO
4, 22% CaCl
2, 11% agarose, 2.5% KCl, 1.5% ALMA blue and 41% distilled water composition.Other are identical with embodiment one to three.
Embodiment five: one of present embodiment and embodiment one to four unlike: by mass percentage by 25% MgSO
4, 25% CaCl
2, 14% agarose, 3% KCl, 2% ALMA blue and 31% distilled water composition.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 measuring behavior platform base of present embodiment carries out according to the following steps:
One, the MgSO of 15% ~ 25% is taken by mass percentage
4, 15% ~ 25% CaCl
2, 3% ~ 15% agarose, 1% ~ 3% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water;
Two, agarose step one taken and KCl put in the distilled water that step one takes, and stir, and be cooled to 40 DEG C ~ 45 DEG C, obtain mixed solution A after being heated to boiling;
Three, the MgSO that step one takes is added in the mixed solution A obtained to step 2
4, CaCl
2blue with ALMA, by the KOH solution that concentration is 5mol/L, pH value is adjusted to 6.8 ~ 7.2 after stirring and dissolving, obtains mixed solution B;
Four, mixed solution B step 3 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 at room temperature are placed on horizontal stand solidify to it, namely obtain C. Elegans Automatic Screening stress response measuring behavior platform base.
Embodiment seven: one of present embodiment and embodiment one to six are cooled to 42 DEG C after being heated to boiling unlike: step 2.Other are identical with one of embodiment one to six.
Embodiment eight: one of present embodiment and embodiment one to seven unlike: by the KOH solution that concentration is 5mol/L, pH value is adjusted to 7.0 after step 3 stirring and dissolving.Other are identical with one of embodiment one to seven.
Embodiment nine: one of present embodiment and embodiment one to eight unlike: described in step 4, the bore dia of 24 porocyte culture plates is 11.0mm, and hole height is 0.5cm.Other are identical with one of embodiment one to eight.
Embodiment ten: one of present embodiment and embodiment one to nine unlike: 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 the MgSO of 25g by mass percentage
4, 25g CaCl
2, the agarose of 15g, KCl, 2g of 3g the distilled water of the blue and 30g of ALMA;
Two, agarose step one taken and KCl put in the distilled water that step one takes, and stir, and be cooled to 45 DEG C, obtain mixed solution A after being heated to boiling;
Three, the MgSO that step one takes is added in the mixed solution A obtained to step 2
4, CaCl
2blue with ALMA, by the KOH solution that concentration is 5mol/L, pH value is adjusted to 7.0 after stirring and dissolving, obtains mixed solution B;
Four, mixed solution B step 3 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 at room temperature are placed on horizontal stand solidify to it, namely obtain C. Elegans Automatic Screening stress response measuring behavior platform base.
C. Elegans Automatic Screening stress response measuring behavior platform base of the present invention is utilized to carry out C. Elegans Automatic Screening acute toxinology experiment, owing to contrasting obviously at the bottom of nematode body colour and medium fast black base, and living nematode shows as standing and swing, what the judgement of dead nematode was become is intuitively simple, and counting efficiency significantly improves.Observation time shortens to 30 ~ 50s, without losing worm, leaking worm phenomenon, improves the accuracy and precision testing result.
Claims (5)
1. a preparation method for C. Elegans Automatic Screening stress response measuring behavior platform base, is characterized in that the preparation method of C. Elegans Automatic Screening stress response measuring behavior platform base carries out according to the following steps:
One, the MgSO of 15% ~ 25% is taken by mass percentage
4, 15% ~ 25% CaCl
2, 3% ~ 15% agarose, 1% ~ 3% KCl, 1% ~ 2% ALMA blue and 30% ~ 65% distilled water;
Two, agarose step one taken and KCl put in the distilled water that step one takes, and stir, and be cooled to 40 DEG C ~ 45 DEG C, obtain mixed solution A after being heated to boiling;
Three, the MgSO that step one takes is added in the mixed solution A obtained to step 2
4, CaCl
2blue with ALMA, by the KOH solution that concentration is 5mol/L, pH value is adjusted to 6.8 ~ 7.2 after stirring and dissolving, obtains mixed solution B;
Four, mixed solution B step 3 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 at room temperature are placed on horizontal stand solidify to it, namely obtain C. Elegans Automatic Screening stress response measuring behavior platform base.
2. the preparation method of a kind of C. Elegans Automatic Screening stress response measuring behavior platform base according to claim 1, is characterized in that step 2 is cooled to 42 DEG C after being heated to boiling.
3. the preparation method of a kind of C. Elegans Automatic Screening stress response measuring behavior platform base according to claim 1, is adjusted to 7.0 by the KOH solution that concentration is 5mol/L by pH value after it is characterized in that step 3 stirring and dissolving.
4. the preparation method of a kind of C. Elegans Automatic Screening stress response measuring behavior platform base according to claim 1, it is characterized in that the bore dia of 24 porocyte culture plates described in step 4 is 11.0mm, hole height is 0.5cm.
5. the preparation method of a kind of C. Elegans Automatic Screening stress response measuring behavior platform base according to claim 1, is characterized in that step 4 liquid level is 0.15cm.
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