CN103868768A - Treatment method of scanning electron microscope samples of insect tentacles and appendages - Google Patents
Treatment method of scanning electron microscope samples of insect tentacles and appendages Download PDFInfo
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- CN103868768A CN103868768A CN201410051505.0A CN201410051505A CN103868768A CN 103868768 A CN103868768 A CN 103868768A CN 201410051505 A CN201410051505 A CN 201410051505A CN 103868768 A CN103868768 A CN 103868768A
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- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000012153 distilled water Substances 0.000 claims description 30
- 206010002091 Anaesthesia Diseases 0.000 claims description 22
- 230000037005 anaesthesia Effects 0.000 claims description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 20
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 20
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- 238000010828 elution Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 6
- 102000002322 Egg Proteins Human genes 0.000 claims description 6
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Images
Abstract
The invention relates to a treatment method of scanning electron microscope samples of insect tentacles and appendages, and belongs to the technical field of treatment of experimental samples. The treatment method provided by the invention comprises the steps of anaesthetizing and dissecting insects, fixing a sample through a glutaraldehyde-formaldehyde phosphate buffer, cleaning the sample and performing gradient dehydration with ethyl alcohol on the sample so as to obtain a scanning electron microscope sample before loading. The treatment method provided by the invention has the advantages that the problems that in the earlier stage treatment of the scanning electron microscope samples of the tentacles and appendages, scales of tentacles and appendages are not fully removed, the surface of the sample is not fully cleaned, the sample is hard to soak in a fixing solution thoroughly, and the sample is crisp and is easy to snap are solved, the surface structure of the treated sample is good, the deformation and pollution are not generated, a tissue surface is fully revealed, the sample has a good conductive property, and the real condition of the surface of the sample is effectively reflected.
Description
Technical field
The scanning electron microscope example disposal route that the present invention relates to a kind of insect feeler and appendage, belongs to laboratory sample processing technology field.
Background technology
Feeler and appendage are the important sense organs of insect, above them have many different types of receptors, and insect receptor is the epidermis part of specialization, be basic structural unit and the insect body perception external environment condition of insect receptor, carry out the information receiver of chemical communication.By insect receptor, insect can experience the volatile compound in host plant, and using this as mating, look for food and find the information in reproduction place, can be used for monitoring and attracting and destroying insects according to this characteristic.Utilizing the behavior of insect pheromone regulation and control insect is a kind of non-harmful insect control technology, insect seeking a spouse, look for food, in the activity such as gathering usually by chemical odor material Transfer information, and the major organs of experiencing these information is the chemocepter being distributed on feeler and appendage.Between the kind of insect and in planting, in chemical communication, voice communication and tactile communication, insect sensor plays an important role, and by feeler and the surperficial dissimilar receptor of appendage, insect body can sensing external environment, seek a spouse and take food.In chemical communication between plant and insect, insect sensor also plays conclusive effect, and it is regulating and controlling the multiple behavior of insect, such as luring insect trend host plant, stimulates insect's food-taking, and guiding insect is selected spawning place, pollinates and defends insect etc.Therefore, specify the sensor kind of insect, feature and distribution, to understanding its chemosensing system in depth, provide histology and morphologic basis for studying its chemical communication system, and be further to develop insect attractant or Behavioral interference agent to lay the foundation, thereby reach the object based theoretical of pest control.
Scanning electron microscope, is a kind of novel electronic optical instrument having developed rapidly since emerging as commercial Electronic Speculum the sixties in last century, thereby is a kind of electron microscope that utilizes electron beam scanning sample surfaces to obtain sample message.It can produce the high-definition picture of sample surfaces, and image is three-dimensional, and scanning electron microscope can be used to identify the surface structure of sample.In scanning electron microscope experiment, most insect imagos are active, the most bodies of larva are less, and the organs such as feeler and appendage are difficult to obtain intact sample, and such sample surfaces covers scale, body wall ossification intensity is high, dense structure, and most insect body surface back ofs the body cover wax coat, cause sample pre-treatments difficulty in Electronic Speculum processing.
Summary of the invention
The object of the present invention is to provide the scanning electron microscope example disposal route of a kind of insect feeler and appendage, solve in insect feeler and the processing in early stage of appendage scanning electron microscope, feeler and appendage scale are removed not exclusively, sample surfaces cleans not thorough, cannot remove insect feeler and appendage wax coat, immobile liquid soaks into difficulty, the sample problems such as frangibility that become fragile.
For achieving the above object, the present invention adopts following technical scheme:
A scanning electron microscope example disposal route for insect feeler and appendage, comprises the following steps:
(1) preparation of sample immobile liquid, cleaning fluid, anesthesia liquid
A. sample immobile liquid: by weight percentage, glutaraldehyde-formaldehyde phosphate buffer consists of glutaraldehyde 1-4%, formaldehyde 30-45%, surplus is phosphate buffer;
B. sample cleaning fluid:
Cleaning fluid I: the sodium bicarbonate solution of preparation 0.05mol/L, adjust PH to 10, then add the egg white that accounts for sodium bicarbonate solution volume 2.5-5%, obtain cleaning fluid I;
Cleaning fluid II: its composition consists of sodium chloride 6.0-7.2g, sodium hypochlorite 0.12-0.16g, lime chloride 0.12-0.14g, potassium chloride 0.11-0.15g and distilled water 1L;
C. sample anesthesia liquid:
The alcohol water blend of preparation 50%, adds ether, makes ether final volume concentration reach 1.2-3.5%;
(2) anesthesia insect: fasten adult with dactylethrae, clamp cotton balls dip in anesthesia liquid with tweezers, cotton balls is blocked the mouth of pipe holonarcosis insect keeping flat;
(3) dissecting insects: under insect anatomy microscope, with complete feeler and the appendage of tweezers and dissecting scissors clip step (2) insect after treatment;
(4) fixed sample: the sample after dissecting is dropped in the centrifuge tube that immobile liquid is housed rapidly centrifugal, until sample arrives below immobile liquid liquid level completely; Then shaking table vibration, makes immobile liquid and sample blending, is placed under room temperature and fixes 2-8h;
(5) clean sample: the sample after step (4) is fixing, gently move on in the cleaning fluid I of step (1) new system, immersion limit, limit vibrates on shaking table, and 20-40min for the first time vibrates; Clean and exchange cleaning fluid II for for the second time and later, the 10-15min that at every turn vibrates, until it is floating to can't see top layer wax coat in can't see the scale on feeler and appendage or clean waste liquid the last time under dissecting microscope; Finally wash 2-4 time each 5-10min with distilled water;
(6) sample dehydration: Gradient elution using ethanol, is then placed in 100% ethanol and preserves;
(7) sticky sample is observed: carefully beat easily feeler or appendage sample with little writing brush, two-sided gluing sample, faces up, complete alcohol volatilizees in fuming cupboard, then insect anatomy microscope preliminary observation, fine setting makes sample expose portion respectively as far as possible at grade, then goes up scanning electron microscopic observation.
Described in the preparation of described step (1) sample immobile liquid, phosphate buffering liquid concentration is 0.01mol/L-0.05 mol/L, and pH is 7.0-7.4, and the concrete preparation method of damping fluid is: dissolving phosphoric acid potassium dihydrogen KH in 500-800mL distilled water
2pO
41.4-7.0g, two hypophosphite monohydrate disodium hydrogen Na
2hPO
42H
2o 1.8-9.0g, sodium chloride 0.58-2.9g and potassium chloride 0.7-3.5g, then add 1 mol/L hydrochloric acid to adjust pH to 7.0-7.4, and autoclave sterilization is placed on 4 DEG C of Refrigerator stores.
In described step (1), the compound method of sample immobile liquid is: in phosphate buffered solution, add the glutaraldehyde solution of mass concentration 25% and the formalin of mass concentration 38%, making glutaraldehyde mass percentage concentration is 1-4%, and formaldehyde mass percentage concentration is 30-45%.
The preparation method of the cleaning fluid II of described step (1) is: successively by sodium chloride 6.0-7.2g, sodium hypochlorite 0.12-0.16g and potassium chloride 0.11-0.15g are dissolved in 60-100mL distilled water, then arrives 940-990mL with distilled water diluting; Finally get 0.12-0.14g lime chloride and be dissolved in 10-60mL distilled water, obtain calcium chloride solution, then calcium chloride solution is dropwise joined in above-mentioned solution, drip on limit, and stir on limit, until dissolve completely.
Described step (2) anesthesia duration 3-5s.
Described step (4) centrifugal condition is: the centrifugal 20-45s of 8000-12000r/min; Oscillating condition is: at 37 degree shaking table 80-100r/min vibration 25-40min; Step (4) sample after treatment need to spend the night, and needs to be placed in 4 DEG C of refrigerators and preserves.
Described step (6) Gradient elution using ethanol is used 50% ethanol dehydration 8-15min, 60% ethanol dehydration 10-15min, 70% ethanol dehydration 14-20min, 80% ethanol dehydration 15-20min, 90% ethanol dehydration 15-25min successively, 100% ethanol dehydration 2-3 time, each 15-25min.
The fuming cupboard wind speed of described step (7) is: 0.4-0.6m/s.
Positive beneficial effect of the present invention:
1. raw material of the present invention and reagent are easy to get, cheap, simple to operate;
2. immobile liquid of the present invention is glutaraldehyde-formaldehyde phosphate buffer, overcome fixedly cause separately with glutaraldehyde or formaldehyde separately fixing not thorough, the sample problem such as easy to break that becomes fragile, more easily soak into sample surfaces scale and wax coat, and add phosphate buffer more balance dissection offspring polypide liquid osmotic pressure shakiness, overcome the deformation effect of sample in fixing, and crystallized ability is strong, in fixation procedure, need not change immobile liquid, step is easy;
3. the adding of egg white in cleaning fluid of the present invention, not only can remove the epidermis dirt of insect feeler and appendage, be more conducive to the removal of sample surfaces scale and wax coat, pass through again the repeatedly rinsing of sample cleaning fluid II, scale and the wax coat that can remove the coating of sample surfaces completely, thoroughly expose sensor;
4. the present invention is after Gradient elution using ethanol, the alcohol that volatilizees in fuming cupboard, and sample is directly gone up sem observation, has saved dry, metal spraying step, and sample extensibility, and stability and electric conductivity are good, and each sensor is sudden and violent leakage thoroughly, clearly visible under scanning electron microscope.
Brief description of the drawings
Fig. 1 is 2 the female adult feeler of committee noctuid electron scanning electromicroscopic photographs that classic method is made;
Fig. 2 is 2 the female adult feeler of committee noctuid electron scanning electromicroscopic photographs of the making of the embodiment of the present invention 1;
Fig. 3 is 2 the female adult feeler of committee noctuid electron scanning electromicroscopic photographs of the making of the embodiment of the present invention 2;
Fig. 4 is 2 the female adult feeler of committee noctuid electron scanning electromicroscopic photographs of the making of the embodiment of the present invention 3.
Embodiment
Below in conjunction with some specific embodiments, the present invention is further described.
Sample source: field searchlight insect trapper lures 2 committee noctuids of collection, and 3 days adults sprout wings.
Embodiment 1
2 committee's noctuid adult feeler scanning electron microscope example preparation processes are as follows:
(1) preparation of sample immobile liquid, cleaning fluid, anesthesia liquid
Preparation phosphate buffer: dissolving phosphoric acid potassium dihydrogen KH in 800mL distilled water
2pO
41.4g, two hypophosphite monohydrate disodium hydrogen Na
2hPO
42H
2o 1.8g, sodium chloride 0.58g and potassium chloride 0.7g, then add 1mol/L hydrochloric acid to adjust pH to 7.4, and autoclave sterilization is placed on 4 DEG C of Refrigerator stores.
The preparation of sample immobile liquid: be configured to glutaraldehyde-formaldehyde phosphate buffer by above-mentioned phosphate buffer and the commercially available glutaraldehyde solution of mass concentration 25% and the formalin of commercially available mass concentration 38%, making glutaraldehyde quality percentage composition is 1%, and formaldehyde quality percentage composition is 30%.
The preparation of sample cleaning fluid: sample cleaning fluid I is: preparation 0.05mol/L sodium bicarbonate solution, adjust PH to 10, then add the egg white that accounts for sodium bicarbonate solution volume 2.5%, obtain cleaning fluid I;
Cleaning fluid II: its composition consists of sodium chloride 6.0g, sodium hypochlorite 0.12g, lime chloride 0.12g, potassium chloride 0.12g and distilled water 1L;
Concrete preparation process is: successively by sodium chloride, sodium hypochlorite and potassium chloride are dissolved in respectively in 100mL distilled water, then arrives 950mL with distilled water diluting; Finally get lime chloride and be dissolved in 50mL distilled water, obtain calcium chloride solution, then calcium chloride solution is dropwise joined in above-mentioned solution, drip on limit, stir on limit, until dissolve completely;
The preparation of insect anesthesia liquid: the alcohol water blend of preparation 50%, then add ether, until ether ultimate density reaches 1.2%;
(2) two committee's noctuid adult anesthesia: be 2-4cm with diameter under dark condition, length is that the sterilizing glass dactylethrae of 5cm is taken off 2 committee noctuid adults from worm cage, clamp cotton balls with tweezers and dip in a little anesthesia liquid, cotton balls is blocked the mouth of pipe 3-4s keeping flat, and insect is anaesthetized completely;
(3) two committee's noctuid adult feelers are dissected: under insect anatomy microscope, with tweezers and dissecting scissors by anaesthetize out of the count insect complete cut feeler;
(4) fixed sample: 2 committee's noctuid feelers after dissecting are dropped into and are equipped with in the centrifuge tube of immobile liquid rapidly, male and female separately, all collection is complete to treat required fixing sample, the centrifugal 45s of 12000 r/min, until sample arrives below immobile liquid liquid level completely, 37 degree shaking table 100r/min vibration 30min, make immobile liquid and sample blending thorough, are placed under room temperature and fix 4h;
(5) clean sample: the sample after fixing, gently move on in fresh special cleaning fluid I, immersion limit, the limit 30min that vibrates for the first time on shaking table, clean and exchange cleaning fluid II for for the second time and later, duration of oscillation is 10min, until can't see the scale on feeler and appendage or clean the last time waste liquid mesexine wax coat floating under dissecting microscope, finally wash 3 times each 10min with distilled water;
(6) sample dehydration: Gradient elution using ethanol is used 50% ethanol dehydration 10min, 60% ethanol dehydration 12min, 70% ethanol dehydration 14min, 80% ethanol dehydration 16min, 90% ethanol dehydration 20min successively, 100% ethanol dehydration 2 times, each 20min.After dehydration, be again placed in 100% ethanol and preserve;
(7) sticky sample is observed: sample is carefully beaten easily feeler or appendage sample from 100% ethanol with little writing brush, two-sided gluing sample, face up, fuming cupboard is placed, and fuming cupboard wind speed is 0.4m/s, treats that alcohol volatilizees totally completely, insect anatomy microscope preliminary observation, fine setting makes sample expose portion respectively as far as possible at grade, then goes up scanning electron microscopic observation, sees Fig. 2.
Embodiment 2
2 committee's noctuid adult feeler scanning electron microscope example preparation processes are as follows:
(1) preparation of sample immobile liquid, cleaning fluid, anesthesia liquid
Preparation phosphate buffer: dissolving phosphoric acid potassium dihydrogen KH in 500mL distilled water
2pO
44.2g, two hypophosphite monohydrate disodium hydrogen Na
2hPO
42H
2o 5.4g, sodium chloride 1.74g and potassium chloride 2.1g, then add 1 mol/L hydrochloric acid to adjust pH to 7.2, and autoclave sterilization is placed on 4 DEG C of Refrigerator stores.
The preparation of sample immobile liquid: be configured to glutaraldehyde-formaldehyde phosphate buffer by above-mentioned phosphate buffer and the commercially available glutaraldehyde solution of mass concentration 25% and the formalin of commercially available mass concentration 38%, making glutaraldehyde quality percentage composition is 3%, and formaldehyde quality percentage composition is 40%.。
The preparation of sample cleaning fluid: sample cleaning fluid I is: preparation 0.05mol/L sodium bicarbonate solution, adjust PH to 10, then add the egg white that accounts for sodium bicarbonate solution volume 4%, obtain cleaning fluid I;
Cleaning fluid II: its composition consists of sodium chloride 6.6g, sodium hypochlorite 0.14g, lime chloride 0.14g, potassium chloride 0.11g and distilled water 1L;
Concrete preparation process is: successively by sodium chloride, sodium hypochlorite and potassium chloride are dissolved in 60mL distilled water, then arrives 940mL with distilled water diluting; Finally get lime chloride and be dissolved in 60mL distilled water,, obtain calcium chloride solution, then calcium chloride solution is dropwise joined in above-mentioned solution, drip on limit, stir on limit, until dissolve completely;
The preparation of insect anesthesia liquid: the alcohol water blend of preparation 50%, then add ether, until ether ultimate density reaches 3.0%;
(2) two committee's noctuid adult anesthesia: be 2-4cm with diameter under dark condition, length is that the sterilizing glass dactylethrae of 5cm is taken off 2 committee noctuid adults from worm cage, clamp cotton balls with tweezers and dip in a little anesthesia liquid, cotton balls is blocked the mouth of pipe 3-3s keeping flat, and insect is anaesthetized completely;
(3) two committee's noctuid adult feelers are dissected: under insect anatomy microscope, with tweezers and dissecting scissors by anaesthetize out of the count insect complete cut feeler;
(4) fixed sample: 2 committee's noctuid feelers after dissecting are dropped into and are equipped with in the centrifuge tube of immobile liquid rapidly, male and female separately, all collection is complete to treat required fixing sample, the centrifugal 30s of 8000r/min, until sample arrives below immobile liquid liquid level completely, 37 degree shaking table 80r/min vibration 40min, make immobile liquid and sample blending thorough, are placed under room temperature and fix 8h;
(5) clean sample: the sample after fixing, gently move on in fresh special cleaning fluid I, immersion limit, the limit 20min that vibrates for the first time on shaking table, clean and exchange cleaning fluid II for for the second time and later, duration of oscillation is 12min, until can't see the scale on feeler and appendage or clean the last time waste liquid mesexine wax coat floating under dissecting microscope, finally wash 2 times each 5min with distilled water;
(6) sample dehydration: Gradient elution using ethanol is used 50% ethanol dehydration 8min, 60% ethanol dehydration 10min, 70% ethanol dehydration 20min, 80% ethanol dehydration 15min, 90% ethanol dehydration 15min successively, 100% ethanol dehydration 2 times, each 16min.After dehydration, be again placed in 100% ethanol and preserve;
(7) sticky sample is observed: sample is carefully beaten easily feeler or appendage sample from 100% ethanol with little writing brush, two-sided gluing sample, face up, fuming cupboard is placed, and fuming cupboard wind speed is 0.6m/s, treats that alcohol volatilizees totally completely, insect anatomy microscope preliminary observation, fine setting makes sample expose portion respectively as far as possible at grade, then goes up scanning electron microscopic observation, sees Fig. 3.
Embodiment 3
2 committee's noctuid adult feeler scanning electron microscope example preparation processes are as follows:
(1) preparation of sample immobile liquid, cleaning fluid, anesthesia liquid
Preparation phosphate buffer: dissolving phosphoric acid potassium dihydrogen KH in 700mL distilled water
2pO
47.0g, two hypophosphite monohydrate disodium hydrogen Na
2hPO
42H
2o 9.0g, sodium chloride 2.9g and potassium chloride 3.5g, then add 1mol/L hydrochloric acid to adjust pH to 7.0, and autoclave sterilization is placed on 4 DEG C of Refrigerator stores.
The preparation of sample immobile liquid: be configured to glutaraldehyde-formaldehyde phosphate buffer by above-mentioned phosphate buffer and the commercially available glutaraldehyde solution of mass concentration 25% and the formalin of commercially available mass concentration 38%, making glutaraldehyde quality percentage composition is 4%, and formaldehyde quality percentage composition is 45%.
The preparation of sample cleaning fluid: sample cleaning fluid I is: preparation 0.05mol/L sodium bicarbonate solution, adjust PH to 10, then add the egg white that accounts for sodium bicarbonate solution volume 5%, obtain cleaning fluid I;
Cleaning fluid II: its composition consists of sodium chloride 7.2g, sodium hypochlorite 0.16g, lime chloride 0.13g, potassium chloride 0.15g and distilled water 1L;
Concrete preparation process is: successively by sodium chloride, sodium hypochlorite and potassium chloride are dissolved in 70mL distilled water, then arrives 990mL with distilled water diluting; Finally get lime chloride and be dissolved in 10mL distilled water,, obtain calcium chloride solution, then calcium chloride solution is dropwise joined in above-mentioned solution, drip on limit, stir on limit, until dissolve completely;
The preparation of insect anesthesia liquid: the alcohol water blend of preparation 50%, then add ether, until ether ultimate density reaches 3.5%;
(2) two committee's noctuid adult anesthesia: be 2-4cm with diameter under dark condition, length is that the sterilizing glass dactylethrae of 5cm is taken off 2 committee noctuid adults from worm cage, clamp cotton balls with tweezers and dip in a little anesthesia liquid, cotton balls is blocked the mouth of pipe 5s keeping flat, and insect is anaesthetized completely;
(3) two committee's noctuid adult feelers are dissected: under insect anatomy microscope, with tweezers and dissecting scissors by anaesthetize out of the count insect complete cut feeler;
(4) fixed sample: 2 committee's noctuid feelers after dissecting are dropped into and are equipped with in the centrifuge tube of immobile liquid rapidly, male and female separately, all collection is complete to treat required fixing sample, the centrifugal 20s of 10000r/min, until sample arrives below immobile liquid liquid level completely, 37 degree shaking table 90r/min vibration 25min, make immobile liquid and sample blending thorough, are placed under room temperature and fix 2h;
(5) clean sample: the sample after fixing, gently move on in fresh special cleaning fluid I, immersion limit, the limit 40min that vibrates for the first time on shaking table, clean and exchange cleaning fluid II for for the second time and later, duration of oscillation is 15min, until can't see the scale on feeler and appendage or clean the last time waste liquid mesexine wax coat floating under dissecting microscope, finally wash 4 times each 8min with distilled water;
(6) sample dehydration: Gradient elution using ethanol is used 50% ethanol dehydration 15min, 60% ethanol dehydration 15min, 70% ethanol dehydration 15min, 80% ethanol dehydration 20min, 90% ethanol dehydration 25min successively, 100% ethanol dehydration 3 times, each 15min.After dehydration, be again placed in 100% ethanol and preserve;
(7) sticky sample is observed: sample is carefully beaten easily feeler or appendage sample from 100% ethanol with little writing brush, two-sided gluing sample, face up, fuming cupboard is placed, and fuming cupboard wind speed is 0.5m/s, treats that alcohol volatilizees totally completely, insect anatomy microscope preliminary observation, fine setting makes sample expose portion respectively as far as possible at grade, then goes up scanning electron microscopic observation, sees Fig. 4.
Interpretation of result
Referring to Fig. 1-4, as seen from the figure: utilize the present invention to process after 2 committee noctuid adults, scale is removed completely, and sensor is clearly visible, the scanning electron microscope example of conventional process, coating scale can not be removed completely, and most of sensor fails to expose.
Claims (8)
1. a scanning electron microscope example disposal route for insect feeler and appendage, is characterized in that, comprises the following steps:
(1) preparation of sample immobile liquid, cleaning fluid, anesthesia liquid
A. sample immobile liquid: by weight percentage, glutaraldehyde-formaldehyde phosphate buffer consists of glutaraldehyde 1-4%, formaldehyde 30-45%, surplus is phosphate buffer;
B. sample cleaning fluid:
Cleaning fluid I: the sodium bicarbonate solution of preparation 0.05mol/L, adjust PH to 10, then add the egg white that accounts for sodium bicarbonate solution volume 2.5-5%, obtain cleaning fluid I;
Cleaning fluid II: its composition consists of sodium chloride 6.0-7.2g, sodium hypochlorite 0.12-0.16g, lime chloride 0.12-0.14g, potassium chloride 0.11-0.15g and distilled water 1L;
C. sample anesthesia liquid:
The alcohol water blend of preparation 50%, adds ether, makes ether final volume concentration reach 1.2-3.5%;
(2) anesthesia insect: fasten adult with dactylethrae, clamp cotton balls dip in anesthesia liquid with tweezers, cotton balls is blocked the mouth of pipe holonarcosis insect keeping flat;
(3) dissecting insects: under insect anatomy microscope, with complete feeler and the appendage of tweezers and dissecting scissors clip step (2) insect after treatment;
(4) fixed sample: the sample after dissecting is dropped in the centrifuge tube that immobile liquid is housed rapidly centrifugal, until sample arrives below immobile liquid liquid level completely; Then shaking table vibration, makes immobile liquid and sample blending, is placed under room temperature and fixes 2-8h;
(5) clean sample: the sample after step (4) is fixing, gently move on in the cleaning fluid I of step (1) new system, immersion limit, limit vibrates on shaking table, and 20-40min for the first time vibrates; Clean and exchange cleaning fluid II for for the second time and later, the 10-15min that at every turn vibrates, until it is floating to can't see top layer wax coat in can't see the scale on feeler and appendage or clean waste liquid the last time under dissecting microscope; Finally wash 2-4 time each 5-10min with distilled water;
(6) sample dehydration: Gradient elution using ethanol, is then placed in 100% ethanol and preserves;
(7) sticky sample is observed: carefully beat easily feeler or appendage sample with little writing brush, two-sided gluing sample, faces up, the complete alcohol that volatilizees in fuming cupboard, and then insect anatomy microscope preliminary observation, then goes up scanning electron microscopic observation.
2. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, it is characterized in that, described in the preparation of step (1) sample immobile liquid, phosphate buffering liquid concentration is 0.01mol/L-0.05 mol/L, pH is 7.0-7.4, and the concrete preparation method of damping fluid is: dissolving phosphoric acid potassium dihydrogen KH in 500-800mL distilled water
2pO
41.4-7.0g, two hypophosphite monohydrate disodium hydrogen Na
2hPO
42H
2o 1.8-9.0g, sodium chloride 0.58-2.9g and potassium chloride 0.7-3.5g, then add 1 mol/L hydrochloric acid to adjust pH to 7.0-7.4, and autoclave sterilization is placed on 4 DEG C of Refrigerator stores.
3. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, it is characterized in that, in step (1), the compound method of sample immobile liquid is: in phosphate buffered solution, add the glutaraldehyde solution of mass concentration 25% and the formalin of mass concentration 38%, making glutaraldehyde mass percentage concentration is 1-4%, and formaldehyde mass percentage concentration is 30-45%.
4. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, it is characterized in that, the preparation method of step (1) cleaning fluid II is: successively by sodium chloride 6.0-7.2g, sodium hypochlorite 0.12-0.16g and potassium chloride 0.11-0.15g are dissolved in 60-100mL distilled water, then arrive 940-990mL with distilled water diluting; Finally get 0.12-0.14g lime chloride and be dissolved in 10-60mL distilled water, obtain calcium chloride solution, then calcium chloride solution is dropwise joined in above-mentioned solution, stir while dripping, until dissolve completely.
5. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, is characterized in that, step (2) anesthesia duration 3-5s.
6. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, is characterized in that, step (4) centrifugal condition is: the centrifugal 20-45s of 8000-12000r/min; Oscillating condition is: at 37 degree shaking table 80-100r/min vibration 25-40min; Step (4) sample after treatment need to spend the night, and needs to be placed in 4 DEG C of refrigerators and preserves.
7. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, it is characterized in that, step (6) Gradient elution using ethanol is used 50% ethanol dehydration 8-15min, 60% ethanol dehydration 10-15min, 70% ethanol dehydration 14-20min, 80% ethanol dehydration 15-20min, 90% ethanol dehydration 15-25min successively, 100% ethanol dehydration 2-3 time, each 15-25min.
8. the scanning electron microscope example disposal route of a kind of insect feeler according to claim 1 and appendage, is characterized in that, in step (7), the wind speed of fuming cupboard is 0.4-0.6m/s.
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