CN106644644A - Quick and low-toxic mosquito paraffin section manufacturing method - Google Patents
Quick and low-toxic mosquito paraffin section manufacturing method Download PDFInfo
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- CN106644644A CN106644644A CN201611261495.9A CN201611261495A CN106644644A CN 106644644 A CN106644644 A CN 106644644A CN 201611261495 A CN201611261495 A CN 201611261495A CN 106644644 A CN106644644 A CN 106644644A
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- paraffin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
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Abstract
The invention discloses a quick and low-toxic mosquito paraffin section manufacturing method and belongs to the technical field of microscopic tissue section. The method includes the steps of artificially feeding mosquitoes, preparing and fixing materials, dehydrating and transparentizing the materials, performing paraffin infiltration and embedding, cutting the materials into sections, and dyeing and sealing the sections. In the paraffin section manufacturing method, the mosquitoes are processed into the sections after being fed, so that intestines and esophagus of the mosquitoes can be observed intuitively. Before fixation, feet and wings of the mosquitoes are removed, so that on one hand, the mosquitoes are convenient to fix, and on the other hand, the follow-up paraffin infiltration process is accelerated and uniformity of the paraffin infiltration is ensured. Meanwhile, the fixing and paraffin infiltration processes in the method are carried out under negative pressure, so that infiltration of a fixing liquid and the paraffin is improved, thereby ensuring successful section cutting. In addition, the method reduces section preparation time at certain degree, and improves work efficiency. Finally, the method is free of xylene, so that the body damage on technical staffs in pathology department and environment pollution are reduced greatly.
Description
Technical field
The present invention relates to medical pathologies field, more particularly to a kind of quick, mosquito paraffin section preparation method of low toxicity.
Background technology
Paraffin method is method most widely used in histology conven tional tabletting techniques, and paraffin section is applied not only to see
The morphosis of normal cell tissue is examined, is also the subjects such as pathology and medical jurisprudence to study, observe and judge cell tissue
The main method of metamorphosis.The basic process of paraffin method be first the censorship fixed tissue Jing is rinsed, dehydration, thoroughly
Bright, waxdip, then with paraffin by organization embedding into wax stone, then sliced machine section and dye, be finally made paraffin section.
Paraffin section technology is also to study the important experimental technique of mosquito tissue morphology and histopathology.For cutting for mosquito
Piece is studied, and the viral distribu-tion and housing construction to grasping in mosquito body has great importance.But, mosquito worm ectoskeleton is more
Hard, water-soluble fixer, dehydrated alcohol etc. are difficult to rapid, homogeneous infiltration in whole mosquito, so as to be difficult to make complete
Paraffin section.Meanwhile, mosquito in-vivo tissue flexible relative is worth the presence of hemolymph, causes tissue as being suspended in fluid
In matter, if waxdip is not enough, it is difficult to cut out complete section.At present mosquito paraffin section method majority is used for the local organization of mosquito
Or organ (such as feeler, alimentary canal, foot), so not it is observed that the overall microstructure of mosquito.Pin is soft for this body
The larva polypide material that soft, content liquid is high, chamber ratio is big, the effect burying, cut into slices etc. is not ideal enough, and final section is to tissue
Discrimination is extremely limited, especially accounts for polypide ratio very large digestive tract and is easily deformed or crushes, it is difficult to it was observed that polypide is complete
Whole space structure.
Meanwhile, formalin, dimethylbenzene employed in traditional flaking method etc. are volatile, have strong impulse and height
Hypertoxicity, all extremely harmful to the working environment and living environment of pathology staff, Long Term Contact formalin may draw
Play chronic respiratory disease, cancer to occur, cause memory and intelligence to decline, gastric-intestinal perforation, liver damage are may occur in which when serious
Even go into a coma；(2) traditional organization processes all substeps are carried out, and the amount of reagent required for each process is big, to operation
The harm of personnel is big；The tissue treatment time is long, and the report from drawing materials to going out needs the time of 3-5d.
At present conventional dicing method not only complex operation, needs to consume a longer time, and often using the diformazan that toxicity is big
Benzene, it is volatile, have strong impulse and high hypertoxicity, the working environment and living environment of pathology staff are all extremely had
Evil, Long Term Contact may cause chronic respiratory disease, cancer to occur, and cause memory and intelligence to decline, and can go out when serious
Existing gastric-intestinal perforation, liver damage are even gone into a coma.More importantly finally the effect of fixed, embedding, section etc. is not ideal enough, finally
Section indexes extremely limited to tissue area, it is difficult to it was observed that polypide holonmic space structure.
The content of the invention
The technical problem to be solved is：In view of the shortcomings of the prior art, a kind of quick, mosquito of low toxicity
Paraffin section preparation method, simple to operate, time-consuming short, toxicity is low, and it is observed that the complete space structure of mosquito body.
To solve above-mentioned technical problem, the technical scheme is that：
A kind of quick, mosquito paraffin section preparation method of low toxicity, comprises the following steps：
1) artificial feeding takes some mosquitoes, and hungry 10-24h is put into mosquito cage, and is put into the sponge for dipping in full mouse blood
Block, takes out mosquito after 30min-60min.
2) to draw materials and freeze fiber crops with the fixed mosquito by after nursing, then remove leg and wing, be put into fixer and fix, negative
The fixed 1h of pressure, continues to fix 12-24h under room temperature, with 60% alcohol washes 2-3 time, is then stored in standby in 60% alcohol.
3) dehydration, it is transparent take it is fixed after polypide, under the conditions of 4 DEG C, successively with concentration gradient be 60%, 70%,
90%th, 100% I, 100% II ethanol solution dehydration, the process time of each gradient is at least 1 hour, is finally added to
Heating in bright dose is boiled, and is carried out transparent.100% I, 100% II is the ethanol that concentration is 100%.
4) thoroughly wax, embedding, by transparent good polypide in 3), are put into Hemo-De and paraffin 1 under condition of negative pressure：1 mixing
20-40min is soaked in solution, immersion 30min-1h tri- times in paraffin are then placed in, wax process is completed, finally, hard wax is put into
In complete embedding process.Hemo-De is a kind of clarifier for being derived from n- limonenes.
5) by embedded paraffin mass in 4) through segmentation, finishing and set, then sliced machine cuts paraffin mass for section
Into 5-8 μm of section, and open up piece.
6) dyeing is concretely comprised the following steps：2-3min is dyeed using haematine dye liquor, running water rinses 5-10min, 1% hydrochloric acid
Alcohol 5-10s, originally washes 3-5min, the aobvious indigo plant 30s of weak alkaline aqueous solution, originally washes 5-10min, and distillation is washed 2-3 time,
0.25% aqueous solution eosin stain dyes 2-3min, and gradient alcohol dehydration is finally used in distillation washing 1-2 time.
7) the gummy mounting that mounting is diluted using turpentine oil.
As a kind of preferred scheme, step 2) in, the fixer is that BouinShi liquid or the PBS containing 4% formaldehyde are molten
Liquid.
Used as a kind of preferred scheme, described BouinShi liquid is formulated by volume by following reagent：Saturation is bitter
15 parts of taste acid, 5 parts of formalin, 1 part of glacial acetic acid.
As a kind of preferred scheme, step 3) in, the clarifier is in hexahydrotoluene, chloroform or n-butanol
Kind.
As a kind of preferred scheme, step 2) in negative pressure be -0.05～-0.3MPa, step 4) in negative pressure for -
0.1～-0.5MPa.
As a kind of preferred scheme, step 4) in, the polypide is put into immersion 30min-1h in paraffin and is specially for tri- times
Soak to be soaked at 30min, 55-58 DEG C at 50-55 DEG C and soak 1h at 30min, 58-60 DEG C.
As a kind of preferred scheme, step 5) in, the slicer is cycle type paraffin slicing machine.
As a result of above-mentioned technical proposal, the invention has the beneficial effects as follows：
Paraffin section preparation method according to the present invention, it is harder for mosquito outer wall, fixer be difficult to it is rapid, uniformly ooze
Thoroughly；Meanwhile, in-vivo tissue is relatively soft, the characteristic for being then difficult section that saturating wax is not enough, takes series of measures.First, take and feed
Mosquito after supporting is cut into slices, more intuitively the intestines and esophagus of observation mosquito.Then, the fixed front pin and wing for removing mosquito, one
Aspect is easy to fix, and on the other hand accelerates follow-up saturating wax process, it is ensured that the uniformity of saturating wax.Meanwhile, this preparation method is consolidated
Fixed and saturating wax process is carried out under negative pressure, and the resistance of tissue and solvent is near minimum, is more beneficial for the leaching of fixer and paraffin
Enter, so as to ensure that what is cut into slices is smoothed out；The also less to a certain extent time of film-making, accelerate operating efficiency.Finally, originally
Preparation method, in fixer, clarifier, the mountant equal solvent of employing and be not used dimethylbenzene, greatly reduce to pathology department
The body harm of technical staff and the pollution to environment.
Specific embodiment
With reference to embodiment, the present invention is expanded on further.It should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Embodiment 1
A kind of quick, mosquito paraffin section preparation method of low toxicity, comprises the following steps：
1) artificial feeding takes some mosquitoes, and hungry 12h is put into mosquito cage, and is put into the sponge block for dipping in full mouse blood,
Mosquito is taken out after 30min.
2) to draw materials and freeze fiber crops with the fixed mosquito by after nursing, leg and wing are then removed, in being put into fixer BouinShi liquid
It is fixed, 1h is fixed under negative pressure, continue to fix 12h under room temperature, with 60% alcohol washes 2 times, then it is stored in 60% alcohol (
Refer to concentration) in it is standby.
3) dehydration, it is transparent take it is fixed after polypide, under the conditions of 4 DEG C, successively with concentration gradient be 60%, 70%,
90%th, 100% I, 100% II ethanol solution dehydration, the process time of each gradient is 1 hour, is finally added to clarifier
Heating in hexahydrotoluene is boiled, and is carried out transparent.
4) thoroughly wax, embedding, by transparent good polypide in 3), are put into Hemo-De and paraffin 1 under condition of negative pressure：1 mixing
20min is soaked in solution, be then placed in paraffin soak three times, three times be specially 30min is soaked at 50 DEG C, at 55 DEG C soak
30min, soaks 1h and completes wax process at 58 DEG C, finally, be put into hard wax and complete embedding process.
5) section by embedded paraffin mass in 4) through segmentation, finishing and set, then Jing cycle types paraffin slicing machine
Paraffin mass is cut into 5 μm of section, and opens up piece.
6) dyeing is concretely comprised the following steps：2min is dyeed using haematine dye liquor, running water rinses 5min, 1% hydrochloride alcohol
5s, originally washes 3min, the aobvious indigo plant 30s of weak alkaline aqueous solution, originally washes 5min, distillation washing 2 times, 0.25% aqueous solution Yihong
Dye liquor dyes 2min, distillation washing 1 time.Section after dyeing still can not be examined under a microscope, and need Jing gradient alcohol dehydrations,
95% and absolute alcohol in time can suitably lengthen it is thorough to ensure dehydration.
7) mounting makes sample using the gummy mounting of turpentine oil dilution.
Embodiment 2
A kind of quick, mosquito paraffin section preparation method of low toxicity, comprises the following steps：
1) artificial feeding takes some mosquitoes, and hungry 18h is put into mosquito cage, and is put into the sponge block for dipping in full mouse blood,
Mosquito is taken out after 60min.
2) to draw materials and freeze fiber crops with the fixed mosquito by after nursing, then remove leg and wing, be put into fixer and fix, negative
Fix 1h under pressure -0.1MPa, continue to fix 15h under room temperature, with 60% alcohol washes 3 times, be then stored in standby in 60% alcohol
With.
3) dehydration, it is transparent take it is fixed after polypide, under the conditions of 4 DEG C, successively with concentration gradient be 60%, 70%,
90%th, 100% I, 100% II ethanol solution dehydration, the process time of each gradient is 1 hour, is finally added to clarifier
Middle heating is boiled, and is carried out transparent.100% I, 100% II is the ethanol that concentration is 100%.
4) thoroughly wax, embedding, by transparent good polypide in 3), are put into Hemo-De and paraffin 1 under the conditions of negative pressure -0.3MPa：
30min is soaked in 1 mixed solution, is then placed in being soaked in paraffin three times, be specially for three times and to soak 30min at 52 DEG C, 55
30min is soaked at DEG C, 1h is soaked at 60 DEG C and is completed wax process, complete wax process, finally, be put into hard wax and complete to embed
Journey.Hemo-De is a kind of clarifier for being derived from n- limonenes.
5) by embedded paraffin mass in 4) through segmentation, finishing and set, then sliced machine cuts paraffin mass for section
Into 8 μm of section, and open up piece.
6) dyeing is concretely comprised the following steps：3min is dyeed using haematine dye liquor, running water rinses 10min, 1% hydrochloride alcohol
10s, originally washes 5min, the aobvious indigo plant 30s of weak alkaline aqueous solution, originally washes 10min, and distillation is washed 3 times, 0.25% aqueous solution she
Red dye liquor dyes 3min, and gradient alcohol dehydration is finally used in distillation washing 2 times.
7) after mounting turpentine oil is transparent, surrounding materials surplus liquid is wiped rapidly, appropriate (1～2 drop) neutral tree is added dropwise
Glue, then the inclination of clean cover glass is put down, in order to avoid there is bubble, permanent slide sample is made after mounting.
Claims (7)
1. a kind of quick, mosquito paraffin section preparation method of low toxicity, it is characterised in that comprise the following steps：
1) artificial feeding takes some mosquitoes, and hungry 10-24h is put into mosquito cage, and is put into the sponge block for dipping in full mouse blood,
Mosquito is taken out after 30min-60min；
2) to draw materials and freeze fiber crops with the fixed mosquito by after nursing, then remove leg and wing, be put into fixer and fix, it is solid under negative pressure
Determine 1h, continue to fix 12-24h under room temperature, with 60% alcohol washes 2-3 time, be then stored in standby in 60% alcohol；
3) dehydration, it is transparent take it is fixed after polypide, under the conditions of 4 DEG C, successively with concentration gradient be 60%, 70%, 90%,
100% I, 100% II ethanol solution dehydration, the process time of each gradient is at least 1 hour, in being finally added to clarifier
Heating is boiled, and is carried out transparent；
4) thoroughly wax, embedding, by transparent good polypide in 3), are put into Hemo-De and paraffin 1 under condition of negative pressure：1 mixed solution
Middle immersion 20-40min, is then placed in immersion 30min-1h tri- times in paraffin, completes wax process, finally, is put into complete in hard wax
Into embedding process；
5) by embedded paraffin mass in 4) through segmentation, finishing and set, then paraffin mass is cut into 5-8 by sliced machine for section
μm section, and open up piece；
6) dyeing is concretely comprised the following steps：2-3min is dyeed using haematine dye liquor, running water rinses 5-10min, 1% hydrochloride alcohol 5-
10s, originally washes 3-5min, the aobvious indigo plant 30s of weak alkaline aqueous solution, originally washes 5-10min, distillation washing 2-3 time, 0.25% water
Solution eosin stain dyes 2-3min, and gradient alcohol dehydration is finally used in distillation washing 1-2 time；
7) the gummy mounting that mounting is diluted using turpentine oil.
2. as claimed in claim 1 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that step 2) in, institute
Fixer is stated for BouinShi liquid or the PBS solution containing 4% formaldehyde.
3. as claimed in claim 1 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that step 2) in
Negative pressure be -0.05～-0.3MPa, step 4) in negative pressure be -0.1～-0.5MPa.
4. as claimed in claim 2 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that described Bouin
Family name's liquid is formulated by volume by following reagent：15 parts of saturation picric acid, 5 parts of formalin, 1 part of glacial acetic acid.
5. as claimed in claim 1 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that step 3) in, institute
Clarifier is stated for the one kind in hexahydrotoluene, chloroform or n-butanol.
6. as claimed in claim 1 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that step 4) in, worm
Body is put into paraffin to soak 30min-1h and be specially for tri- times and soaks 30min at 50-55 DEG C respectively, soaks at 55-58 DEG C
1h is soaked at 30min, 58-60 DEG C.
7. as claimed in claim 1 quickly, the mosquito paraffin section preparation method of low toxicity, it is characterised in that step 5) in, institute
Slicer is stated for cycle type paraffin slicing machine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611261495.9A CN106644644A (en) | 2016-12-30 | 2016-12-30 | Quick and low-toxic mosquito paraffin section manufacturing method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611261495.9A CN106644644A (en) | 2016-12-30 | 2016-12-30 | Quick and low-toxic mosquito paraffin section manufacturing method |
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| Publication Number | Publication Date |
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| CN106644644A true CN106644644A (en) | 2017-05-10 |
Family
ID=58837946
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611261495.9A Pending CN106644644A (en) | 2016-12-30 | 2016-12-30 | Quick and low-toxic mosquito paraffin section manufacturing method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389412A (en) * | 2017-08-31 | 2017-11-24 | 山东农业大学 | A kind of paraffin section preparation method of Shrimp waste watery tissue |
| CN107843470A (en) * | 2017-10-13 | 2018-03-27 | 济南金域医学检验中心有限公司 | A kind of preparation method of Skin slice |
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101390510A (en) * | 2008-11-12 | 2009-03-25 | 中国农业大学 | Preprocessing method of Orthoptera insect integer serial section |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
-
2016
- 2016-12-30 CN CN201611261495.9A patent/CN106644644A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101390510A (en) * | 2008-11-12 | 2009-03-25 | 中国农业大学 | Preprocessing method of Orthoptera insect integer serial section |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
Non-Patent Citations (1)
| Title |
|---|
| 谢超等: "蚊虫连续石蜡切片制备及染色", 《昆虫知识》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389412A (en) * | 2017-08-31 | 2017-11-24 | 山东农业大学 | A kind of paraffin section preparation method of Shrimp waste watery tissue |
| CN107389412B (en) * | 2017-08-31 | 2019-10-22 | 山东农业大学 | A kind of paraffin section production method of Shrimp waste watery tissue |
| CN107843470A (en) * | 2017-10-13 | 2018-03-27 | 济南金域医学检验中心有限公司 | A kind of preparation method of Skin slice |
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN109556933B (en) * | 2018-12-12 | 2021-08-17 | 宁波中盛产品检测有限公司 | Method for quickly preparing perineal pattern of root-knot nematode |
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Application publication date: 20170510 |