CN105806680A - Quick paraffin sectioning method of plant tissues - Google Patents
Quick paraffin sectioning method of plant tissues Download PDFInfo
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- CN105806680A CN105806680A CN201610387172.8A CN201610387172A CN105806680A CN 105806680 A CN105806680 A CN 105806680A CN 201610387172 A CN201610387172 A CN 201610387172A CN 105806680 A CN105806680 A CN 105806680A
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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
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Abstract
The invention discloses a quick paraffin sectioning method of plant tissues. The quick paraffin sectioning method includes the steps of assembling a reaction unit which is assembled by a numerical control thermostat heating jacket and a round-bottom flask, and filling in fixative; placing a small fragment of plant into the unit, and fixing at 60-65 DEG C for 5-10min; performing gradient dehydration with alcohol for 1-2 min respectively; performing dimethylbenzene transparency treatment for 1-2min; adding molten paraffin, performing numerical control thermostat heating at the temperature of 65-75 DEG C for 10-15min to obtain a paraffin-impregnated plant fragment; performing embedding treatment; performing sectioning, drying, and dyeing to obtain plant tissue paraffin sections. Compared with the traditional paraffin sectioning method, the quick paraffin sectioning method of plant tissues greatly shortens the production cycle, which is shortened from 9 days to 7 days.
Description
Technical field
The present invention relates to plant technology field, be more particularly to a kind of quick paraffin system of plant tissue
The method of sheet.
Background technology
Paraffin wax flaking technology is a kind of technology the most frequently used in organic section making technology, typically can be through
The material of various agent treated in paraffin must be lived can use.Connect owing to it can be cut into relatively thin
Continuous section, plant anatomy scholar mainly by its observation of cell tissue and the form of organ, makes
Permanent slide preserves for a long time.But prior paraffin method of tableting step includes drawing materials, fixes, is dehydrated,
Transparent, waxdip, embedding, cut into slices and dyeing etc., the cycle substantially needs 9 days, uses chemical reagent
More, and some reagent has certain murder by poisoning to human body.Therefore, improve prior paraffin method,
Shortening the film-making cycle, finding a kind of quick paraffin wax flaking technology is to have certain meaning.
Summary of the invention
(1) to solve the technical problem that
The technical problem to be solved in the present invention is how to shorten and makes plant tissue paraffin wax flaking
Time, reduce toxic pharmaceuticals usage amount, and the side of the quick paraffin wax flaking of a kind of plant tissue is provided
Method.
(2) technical scheme
In order to solve above-mentioned technical problem, the invention provides a kind of quick paraffin system of plant tissue
The method of sheet (the equal city of material therefor available from), the method comprises the steps:
Step one: preparation: assemble reaction unit, adds fixative, and described fixative is joined
Putting weight ratio is formaldehyde: glacial acetic acid: ethanol=3-5: 5-8: 80-95；
Step 2: fixing process: by the fresh plant tissue section of cutting into pieces, piece size is
0.5-1.5cm, is then placed in reaction unit, and digital control constant temperature heats, and temperature range is 60-65 DEG C,
The process time is 5-10min, changes fixative, repeats process 2 times；
Step 3: processed: outwell the fixative used in step 2, change weight successively
Percent concentration is respectively 70%, the alcohol water blend of 85%, 95%, 100%, 100% II,
Temperature is transferred to 100-110 DEG C, under every kind of concentration alcohol water blend, processes 1-2min, planted
Thing fragment carries out ethanol serial dehydration；
Step 4: transparent processing: outwell the ethanol of step 3 processed, is added by 1:1 volume ratio
Enter ethanol and dimethylbenzene, process 1-2min；Pour out ethanol and dimethylbenzene, change pure dimethylbenzene into,
Reprocessing 1-2min, obtains sciophiles fragment；
Step 5: waxdip processes: outwell the pure dimethylbenzene of step 4 transparent processing, adds fusing
Paraffin, temperature is 65-75 DEG C, processes time 10-15min, changes the paraffin of fusing, then locates
Reason 10-15min, obtains waxdip plant fragments；
Step 6: embedding treatment: the paraffin of fusing is inclined in the lump together with waxdip plant fragments bag
Bury in box, rapidly plant fragments is established row side by side neat, then by its average putting into
In cold water so that it is rapid solidification, obtain embedded plant fragments；
Step 7: slicing treatment: embedded plant fragments step 6 obtained just is trimmed to
Square wax stone, is sticked on wood particle, is then cut into slices, and the section obtained is bonded at microscope slide
On, toast 0.5-1.5 hour at a temperature of 30-40 DEG C；
Step 8: dyeing processes: use sarranine-fast green colouring method to dye, then mounting.
Preferably, described plant tissue is the root of plant, stem, leaf, flower or fruit.
Preferably, in step one, described reaction unit is by digital control constant temperature heating mantle and circle
End flask forms.
Preferably, in step 3, described processed temperature is 105 DEG C, and the time is respectively
2min。
Preferably, in step 5, described waxdip treatment temperature is 70 DEG C, processes the time
10min。
Preferably, in step 8, described sarranine is the weight hundred with 95% alcoholic solution preparation
Proportion by subtraction concentration 2% sarranine, described fast green be that the percentage by weight with 95% alcoholic solution preparation is dense
Spend 1% fast green.
Present invention also offers planting obtained by the method for the quick paraffin wax flaking of described plant tissue
Fabric texture paraffin wax flaking
(3) beneficial effect
The method of the quick paraffin wax flaking of plant tissue of the present invention compared with prior paraffin flaking method,
Fabrication cycle is greatly shortened, and was shortened to 7 hours by 9 days, and decreases toxic pharmaceuticals usage amount.
The plant tissue slice that the inventive method obtains, histiocyte is complete, clear, can effectively see
Examine tissue morphology.The paraffin wax flaking method that table 1 lists the present invention and provides respectively walks with prior paraffin method
Required time compares:
Table 1:
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below
The accompanying drawing used required in embodiment or description of the prior art will be briefly described, aobvious and
Easily insight, the accompanying drawing in describing below is only some embodiments of the present invention, for this area
From the point of view of those of ordinary skill, on the premise of not paying creative work, it is also possible to according to these
Accompanying drawing obtains other accompanying drawing.
Fig. 1 is Herba stellariae mediae leaf texture (× 100) picture that the inventive method film-making obtains；
Fig. 2 is polyphyll Jasminum mesnyi Hance flower tissue (× 100) picture that the inventive method film-making obtains；
Detailed description of the invention
With embodiment, embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings.
Following example are used for illustrating the present invention, but can not be used for limiting the scope of the present invention.The present invention
The equal city of material therefor available from.
Embodiment 1
The step of the plant tissue quick paraffin wax flaking method of the present embodiment is as follows:
1) assembling device: reaction unit is by digital control constant temperature heating mantle and isopyknic round-bottomed flask group
Dress forms, and adds fixative, and the configuration of described fixative is by weight for formaldehyde: glacial acetic acid: wine
Essence=5: 5: 90；
2) fixing: by the fresh Herba stellariae mediae leaf section of cutting into pieces, in embedding device and guarantee that fragment is complete
Entirely it is immersed in fixative.Temperature is set as 65 DEG C, processes 5min, changes fixative, repeats 2
Secondary.
3) dehydration: after fixing process, outwell fixative, be replaced by variable concentrations ethanol (70%
85% 95% 100% 100% II).Temperature is transferred to 100 DEG C of process, and material is in device
Carry out ethanol serial dehydration, each 2min under every kind of concentration.Add a little when changing 95% ethanol
Sarranine.
4) transparent: in the round-bottomed flask equipped with dehydration good material, to pour 1/2 ethanol+1/2 diformazan into
Benzene, processes 2min in reaction unit.Pour out 1/2 ethanol+1/2 dimethylbenzene, change pure diformazan into
Benzene, processes 2min in reaction unit.
5) waxdip: pour in device and dissolve paraffin, processes 10min, temperature in reaction unit
Degree is set as 75 DEG C.Paraffin is dissolved in replacing, reprocesses 10min.
6) embed, cut into slices and dry sheet: inclined in the lump in embedded box together with material by the paraffin of fusing.
Then by tweezers burn-out on alcohol burner, rapidly material is established marshalling.Then put down
Keep flat in cold water so that it is rapid solidification.The wax stone of the most embedded material is accomplished little square
Comprise a material.Little wax stone is sticked on wood particle, rotary microtome is cut into slices.Section
After cutting, glued and scribbled on the microscope slide of bonding die agent.Be put into baking sheet machine on 40 DEG C dry 1h,
Then 1h in 80 DEG C of incubators it is put in.
6) sarranine-fast green dyeing, mounting: use sarranine-fast green dyeing.Slide is put into equipped with two
Dewaxing treatment 3-5min in the reaction unit of toluene, temperature is set as 75-80 DEG C；Then dyeing is each
Step processes and the time is: dimethylbenzene I (more than 30min) dimethylbenzene II (3min) 1/2 wine
Essence+1/2 dimethylbenzene (3min) 100% ethanol I (3min) 95% ethanol (3min) 2%
Sarranine (95% alcohol, 3min).Microscope slide puts into 95% ethanol (2min) 95% ethanol
(2min) 1% fast green (95% alcohol, 10s) 95% ethanol (2min) 100% ethanol
I (2min) 100% ethanol II (2min) 1/2 ethanol+1/2 dimethylbenzene (2min) pure two
Toluene I (2min) pure dimethylbenzene II (2min) mounting.
Embodiment 2
The step of the plant tissue quick paraffin wax flaking method of the present embodiment is as follows:
1) assembling device: reaction unit is by digital control constant temperature heating mantle and isopyknic round-bottomed flask group
Dress forms, and adds fixative, and the configuration of described fixative is by weight for formaldehyde: glacial acetic acid: wine
Essence=4: 6: 90；
2) fixing: by fresh polyphyll Jasminum mesnyi Hance alabastrum, to put in round-bottomed flask, digital control constant temperature
In heating mantle, temperature is set as 60 DEG C, processes 8min, changes fixative, is repeated 2 times.
3) dehydration: after fixing process, outwell fixative, be replaced by variable concentrations ethanol (70%
85% 95% 100% 100% II).Temperature is transferred to 110 DEG C of process, and material is in device
Carry out ethanol serial dehydration, each 1min under every kind of concentration.Add a little when changing 95% ethanol
Sarranine.
4) transparent: in the round-bottomed flask equipped with dehydration good material, to pour 1/2 ethanol+1/2 diformazan into
Benzene, processes 1min in reaction unit.Pour out 1/2 ethanol+1/2 dimethylbenzene, change pure diformazan into
Benzene, processes 1min in reaction unit.
5) waxdip: pour in round-bottomed flask and dissolve paraffin, processes 15min in reaction unit,
Temperature is set as 70 DEG C.Paraffin is dissolved in replacing, reprocesses 15min.
6) embed, cut into slices and dry sheet: inclined in the lump in embedded box together with material by the paraffin of fusing.
Then by tweezers burn-out on alcohol burner, rapidly material is established marshalling.Then put down
Keep flat in cold water so that it is rapid solidification.The wax stone of the most embedded material can be accomplished little just
Square comprise a material.Little wax stone is sticked on wood particle, rotary microtome is cut into slices.
After cutting, glued and scribbled on the microscope slide of bonding die agent.Be put into baking sheet machine on 40 DEG C bake
Dry, then it is put in 1h in 80 DEG C of incubators.
7) sarranine-fast green dyeing, mounting: use sarranine-fast green dyeing.Microscope slide put into equipped with
Dewaxing treatment 3-5min in the reaction unit of dimethylbenzene, temperature is set as 75-80 DEG C；Then dye
Each step processes and the time is: dimethylbenzene II (3min) 1/2 ethanol+1/2 dimethylbenzene
(3min) 100% ethanol I (3min) 95% ethanol (3min) 2% sarranine (95% ethanol
Preparation, 3min).It is solid that microscope slide puts into 95% ethanol (2min) 95% ethanol (2min) 1%
Green (95% alcohol, 10s) 95% ethanol (2min) 100% ethanol I (2min) 100%
Ethanol II (2min) 1/2 ethanol+1/2 dimethylbenzene (2min) pure dimethylbenzene I (2min) is pure
Dimethylbenzene II (2min) mounting.
Comparative example 1
The step that this comparative example prepares plant tissue paraffin wax flaking according to prior paraffin method is as follows:
1) fixing: by the fresh Herba stellariae mediae leaf section of cutting into pieces, to put in round-bottomed flask, add FAA
Fixative (formaldehyde: glacial acetic acid: 70% ethanol=4: 6: 90) also guarantees that fragment is completely submerged in
In fixative, process 48h.
2) dehydration: after fixing process, outwell fixative, be replaced by variable concentrations ethanol (70%
85% 95% 100% 100% II).Temperature is transferred to 100 DEG C of process, and material is in device
Carry out ethanol serial dehydration, each 2h under every kind of concentration.A little kind is added when changing 95% ethanol
Red.
3) transparent: in the round-bottomed flask equipped with dehydration good material, to pour 1/2 ethanol+1/2 diformazan into
Benzene, processes 2h.Pour out 1/2 ethanol+1/2 dimethylbenzene, change pure dimethylbenzene into, process 3h, phase
Between change a dimethylbenzene.
4) waxdip: pour in the round-bottomed flask of transparent good material and dissolve paraffin, processes 24h,
Temperature is set as 40 DEG C.Paraffin is dissolved in replacing, reprocesses 12h.
5) embed, cut into slices and dry sheet: inclined in the lump in embedded box together with material by the paraffin of fusing.
Then by tweezers burn-out on alcohol burner, rapidly material is established marshalling.Then put down
Keep flat in cold water so that it is rapid solidification.The wax stone of the most embedded material can be accomplished little just
Square comprise a material.Little wax stone is sticked on wood particle, rotary microtome is cut into slices.
After cutting, glued and scribbled on the microscope slide of bonding die agent.Be put into baking sheet machine on 40 DEG C bake
Dry, then it is put in 2h in 40 DEG C of incubators.
6) sarranine-fast green dyeing, mounting: use sarranine-fast green dyeing.Each step that dyes process and
Time is: dimethylbenzene I (more than 30min) dimethylbenzene II (3min) 1/2 ethanol+1/2 two
Toluene (3min) 100% ethanol I (3min) 95% ethanol (3min) 2% sarranine (50%
Alcohol, 12h).Microscope slide puts into 95% ethanol (2min) 95% ethanol (2min) 1%
Fast green (95% alcohol, 30s) 95% ethanol (2min) 100% ethanol I
(2min) 100% ethanol II (2min) 1/2 ethanol+1/2 dimethylbenzene (2min) pure diformazan
Benzene I (2min) pure dimethylbenzene II (2min) mounting.
Comparative example 2
The step that this comparative example prepares plant tissue paraffin wax flaking according to prior paraffin method is as follows:
1) fixing: by fresh polyphyll Jasminum mesnyi Hance alabastrum, to put in round-bottomed flask, add FAA
Fixative (formaldehyde: glacial acetic acid: 70% ethanol=4: 6: 90) also guarantees that fragment is completely submerged in
In fixative, process 48h.
2) dehydration: after fixing process, outwell fixative, be replaced by variable concentrations ethanol (70%
85% 95% 100% 100% II).Temperature is transferred to 100 DEG C of process, and material is in device
Carry out ethanol serial dehydration, each 2h under every kind of concentration.A little kind is added when changing 95% ethanol
Red.
3) transparent: in the round-bottomed flask equipped with dehydration good material, to pour 1/2 ethanol+1/2 diformazan into
Benzene, processes 2h.Pour out 1/2 ethanol+1/2 dimethylbenzene, change pure dimethylbenzene into, process 3h, phase
Between change a dimethylbenzene.
4) waxdip: pour in the round-bottomed flask of transparent good material and dissolve paraffin, processes 24h,
Temperature is set as 40 DEG C.Paraffin is dissolved in replacing, reprocesses 12h.
5) embed, cut into slices and dry sheet: inclined in the lump in embedded box together with material by the paraffin of fusing.
Then by tweezers burn-out on alcohol burner, rapidly material is established marshalling.Then put down
Keep flat in cold water so that it is rapid solidification.The wax stone of the most embedded material can be accomplished little just
Square comprise a material.Little wax stone is sticked on wood particle, rotary microtome is cut into slices.
After cutting, glued and scribbled on the microscope slide of bonding die agent.Be put into baking sheet machine on 40 DEG C bake
Dry, then it is put in 2h in 40 DEG C of incubators.
6) sarranine-fast green dyeing, mounting: use sarranine-fast green dyeing.Each step that dyes process and
Time is: dimethylbenzene I (more than 30min) dimethylbenzene II (3min) 1/2 ethanol+1/2 two
Toluene (3min) 100% ethanol I (3min) 95% ethanol (3min) 2% sarranine (50%
Alcohol, 12h).Microscope slide puts into 95% ethanol (2min) 95% ethanol (2min) 1%
Fast green (95% alcohol, 30s) 95% ethanol (2min) 100% ethanol I
(2min) 100% ethanol II (2min) 1/2 ethanol+1/2 dimethylbenzene (2min) pure diformazan
Benzene I (2min) pure dimethylbenzene II (2min) mounting.
Embodiment of above is merely to illustrate the present invention, rather than limitation of the present invention.Although ginseng
Being described in detail the present invention according to embodiment, those of ordinary skill in the art should manage
Solve, technical scheme is carried out various combination, amendment or equivalent, does not takes off
From the spirit and scope of technical solution of the present invention, all should contain in scope of the presently claimed invention
In the middle of.
Claims (7)
1. the method for the quick paraffin wax flaking of plant tissue, it is characterised in that the method includes
Following steps:
Step one: preparation: assemble reaction unit, adds fixative, and described fixative is joined
Putting weight ratio is formaldehyde: glacial acetic acid: ethanol=3-5: 5-8: 80-95；
Step 2: fixing process: by the fresh plant tissue section of cutting into pieces, piece size is
0.5-1.5cm, is then placed in reaction unit, and digital control constant temperature heats, and temperature range is 60-65 DEG C,
The process time is 5-10min, changes fixative, repeats process 2 times；
Step 3: processed: outwell the fixative used in step 2, change weight successively
Percent concentration is respectively 70%, the alcohol water blend of 85%, 95%, 100%, 100% II,
Temperature is transferred to 100-110 DEG C, under every kind of concentration alcohol water blend, processes 1-2min, planted
Thing fragment carries out ethanol serial dehydration；
Step 4: transparent processing: outwell the ethanol of step 3 processed, is added by 1:1 volume ratio
Enter ethanol and dimethylbenzene, process 1-2min；Pour out ethanol and dimethylbenzene, change pure dimethylbenzene into,
Reprocessing 1-2min, obtains sciophiles fragment；
Step 5: waxdip processes: outwell the pure dimethylbenzene of step 4 transparent processing, adds fusing
Paraffin, temperature is 65-75 DEG C, processes time 10-15min, changes the paraffin of fusing, then locates
Reason 10-15min, obtains waxdip plant fragments；
Step 6: embedding treatment: the paraffin of fusing is inclined in the lump together with waxdip plant fragments bag
Bury in box, rapidly plant fragments is established row side by side neat, then by its average putting into
In cold water so that it is rapid solidification, obtain embedded plant fragments；
Step 7: slicing treatment: embedded plant fragments step 6 obtained just is trimmed to
Square wax stone, is sticked on wood particle, is then cut into slices, and the section obtained is bonded at microscope slide
On, toast 0.5-1.5 hour at a temperature of 30-40 DEG C；
Step 8: dyeing processes: use sarranine-fast green colouring method to dye, then mounting.
The method of the quick paraffin wax flaking of plant tissue the most according to claim 1, its feature
Being, described plant tissue is the root of plant, stem, leaf, flower or fruit.
The method of the quick paraffin wax flaking of plant tissue the most according to claim 1, its feature
Being, in step one, described reaction unit is by digital control constant temperature heating mantle and round-bottomed flask
Composition.
The method of the quick paraffin wax flaking of plant tissue the most according to claim 1, its feature
Being, in step 3, described processed temperature is 105 DEG C, and the time is respectively 2min.
The method of the quick paraffin wax flaking of plant tissue the most according to claim 1, its feature
Being, in step 5, described waxdip treatment temperature is 70 DEG C, processes time 10min.
6. according to the method for the quick paraffin wax flaking of plant tissue described in any one of claim 1-5,
It is characterized in that, in step 8, described sarranine is the weight with 95% alcoholic solution preparation
Percent concentration 2% sarranine, described fast green be the weight percent with 95% alcoholic solution preparation
Specific concentration 1% is fast green.
7. the method for the quick paraffin wax flaking of plant tissue described in any one of claim 1-6 is made
The plant tissue paraffin wax flaking obtained.
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| CN201610387172.8A CN105806680A (en) | 2016-06-02 | 2016-06-02 | Quick paraffin sectioning method of plant tissues |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610387172.8A CN105806680A (en) | 2016-06-02 | 2016-06-02 | Quick paraffin sectioning method of plant tissues |
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| CN107702955A (en) * | 2017-08-24 | 2018-02-16 | 胡雨逸 | A kind of preparation method of paraffin section |
| CN109211606A (en) * | 2018-09-06 | 2019-01-15 | 吉林省农业科学院 | A kind of fast method for preparing of pears tissue paraffin section de |
| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109374373A (en) * | 2018-10-09 | 2019-02-22 | 黑龙江省农业科学院牡丹江分院 | A kind of Apple paraffin section fast method for preparing |
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Application publication date: 20160727 |