CN107202720B - A kind of paraffin section method of pomegranate seed - Google Patents
A kind of paraffin section method of pomegranate seed Download PDFInfo
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- CN107202720B CN107202720B CN201710342468.2A CN201710342468A CN107202720B CN 107202720 B CN107202720 B CN 107202720B CN 201710342468 A CN201710342468 A CN 201710342468A CN 107202720 B CN107202720 B CN 107202720B
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- dimethylbenzene
- paraffin
- ethyl alcohol
- waxdip
- volume ratio
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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
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
Abstract
The present invention discloses a kind of paraffin section method of pomegranate seed, includes the following steps: (1) materials and fixes；(2) dehydration with it is transparent: serial dehydration, every gradient handle each 2h, are transferred to dehydrated alcohol later and handle 1.5h twice；Successively enter the dehydrated alcohol that volume ratio is 2:1 and dehydrated alcohol and dimethylbenzene mixed solution 2h that dimethylbenzene mixed solution 2h, volume ratio are 1:2, is transferred to pure dimethylbenzene processing 1.5h later twice；(3) waxdip and embedding: the material after will be transparent is placed in the dimethylbenzene and paraffin mixed liquor waxdip 4-5h that volume ratio is 1:1, it is subsequently placed in the dimethylbenzene and paraffin mixed liquor waxdip 4-5h that volume ratio is 1:2, paraffin refined wax waxdip 4-5h is used later, and replacement paraffin refined wax continues waxdip 3-4d；(4) slice and exhibition piece；(5) dyeing, transparent, mounting.Biopsy tissues structural integrity, the cell boundary of the method for the present invention production understand that slice is clean, are easy to observe.
Description
Technical field
The present invention relates to plant paraffin microtomy fields, and in particular to a kind of paraffin section method of pomegranate seed.
Background technique
Pomegranate (Punica granatum) is Lythraceae (Lythraceae) Punica (Punica) machaka or small
Arbor, originates in Iran and Afghanistan etc. is regional, until Western Han Dynastry Zhang Qian sends as an envoy to, the Western Regions introduce China, has cultivation in more than 2 000 years
History.Pomegranate fruit is the round or ellipse fruit wrapped up by keratin pericarp, has 5-7 ventricle, Mei Gexin inside pericarp
Room is made of tens to several hundred seeds, and seed is the Leaf-feeding insects of fruit, this and apple, pears, strawberry, peach, Lee, apricot and dragon
Eyes etc. are all different.Pomegranate kind skin is made of inside and outside two layers kind of skin, exosper plumpness succulence, rich in sugar, organic acid, polyphenol etc., is
Main Leaf-feeding insects；Endotesta is the main composition factor of grain hardness rich in lignin, cellulose, hemicellulose etc..Seed
An important factor for hardness is as fruit quality drastically influences the consumption and commodity of pomegranate fruit, it has also become pomegranate production
With the emphasis of consumer's concern and the hot spot of pomegranate breeder concern.The research of pomegranate seeds is concentrated mainly on point at present
Class, hard soft Grain formation key gene clone etc. research, specifying pomegranate kernel structure feature can send out for pomegranate seed
It educates and lays a good foundation with study on regulation.
Paraffin section is a kind of widely used plant histology method.Since there is paraffin section slice thickness to be easy to control
System, the advantages that clarity is good and be widely used in the film-making and cytology research of plant tissue.Though paraffin section whole process
It is right and uncomplicated, but the slice of high quality is produced, the operation of each step is critically important, and with different tissues structure
Processing needed for the institutional framework of plant and plant different parts is different, is groped and is improved.Currently, being used for paraffin
The vegetable material of slice includes ovule, bud, pulp and rhizome etc..And in pomegranate exosper due to architectural difference it is larger, outer kind
Pachyhymenia succulence, endotesta is rich in lignin, cellulose, hemicellulose etc., and keratin is hard, very according to prior paraffin dicing method
Hardly possible obtains intact, clearly microsection, and pomegranate seed coat structure observation band is given to carry out certain challenge.The present invention will be by improving stone
Wax dicing method establishes a kind of paraffin section method for being suitable for pomegranate kind skin displaing microstructure observing, with inside and outside clear pomegranate kinds
The micro-structural feature of skin, to lay the foundation for further pomegranate Grain Development and study on regulation.
Summary of the invention
The object of the present invention is to provide a kind of paraffin section methods of pomegranate seed, so as to solve the deficiencies in the prior art.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of paraffin section method of pomegranate seed, includes the following steps:
Step 1 is drawn materials and is fixed: choosing fresh, undamaged pomegranate, takes out complete, unabroken pomegranate seed；As early as possible
It puts into FAA fixer and fixes, fix more than for 24 hours；
Step 2, dehydration with it is transparent: the pomegranate seed after fixation is sequentially placed into 70% ethyl alcohol, 80% ethyl alcohol, 90% second
Alcohol, 95% ethyl alcohol carry out serial dehydration, and every gradient handles each 2h, are transferred to dehydrated alcohol processing 1.5h later, then replace anhydrous second
Alcohol handles 1.5h, and dehydration terminates；Dewatered pomegranate seed is sequentially placed into volume ratio to mix for the dehydrated alcohol and dimethylbenzene of 2:1
It closes solution 2h, the dehydrated alcohol and dimethylbenzene mixed solution 2h that volume ratio is 1:2, is transferred to pure dimethylbenzene processing 1.5h later, then
Replace pure dimethylbenzene processing 1.5h, transparent end；
Step 3, waxdip and embedding: be higher than 2 DEG C of paraffin melting point under the conditions of, will be transparent after pomegranate seed be placed in volume ratio
For the dimethylbenzene and paraffin mixed liquor waxdip 4-5h of 1:1, it is subsequently placed in dimethylbenzene and paraffin mixed liquor waxdip that volume ratio is 1:2
4-5h uses paraffin refined wax waxdip 4-5h later, replaces pure wax and continues waxdip 3-4d；The paraffin refined wax melted is taken out from incubator, slowly
It pours into the carton of embedding, is put in carton after slightly cooling down with the pomegranate seed that the tweezers of warm clamp waxdip, simultaneously
Surrounding materials bubble is driven away with the tweezers of warm；When carton surface, paraffin slightly solidifies, translation carton is put on the cold water water surface,
Make its quick solidification；
Step 4, slice with exhibition piece: embedded wax stone is taken out from carton, is cut into small pieces, accomplish it is trapezoidal, by wax
Block is cut into 5~10um wax band, and the wax band cut opens up piece in 40 DEG C of distilled water；Clean glass slide is put into bonding die agent, number
It picks up after minute, is dried in 37 DEG C of baking ovens, clamp wax band with tweezers, be put on the above-mentioned glass slide handled well, glass slide is set
In on pre-heated electric hot plate, wax band is put in 37 DEG C of incubator overnight after by hot stretched, obtains dry slice；
Step 5, dyeing, transparent, mounting: dry slice is dyed, is transparent；After mounting.
Further, FAA fixer presses 70% ethyl alcohol of 90 parts by volume, the glacial acetic acid of 5 parts by volume, 5 parts by volume in step 1
38% formalin configured.
Further, angle and the position between wax stone and the edge of a knife are adjusted in step 4, are made between blade and wax stone about at 15
Wax stone is cut into 5~10um wax band by degree, rocking-turn 40~50r/min of speed.
Further, bonding die agent is configured in the ratio of 100ml distilled water, 1g gelatin and 15ml glycerol in step 4.
Further, dyeing, transparent concrete operations are as follows in step 5: dry slice is sequentially placed into dimethylbenzene
30min, the dehydrated alcohol and dimethylbenzene mixed liquor 15min, dehydrated alcohol 2min, 95% ethyl alcohol 2min, 85% that volume ratio is 1:1
Ethyl alcohol 2min, 70% ethyl alcohol 2min, 50% ethyl alcohol 2min are placed in 48h in 1% sarranine dye liquor later under the conditions of 37 DEG C；Sarranine
Slice after dyeing is sequentially placed into 50% ethyl alcohol 60s, 70% ethyl alcohol 60s, 85% ethyl alcohol 60s, be placed on 0.5% fast green dye liquor
15s；Slice after fast green dyeing is sequentially placed into 95% ethyl alcohol 60s, dehydrated alcohol 30s, the dehydrated alcohol and two that volume ratio is 1:1
Toluene mixture liquid 30s, dimethylbenzene 60s carry out transparent processing.
Further, neutral gum mounting is used in step 5.
Beneficial effects of the present invention:
The present invention has found a kind of paraffin section method of suitable pomegranate seed, the biopsy tissues structural integrity of production, thin
Born of the same parents' boundary clear, slice is clean, is easy to observe.
Detailed description of the invention
Fig. 1: the microstructure of pomegranate kind skin；
PL palisade layer, VB vascular bundle, CK phellem layer, StL prothenchyma (of wood) layer, PC parenchyma cell.
Specific embodiment
The present invention is done below with reference to embodiment and attached drawing and is further explained.The following example is merely to illustrate this hair
It is bright, but be not used to limit practical range of the invention.
Embodiment 1
A kind of paraffin section method of pomegranate seed, includes the following steps:
Step 1 is drawn materials and is fixed: choosing fresh, undamaged pomegranate fruit 3~4, is placed in ice chest, transports laboratory back.
Complete, unabroken pomegranate seed 100 are taken out, puts into FAA fixer fix immediately, the set time is more than for 24 hours.Its
In, FAA fixer is matched by 38% formalin of 70% ethyl alcohol of 90 parts by volume, the glacial acetic acid of 5 parts by volume, 5 parts by volume
It sets.
Step 2, dehydration with it is transparent: the pomegranate seed after fixation is sequentially placed into 70% ethyl alcohol, 80% ethyl alcohol, 90% second
Alcohol, 95% ethyl alcohol carry out serial dehydration, and every gradient handles each 2h, are transferred to dehydrated alcohol processing 1.5h later, then replace anhydrous second
Alcohol handles 1.5h, and dehydration terminates；Dewatered pomegranate seed is sequentially placed into volume ratio to mix for the dehydrated alcohol and dimethylbenzene of 2:1
It closes solution 2h, the dehydrated alcohol and dimethylbenzene mixed solution 2h that volume ratio is 1:2, is transferred to pure dimethylbenzene processing 1.5h later, then
Replace pure dimethylbenzene processing 1.5h, transparent end.Dehydration is to harden tissue further, creates item for transparent in next step and waxdip
Part, dehydration, which is not thorough, can make clarifier white opacity phenomenon occur, and clearing time is too long and tissue can be made to become fragile be hardened.This step
Ensure transparency of organization and structural integrity, hardness is moderate, and when slice organizes non-friable.
Step 3, waxdip and embedding: with paraffin replace clarifier, when dimethylbenzene immersion group is woven in slice from supporting function claim
For waxdip, carried out usually in insulating box.Be higher than 2 DEG C of paraffin melting point under the conditions of, will be transparent after pomegranate seed be placed in volume
Than the dimethylbenzene and paraffin mixed liquor waxdip 4-5h for 1:1, it is subsequently placed in the dimethylbenzene and the leaching of paraffin mixed liquor that volume ratio is 1:2
Wax 4-5h, uses paraffin refined wax waxdip 4-5h later, and replacement paraffin refined wax continues waxdip 3-4d；The paraffin refined wax melted is taken out from incubator,
It slowly pours into the carton of embedding, is put in carton after slightly cooling down with the pomegranate seed that the tweezers of warm clamp waxdip,
Surrounding materials bubble is driven away with the tweezers of warm simultaneously.When carton surface, paraffin slightly solidifies, translation carton is put into cold water water
On face, make its quick solidification.The waxdip time directly affects dicing effect.Pomegranate exosper plumpness succulence, endotesta is rich in wooden
Element, cellulose, hemicellulose etc., keratin is hard, it is necessary to extend the waxdip time, ability waxdip is complete.Waxdip is incomplete, and there are skies
Hole can not be sliced.
Step 4, slice are with exhibition piece: embedded wax stone being taken out from carton, is cut into small pieces, trapezoidal, adjustment is accomplished
Angle and position between wax stone and the edge of a knife make between blade and wax stone about into 15 degree, rocking-turn 40~50r/min of speed, by wax stone
It is cut into 5~10um wax band, the wax band cut opens up piece in 40 DEG C of distilled water；Clean glass slide is put into bonding die agent (by 100ml
The ratio of distilled water, 1g gelatin and 15ml glycerol is configured) in, it is picked up after several minutes, is dried in 37 DEG C of baking ovens, use tweezers
Clamp wax band, be about 5cm, be put on the above-mentioned glass slide handled well, glass slide is placed on pre-heated electric hot plate, wax band by
It is put in after hot stretched in 37 DEG C of incubator overnight, obtains dry slice.
Step 5, dyeing, transparent, mounting: by the nothing that dry slice is sequentially placed into dimethylbenzene 30min, volume ratio is 1:1
Water-ethanol and dimethylbenzene mixed liquor 15min, dehydrated alcohol 2min, 95% ethyl alcohol 2min, 85% ethyl alcohol 2min, 70% ethyl alcohol
2min, 50% ethyl alcohol 2min are placed in 1% sarranine dye liquor and (weigh 1g sarranine, be dissolved in 100ml50%'s later under the conditions of 37 DEG C
In alcohol, used after filtering) in 48h；Slice after sarranine dyeing is sequentially placed into 50% ethyl alcohol 60s, 70% ethyl alcohol 60s, 85%
Ethyl alcohol 60s, be placed on 0.5% fast green dye liquor (weigh 0.5g is fast green, be dissolved in the alcohol of 100ml95%, use after filtering)
15s；Slice after fast green dyeing is sequentially placed into 95% ethyl alcohol 60s, dehydrated alcohol 30s, the dehydrated alcohol and two that volume ratio is 1:1
Toluene mixture liquid 30s, dimethylbenzene 60s carry out transparent processing.After use neutral gum mounting.
The slice of production as microscopically observation and is taken pictures, as a result as shown in Figure 1, its institutional framework is complete, cell
Boundary clear, slice are clean.
Claims (4)
1. a kind of paraffin section method of pomegranate seed, which comprises the steps of:
Step 1 is drawn materials and is fixed: choosing fresh, undamaged pomegranate, takes out complete, unabroken pomegranate seed；It puts into as early as possible
It is fixed in FAA fixer, it fixes more than for 24 hours；
Step 2, dehydration with it is transparent: by the pomegranate seed after fixation be sequentially placed into 70% ethyl alcohol, 80% ethyl alcohol, 90% ethyl alcohol,
95% ethyl alcohol carries out serial dehydration, and every gradient handles each 2h, is transferred to dehydrated alcohol processing 1.5h later, then replace at dehydrated alcohol
1.5h is managed, dehydration terminates；It is molten for dehydrated alcohol and the dimethylbenzene mixing of 2:1 that dewatered pomegranate seed is sequentially placed into volume ratio
Liquid 2h, the dehydrated alcohol and dimethylbenzene mixed solution 2h that volume ratio is 1:2 are transferred to pure dimethylbenzene processing 1.5h later, then replace
Pure dimethylbenzene handles 1.5h, transparent end；
Step 3, waxdip and embedding: be higher than 2 DEG C of paraffin melting point under the conditions of, will be transparent after pomegranate seed be placed in volume ratio be 1:
1 dimethylbenzene and paraffin mixed liquor waxdip 4-5h is subsequently placed in dimethylbenzene and paraffin mixed liquor waxdip 4- that volume ratio is 1:2
5h, uses paraffin refined wax waxdip 4-5h later, and replacement paraffin refined wax continues waxdip 3-4d；The paraffin refined wax melted is taken out from incubator, slowly
It pours into the carton of embedding, is put in carton after slightly cooling down with the pomegranate seed that the tweezers of warm clamp waxdip, simultaneously
Surrounding materials bubble is driven away with the tweezers of warm；When carton surface, paraffin slightly solidifies, translation carton is put on the cold water water surface,
Make its quick solidification；
Step 4, slice are with exhibition piece: embedded wax stone being taken out from carton, is cut into small pieces, trapezoidal, adjustment wax stone is accomplished
Angle and position between the edge of a knife make about at 15 degree between blade and wax stone, and wax stone is cut into 5 by rocking-turn 40~50r/min of speed
~10um wax band, the wax band cut open up piece in 40 DEG C of distilled water；Clean glass slide is put into bonding die agent, is fished out after several minutes
It rises, is dried in 37 DEG C of baking ovens, clamp wax band with tweezers, be put on the above-mentioned glass slide handled well, glass slide is placed in be added in advance
On the electric hot plate of heat, wax band is put in 37 DEG C of incubator overnight after by hot stretched, obtains dry slice；
Step 5, dyeing, transparent, mounting: by the anhydrous second that dry slice is sequentially placed into dimethylbenzene 30min, volume ratio is 1:1
Pure and mild dimethylbenzene mixed liquor 15min, dehydrated alcohol 2min, 95% ethyl alcohol 2min, 85% ethyl alcohol 2min, 70% ethyl alcohol 2min,
50% ethyl alcohol 2min is placed in 48h in 1% sarranine dye liquor later under the conditions of 37 DEG C；Slice after sarranine dyeing is sequentially placed into
50% ethyl alcohol 60s, 70% ethyl alcohol 60s, 85% ethyl alcohol 60s, be placed on 0.5% fast green dye liquor 15s；Slice after fast green dyeing
It is sequentially placed into 95% ethyl alcohol 60s, dehydrated alcohol 30s, the dehydrated alcohol and dimethylbenzene mixed liquor 30s, dimethylbenzene that volume ratio is 1:1
60s carries out transparent processing；After mounting.
2. the paraffin section method of pomegranate seed according to claim 1, which is characterized in that FAA fixer is pressed in step 1
70% ethyl alcohol of 90 parts by volume, the glacial acetic acid of 5 parts by volume, 5 parts by volume 38% formalin configured.
3. the paraffin section method of pomegranate seed according to claim 1, which is characterized in that bonding die agent is pressed in step 4
The ratio of 100ml distilled water, 1g gelatin and 15ml glycerol is configured.
4. the paraffin section method of pomegranate seed according to claim 1, which is characterized in that use neutral gum in step 5
Mounting.
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| CN201710342468.2A CN107202720B (en) | 2017-05-16 | 2017-05-16 | A kind of paraffin section method of pomegranate seed |
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| CN201710342468.2A CN107202720B (en) | 2017-05-16 | 2017-05-16 | A kind of paraffin section method of pomegranate seed |
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| CN107202720B true CN107202720B (en) | 2019-10-11 |
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| CN110793829A (en) * | 2019-11-22 | 2020-02-14 | 西藏自治区农牧科学院农业资源与环境研究所 | Method for slicing highland barley stem profile paraffin suitable for plateau environment |
| CN112033723B (en) * | 2020-10-10 | 2024-03-29 | 北华大学 | Paraffin slicing method of tilia amurensis seeds |
| CN113702131A (en) * | 2021-09-06 | 2021-11-26 | 海南大学 | Paraffin section of camellia oleifera flower bud and preparation method and application thereof |
Citations (3)
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|---|---|---|---|---|
| CN103630421A (en) * | 2013-12-03 | 2014-03-12 | 沈阳农业大学 | Production method for paraffin section of paeonia lactiflora mature embryo |
| CN104614211A (en) * | 2015-01-19 | 2015-05-13 | 沈阳农业大学 | Paraffin sectioning method of Magnolia sieboldii K.Koch seeds |
| CN105973673A (en) * | 2016-06-30 | 2016-09-28 | 中国林业科学研究院热带林业研究所 | Paraffin sectioning method for eucalyptus tissue |
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- 2017-05-16 CN CN201710342468.2A patent/CN107202720B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630421A (en) * | 2013-12-03 | 2014-03-12 | 沈阳农业大学 | Production method for paraffin section of paeonia lactiflora mature embryo |
| CN104614211A (en) * | 2015-01-19 | 2015-05-13 | 沈阳农业大学 | Paraffin sectioning method of Magnolia sieboldii K.Koch seeds |
| CN105973673A (en) * | 2016-06-30 | 2016-09-28 | 中国林业科学研究院热带林业研究所 | Paraffin sectioning method for eucalyptus tissue |
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| 费约果花芽分化及胚胎学研究;李泽华;《中国优秀硕士学位论文全文数据库 农业科技辑》;20170315(第3期);全文 * |
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