CN107576552A - A kind of paraffin section colouring method for observing Chinese Rose infection processs - Google Patents
A kind of paraffin section colouring method for observing Chinese Rose infection processs Download PDFInfo
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- CN107576552A CN107576552A CN201710756558.6A CN201710756558A CN107576552A CN 107576552 A CN107576552 A CN 107576552A CN 201710756558 A CN201710756558 A CN 201710756558A CN 107576552 A CN107576552 A CN 107576552A
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Abstract
The present invention relates to a kind of paraffin section colouring method for observing Chinese Rose infection processs, after section dewaxing to water, is first dyed with the 1% crystal violet aqueous solution, recycles iodine solution to carry out mordant dyeing, finally decolourized with 95% alcohol.Above two dye liquor and discoloration method are selected, histocyte can be contaminated for blueness, be red by mycelia dyeing, method is provided for observe the observation of black spot disease process.And the method for the present invention is substantially reduced the dyeing time that blade process is infected using paraffin section observation Chinese Rose, improves test efficiency by improved colouring method.
Description
Technical field
The present invention relates to microscopic examination technique field, specifically a kind of observation of plant leaf portion fungal disease histopathology mistake
The colouring method of journey
Background technology
Plant leaf portion fungal disease often results in plant leaf portion scab, yellow, early leaf fall, and severe patient directly results in plant
Death, plant normal growth development is have impact on, adds cultivation management maintenance cost.
Chinese rose is first of four big cut-flowers, is rose family Rosa perennial woody flowers, there is florescence length, pattern to enrich,
The advantages that plant type is different.And Chinese Rose is that incidence is high and destructive extremely strong fungal disease in a kind of cultivation of garden,
Often result in blade surface and the symptom such as raw blackspot, yellow, come off.The disease was typically broken out in high temperature and rainy season, was had a strong impact on
Appreciation effect.The disease is caused by rose Marssonina (Marssonina rosae), and the fungi can invade the blade of host
Disease is caused, the disease fungus can produce substantial amounts of conidium in the later stage history of life, and expansion is very fast.Disease-resistant in Chinese rose is ground
It is disease resistance of Tester etc., often using Chinese Rose as research object in studying carefully.In addition, in ornamental plant pathogen
It is very high with the field of host's interaction, the researching value of Chinese Rose.Although Chinese Rose accounts in the disease research of Chinese rose
There is consequence, but the histopathology about the disease is not clear, constrains the development of Chinese Rose research.This hair
Bright is that the research of the plant leaf diseases histopathology including Chinese Rose directly provides reliable method, to solve
The key content of pathological research provides technical support.
Research for the histopathology phagocytic process of leaf portion fungal disease, traditional research method have a lot, including
The methods of adhesive tape is pasted, bleaching is observed and organizes whole clearing.But these methods are in the research of Chinese rose foliage fungal disease
There is certain limitation, in Chinese Rose research process, the cause of disease of some host surfaces can only be obtained by conventional method
Bacterium structure, it is impossible to clearly show the fungi of inside plant tissues, and dyed to it.The present invention can make up this well
A little deficiencies, solve scientific research problem on technological layer.
The content of the invention
Present invention aim to address conventional section technology prepare infect plant cell wall in blade blueness, plant it is thin
Karyon and the common red coloration of pathogen, the problem of leading to not clearly distinguish fungi and plant nucleolus, there is provided one kind observation
The colouring method of plant leaf portion fungal disease histopathology process.Dyeing course of the invention is efficient and convenient, quality is good, can
Technical support is provided for related science research.
The method of the present invention is after section dewaxing to water, first to be dyed with the 1% crystal violet aqueous solution, recycle iodine
Liquid carries out mordant dyeing, is finally decolourized with 95% alcohol.
Preferably, method of the invention comprises the following steps：
1) after section dewaxing to water, section is placed in 5~10min of dyeing in the 1% crystal violet aqueous solution；
2) crystal violet of the slice surface after washing away dyeing with distilled water；
3) 30~60s of section after rinsing is handled with iodine solution, carries out mordant dyeing；
4) with 15~60s of section after 95% alcohol washes mordant dyeing, decolourized；
5) it is dehydrated 5~10s with absolute ethyl alcohol.
In aforesaid operations, the crystal violet of slice surface, can avoid crystal violet from persistently dyeing after washing away dyeing with distilled water, real
Now appropriateness dyeing, with the section after 95% alcohol washes mordant dyeing, can slough the dye liquor combined with plant tissue, realize fungi bacterium
Silk red coloration, plant tissue are blue effect.
Preferably, the concrete operations in the step 2) are to get slide express developed twice with distilled water, each 5s.
Preferably, the mordant dyeing time in the step 3) is 30s.
During operation, after two kinds of optimum conditions that the solution of the present invention is stated in the choice, hypha,hyphae can be optimized
Red coloration, plant tissue are blue Color.
Preferably, section is placed in the 1% crystal violet aqueous solution and dyed by after section dewaxing to water in the step 1)
5min；
Preferably, cleaned 2 times with the section 60s after 95% alcohol Rapid Cleaning mordant dyeing in the step 4).
After stating two kinds of optimum conditions in the choice, it can further optimize the effect of dyeing.
Preferably, the described 1% crystal violet aqueous solution is prepared by the following method, and 1g crystal violet is dissolved in into 100ml
In distilled water, fully after dissolving, filtered, produced in filter paper.
Preferably, the iodine solution is prepared by the following method, takes iodine and each 1g of KI to be dissolved in the alcohol of 100ml 80%
In, fully dissolving, produce.
Most preferably, method of the invention comprises the following steps：
1) after section dewaxing to water, will be dyed in the crystal violet aqueous solution after the filtering for the Fresh for being placed in 1% of cutting into slices
5min；
2) the section 5s after getting dyeing express developed with distilled water, rinse 2 times；
3) the section 60s after rinsing is handled with iodine solution, carries out mordant dyeing；
4) with the section 5s after 95% alcohol Rapid Cleaning mordant dyeing, clean 2 times；
5) it is dehydrated 5~10s with absolute ethyl alcohol.
Using above-mentioned optimal scheme, the paraffin section texture of formation is clear-cut clearly demarcated, and only hypha,hyphae it is red
Color, plant tissue are blueness, can substantially distinguish fungi state.
It is another object of the present invention to protect a kind of preparation method for the paraffin section for observing Chinese Rose infection processs,
Its step is：Sample is fixed, is dehydrated, be transparent, waxdip, embedding, section are handled with exhibition piece, dewaxing, dyeing and mounting；
The dyeing is carried out using method described herein；
The operation of the mounting is：
Section after dehydration is carried out with transparent liquid under an optical microscope it is transparent, when observing fungi and host's group
When knitting display and understanding, handled with dimethylbenzene, carry out mounting.
Preferably, the transparent liquid is by cloves oil, absolute ethyl alcohol and dimethylbenzene by volume 50：25：25 are formulated.
Final object of the present invention is the paraffin section that protection is prepared using herein described method.
The method of the present invention has the advantages that：
(1) present invention can ensure to be attached to the unnecessary crystal violet on plant tissue using the alcohol bleaching time of optimization
Dye liquor decolourizes to complete, and has no effect on the Color to fungi, realizes the single dyeing of pathogen in the blade of fungal infection,
Method is provided to observe the observation of black spot disease process.
(2) time that blade process is infected using paraffin section observation Chinese Rose is substantially reduced, improves experiment
Efficiency.
Brief description of the drawings
Fig. 1 is the paraffin section figure of the embodiment of the present invention 1
Fig. 2 is the paraffin section figure of the embodiment of the present invention 2
Fig. 3 is the paraffin section figure of the embodiment of the present invention 3
Fig. 4 is the paraffin section figure of the embodiment of the present invention 4
Fig. 5 is the paraffin section figure of the embodiment of the present invention 5
Fig. 6 is the paraffin section figure of the embodiment of the present invention 6
Fig. 7 is the paraffin section figure of comparative example 1.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
Experimental method：
1st, film-making specimen in use and source：Sample is the leaf spot lesion portion of 12dpi after rose Marssonina bacterium solution artificial spraying
Position.Source：Plant origin is by Beijing Forestry University artificial hybridization offspring, and pathogen source is in country using tissue isolation
The single bacterial strain of acquisition is separated on the black spot disease plant of flowers engineering center little Tang mountains base.
2nd, fixing means：The blade or scalpel of alcohol disinfecting, the blade of inoculation is cut into and is about 10mm, wide about
5mm or so fritter sample.It is fixed in FAA fixers of the volumetric concentration for the configuration of 70% alcohol, 24h is fixed after vacuumizing,
It is stand-by to be stored in 4 DEG C of refrigerators.FAA fixers by volumetric concentration be 70% alcohol：Glacial acetic acid：Formaldehyde volume ratio is 90:5:5 match somebody with somebody
System forms.
3rd, it is dehydrated：Material is taken out from FAA fixers, filter paper blots unnecessary FAA fixers, is put into band rubber stopper mould
In plain bottle, it is in 80% alcohol to add volumetric concentration, vacuum suction 10 minutes, stands 10 minutes；Subsequent replacing 85%,
90%th, 95%, 100%, 100% alcohol, identical vacuum filtration is all carried out after changing every time and stands process.
4th, it is transparent：Dimethylbenzene/alcohol that absolute alcohol is 1/3 volume ratio is changed, after standing 30min；Change 2/3 volume ratio
Dimethylbenzene/alcohol, stand 30min；After changing pure dimethylbenzene, 20min is stood, after the repetition of this process is secondary, is completed transparent.
5th, waxdip：Sample after will be transparent is placed in the atoleine heated in advance in 60 DEG C of baking ovens with dimethylbenzene with volume
Than 1:In 1/2 paraffin after 1 mixing, bottle stopper is stoppered, is put into 45 DEG C of insulating boxs overnight；Next day sample is placed in 60 DEG C of baking ovens,
Bottle stopper is opened, after treating dimethylbenzene volatilization completely, the paraffin that the molten point melted is 56 DEG C is changed to, is placed in 60 DEG C of insulating boxs
4h；Then identical paraffin is changed to again, is so repeated 3 times；After last time changes paraffin, it can be wrapped after standing 2h
Bury.
6th, embed：Taken out from insulating box and fill the vial of sample, the paraffin for having sample is poured into fill it is identical
In the embedding capsule of temperature paraffin, and sample position is adjusted with preheated tweezers, put well according to slice direction, stand cooling.
It is put into after surface solidification in basin, after complete solidification, removes embedded box, room temperature is dried, is in store for.
7th, section and exhibition piece：Embedded box is fixed on slicer, slice thickness is mixed up and is cut into slices for 8-10 μm, use is small
Thumb applies very small amount gelatin bonding die agent on slide, and slide is placed on 42 scholar, 1 DEG C of warm platform of exhibition, 3-5 is added on slide
Distilled water is dripped, takes cover glass length 1/5-2/5 wax band to be placed on the water surface of slide and is allowed to rapid open and flat, blotting paper sucks more
Remaining moisture is placed on Zhan Wentai again, carries out mark, and being put into 42 DEG C of baking ovens after toasting day can just be carried out in next step.
8th, dewax：The slice, thin piece that will be dyed is put into 100% dimethylbenzene at twice, each 30min, sloughs paraffin.
9th, rehydration：By complete dewaxing slice, thin piece successively the alcohol of 1/2 dimethylbenzene+1/2,100% alcohol, 95% dimethylbenzene,
In 90% dimethylbenzene, 80% dimethylbenzene, 70% dimethylbenzene, 50% dimethylbenzene, water, per link 3min.
10th, mounting is dyed：
The slice, thin piece for completing rehydration is placed in the 1% crystal violet aqueous solution of filtered Fresh and dyes 5min；Then,
Slide is got express developed with distilled water, each 5s, is total to twice；After iodine solution mordant dyeing 30s, 95% alcohol is taken to carry out decolouring 15s；With
Take absolute ethyl alcohol fast dewatering.After dehydration, under light microscope, carried out with transparent liquid it is suitably transparent, when fungi and host
When tissue display understands, handled with dimethylbenzene.Mounting then is carried out with face cream, the clarifier is by cloves oil, absolute ethyl alcohol
With dimethylbenzene by volume 50：25：25 are formulated.
Embodiment 2：
In the dyeing mounting stage of embodiment 1, the slice, thin piece for completing rehydration is placed in 1% crystallization of filtered Fresh
5min is dyed in the purple aqueous solution；Then, get slide express developed with distilled water, each 5s, be total to twice；After iodine solution mordant dyeing 30s,
95% alcohol is taken to carry out decolouring 30s；Absolute ethyl alcohol fast dewatering is taken immediately.After dehydration, under light microscope, entered with transparent liquid
Row is suitably transparent, when fungi and host tissue are shown when understanding, is handled with dimethylbenzene.Then mounting is carried out with face cream.
Embodiment 3：
In the dyeing mounting stage of embodiment 1, the slice, thin piece for completing rehydration is placed in 1% crystallization of filtered Fresh
5min is dyed in the purple aqueous solution；Then, get slide express developed with distilled water, each 5s, be total to twice；After iodine solution mordant dyeing 30s,
95% alcohol is taken to carry out decolouring 60s；Absolute ethyl alcohol fast dewatering is taken immediately.After dehydration, under light microscope, entered with transparent liquid
Row is suitably transparent, when fungi and host tissue are shown when understanding, is handled with dimethylbenzene.Then mounting is carried out with face cream.
Embodiment 4：
In the dyeing mounting stage of embodiment 1, the slice, thin piece for completing rehydration is placed in 1% crystallization of filtered Fresh
10min is dyed in the purple aqueous solution；Then, get slide express developed with distilled water, each 5s, be total to twice；Through iodine solution mordant dyeing 30s
Afterwards, 95% alcohol is taken to carry out decolouring 15s；Absolute ethyl alcohol fast dewatering is taken immediately.After dehydration, under light microscope, use is transparent
Liquid carries out suitably transparent, when fungi and host tissue are shown when understanding, is handled with dimethylbenzene.Then mounting is carried out with face cream.
Embodiment 5：
In the dyeing mounting stage of embodiment 1, the slice, thin piece for completing rehydration is placed in 1% crystallization of filtered Fresh
10min is dyed in the purple aqueous solution；Then, get slide express developed with distilled water, each 5s, be total to twice；Through iodine solution mordant dyeing 30s
Afterwards, 95% alcohol is taken to carry out decolouring 30s；Absolute ethyl alcohol fast dewatering is taken immediately.After dehydration, under light microscope, use is transparent
Liquid carries out suitably transparent, when fungi and host tissue are shown when understanding, is handled with dimethylbenzene.Then mounting is carried out with face cream.
Embodiment 6：
In the dyeing mounting stage of embodiment 1, the slice, thin piece for completing rehydration is placed in 1% crystallization of filtered Fresh
10min is dyed in the purple aqueous solution；Then, get slide express developed with distilled water, each 5s, be total to twice；Through iodine solution mordant dyeing 30s
Afterwards, 95% alcohol is taken to carry out decolouring 60s；Absolute ethyl alcohol fast dewatering is taken immediately.After dehydration, under light microscope, use is transparent
Liquid carries out suitably transparent, when fungi and host tissue are shown when understanding, is handled with dimethylbenzene.Then mounting is carried out with face cream.
Comparative example 1
Colouring method in comparative example 1 is conventional general red-fast green decoration method, and concrete operations are that will dewax to the section of water
Be placed in using 50% ethanol as 0.5% sarranine solution of solvent in 4-5h, then successively in 50%, 70%, 80%, 90%, 95%
Ethanol in be dehydrated, each 1min；Then using 95% Yichuan as solvent 0.3% it is fast green in carry out redying 30s after, put
Further dehydration 2 times, each 30s in 100% ethanol.After the completion of dehydration, respectively at the dimethylbenzene of isometric ratio：It is alcohol, pure
Transparent, each 3min is carried out in dimethylbenzene, is carried out 2 times wherein pure dimethylbenzene is transparent.Then complete transparent.Hereafter conventional envelope is carried out
Piece.
In order to illustrate the effect of the present invention, applicant as a control group, then takes implementation with routine paraffin wax section statining method
Example 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5, embodiment 6 and paraffin section made from comparative example 1, by above-mentioned 7
Group material is taken pictures respectively at Lycra biology microscope Microscopic observation, and it is (real to respectively obtain Fig. 1 (embodiment 1), Fig. 2 (embodiment 2), Fig. 3
Apply example 3), Fig. 4 (embodiment 4), Fig. 5 (embodiment 5), Fig. 6 (embodiment 6) and Fig. 7 (control group).As seen from the figure, Fig. 1,
The decolouring of Fig. 2 plant tissues is weaker, and plant tissue blueness；Fig. 4, Fig. 5 and Fig. 6 dyeing are deeper, and fungi result part
Blueness；Fig. 3 paraffin section textures are clear-cut clearly demarcated, and only hypha,hyphae red coloration, and plant tissue is blueness, can substantially be distinguished
Other fungi state.
And only have the single coloring of mycelia in Fig. 3 it can be seen from Fig. 7 is compared with Fig. 3, and mycelia and plant cell in Fig. 7
With equal red coloration, and plant cell wall blueness, can not clearly distinguish and distinguish fungi and plant cell nucleus.
Although above the present invention is made to retouch in detail with general explanation, embodiment and experiment
State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
Claims (10)
1. a kind of paraffin section colouring method for observing Chinese Rose infection processs, it is characterised in that in section dewaxing to water
Afterwards, first dyed with the 1% crystal violet aqueous solution, recycle iodine solution to carry out mordant dyeing, finally decolourized with 95% alcohol.
2. according to the method for claim 1, it is characterised in that comprise the following steps：
1) after section dewaxing to water, section is placed in 5~10min of dyeing in the 1% crystal violet aqueous solution；
2) crystal violet of the slice surface after washing away dyeing with distilled water；
3) 30~40s of section after rinsing is handled with iodine solution, carries out mordant dyeing；
4) with 15~60s of section after 95% alcohol washes mordant dyeing, decolourized；
5) it is dehydrated 5~10s with absolute ethyl alcohol.
3. according to the method for claim 2, it is characterised in that the concrete operations in the step 2) are quick with distilled water
Rinse slide twice, each 5s.
4. the method according to claim 1 or 3, it is characterised in that the mordant dyeing time in the step 3) is 30s.
5. the method according to claim 2 or 4, it is characterised in that by after section dewaxing to water in the step 1), will cut
Piece is placed in the 1% crystal violet aqueous solution and dyes 5min；
And/or cleaned 2 times with the section 60s after 95% alcohol Rapid Cleaning mordant dyeing in the step 4).
6. according to the method described in any one of Claims 1 to 5, it is characterised in that the described 1% crystal violet aqueous solution is by as follows
Method is prepared, and 1g crystal violet is dissolved in 100ml distilled water, fully after dissolving, is filtered, produced in filter paper；
And/or the iodine solution is prepared by the following method, takes iodine and each 1g of KI to be dissolved in the alcohol of 100ml 80%, fill
Divide dissolving, produce.
7. according to the method described in any one of claim 1~6, it is characterised in that comprise the following steps：
1) after section dewaxing to water, 5min will be dyed in the crystal violet aqueous solution after the filtering for the Fresh for being placed in 1% of cutting into slices；
2) the section 5s after getting dyeing express developed with distilled water, rinse 2 times；
3) the section 30s after rinsing is handled with iodine solution, carries out mordant dyeing；
4) with the section 60s after 95% alcohol Rapid Cleaning mordant dyeing, clean 2 times；
5) it is dehydrated 5~10s with absolute ethyl alcohol.
8. a kind of preparation method for the paraffin section for observing Chinese Rose infection processs, it is characterised in that consolidated sample
Fixed, dehydration, transparent, waxdip, embedding, section and exhibition piece, dewaxing, dyeing and mounting are handled, and the dyeing uses claim 1
Method described in~7 any one is carried out, and the operation of the mounting is：
Section after dehydration is carried out with transparent liquid under an optical microscope it is transparent, when observing that fungi and host tissue show
Show when understanding, handled with dimethylbenzene, carry out mounting.
9. according to the method for claim 8, it is characterised in that the transparent liquid is by cloves oil, absolute ethyl alcohol and diformazan
Benzene by volume 50：25：25 are formulated.
10. the paraffin section that claim 9 methods described is prepared.
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| CN109374376A (en) * | 2018-11-09 | 2019-02-22 | 上海市农业科学院 | A kind of slice preparation method suitable for mushroom lamella Basidium morphologic observation |
| CN109511415A (en) * | 2019-01-18 | 2019-03-26 | 江苏省农业科学院宿迁农科所 | A kind of inoculation cultural method of day lily rust |
| CN110455604A (en) * | 2019-08-28 | 2019-11-15 | 昆氏(深圳)生物科技有限公司 | Epidermis fungi samples colouring method |
| CN110487615A (en) * | 2019-08-29 | 2019-11-22 | 沈阳农业大学 | A kind of composite fluorescence colouring method for identifying ten Zi Hua section plant clubroots |
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