CN106383047A - Staining method for observing histopathologic process of fungus disease in leaf segment of plant - Google Patents
Staining method for observing histopathologic process of fungus disease in leaf segment of plant Download PDFInfo
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- CN106383047A CN106383047A CN201610985799.3A CN201610985799A CN106383047A CN 106383047 A CN106383047 A CN 106383047A CN 201610985799 A CN201610985799 A CN 201610985799A CN 106383047 A CN106383047 A CN 106383047A
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- 238000000034 method Methods 0.000 title claims abstract description 69
- 230000008569 process Effects 0.000 title claims abstract description 24
- 208000031888 Mycoses Diseases 0.000 title claims abstract description 20
- 230000003118 histopathologic effect Effects 0.000 title abstract description 4
- 238000007447 staining method Methods 0.000 title abstract 3
- 239000000463 material Substances 0.000 claims abstract description 10
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- 238000004043 dyeing Methods 0.000 claims description 35
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 33
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- 241000196324 Embryophyta Species 0.000 claims description 24
- 201000010099 disease Diseases 0.000 claims description 22
- 241000219000 Populus Species 0.000 claims description 19
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 claims description 16
- 229960002327 chloral hydrate Drugs 0.000 claims description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 206010027146 Melanoderma Diseases 0.000 claims description 13
- 241000124033 Salix Species 0.000 claims description 13
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 claims description 11
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 240000008254 Rosa chinensis Species 0.000 claims description 6
- 235000000664 Rosa chinensis Nutrition 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 6
- 241000193738 Bacillus anthracis Species 0.000 claims description 5
- 240000005001 Paeonia suffruticosa Species 0.000 claims description 5
- 235000003889 Paeonia suffruticosa Nutrition 0.000 claims description 5
- 241000221785 Erysiphales Species 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
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- 244000025254 Cannabis sativa Species 0.000 claims 1
- 239000011259 mixed solution Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 238000010186 staining Methods 0.000 abstract description 5
- 238000010438 heat treatment Methods 0.000 abstract description 4
- 244000053095 fungal pathogen Species 0.000 abstract description 3
- 238000002791 soaking Methods 0.000 abstract 1
- 238000011160 research Methods 0.000 description 15
- 241000233866 Fungi Species 0.000 description 10
- RSGINGXWCXYMQU-UHFFFAOYSA-N 2,3-dihydroxypropyl 2-hydroxypropanoate Chemical compound CC(O)C(=O)OCC(O)CO RSGINGXWCXYMQU-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
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- 239000007788 liquid Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
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- 241000628997 Flos Species 0.000 description 3
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- 206010039509 Scab Diseases 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
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- 230000035613 defoliation Effects 0.000 description 2
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- 239000012362 glacial acetic acid Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
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- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000068958 Marssonina brunnea Species 0.000 description 1
- 208000026062 Tissue disease Diseases 0.000 description 1
- 229910000004 White lead Inorganic materials 0.000 description 1
- RZUBARUFLYGOGC-MTHOTQAESA-L acid fuchsin Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=C(N)C(C)=CC(C(=C\2C=C(C(=[NH2+])C=C/2)S([O-])(=O)=O)\C=2C=C(C(N)=CC=2)S([O-])(=O)=O)=C1 RZUBARUFLYGOGC-MTHOTQAESA-L 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
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- 210000004247 hand Anatomy 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- HFFLGKNGCAIQMO-UHFFFAOYSA-N trichloroacetaldehyde Chemical compound ClC(Cl)(Cl)C=O HFFLGKNGCAIQMO-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a staining method for observing the histopathologic process of a fungus disease in a leaf segment of a plant. The method comprises the following steps of (1): the preparation of a sample block: selecting a leaf blade of the plant, which is suffered from the fungus disease, as a to-be-treated material, and making the to-be-treated material into the sample block; (2), fixed destaining: soaking the sample block in a fixed destaining solution, carrying out the fixed destaining for 4h to 6h, then replacing the fixed destaining solution, and carrying out the fixed destaining for 4h to 6h; (3), transparent staining: immersing the sample block subjected to the fixed destaining in a transparent staining agent, carrying out heating treatment on the sample block for 4h to 6h at 40 DEG C to 60 DEG C; (4), flaking and observation. Compared with other histopathologic observation methods of the fungus disease in the leaf segment, the staining method provided by the invention is simple and quick to use, and moreover, is quite good in effect; a mycelium which is formed inside a cell of the leaf blade by a pathogenic fungus can be clearly observed; a high-quality observation result in a field can be obtained.
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
The leaf portion fungal disease of plant is very big to the health of plant and infringement attractive in appearance, often results in plant untimely defoliation, very
To the death leading to plant, very serious impact is caused to national agricultural economy.
Willow is the essentialspecies tree planting kind of China, has certain distribution in each area national.Black spot disease in poplar is poplar
A kind of important disease of tree, extensively, the place of nearly all cultivation willow is all distributed this sick occurrence scope.This disease typically exists
Occur before and after at the bottom of June, cause aggrieved willow untimely defoliation, photosynthetic rate is decreased obviously, and has had a strong impact on its wood quality and life
Long amount, particularly in the simple nursery of some standing forests, especially severe of falling ill, or even the death that can result in poplar seedlings.This disease
Evil is caused by ascomycetess poplar Marssonina (Marssonina brunnea), and the blade that this fungus can invade host willow causes disease
Evil, this pathogenic fungi can produce substantial amounts of conidium in the life cycle later stage, and expansion is very fast.In the disease-resistant gene of willow with educate
Kind research in, be disease resistance of Tester etc., also often using Black spot disease in poplar as object of study.Additionally, in forestry disease
Opportunistic pathogen and the field of host's interaction, the researching value of Black spot disease in poplar is very high.Although however, Black spot disease in poplar is in the disease of willow
Occupy an important position in research, but the histopathology about this disease is not clear, this just seriously hinders its cause
The research work of Anttdisease Mechanism, governs the development about other researchs of Black spot disease in poplar, including molecule Plant Pathology.The present invention
It is that the histopathologic research of plant leaf diseases including Black spot disease in poplar directly provides effective reliability side
Method, provides technical support for solving the key content in pathology.
For the research of the histopathology phagocytic process of leaf portion fungal disease, traditional research method have a lot, including
Adhesive tape is pasted, bleaching is observed and organizes the methods such as whole clearing.But, these methods are studied in poplar leaf fungal disease
In, there is certain limitation, in the research process of Black spot disease in poplar, some host surfaces can only be obtained by traditional method
Pathogenic bacteria is it is impossible to enough clearly represent the funguses architectural feature of inside plant tissues and it is dyeed it is impossible to complete
Expected resultss, could not solve actual Scientific Research Problem.It is not enough, in technological layer solution scientific research that the present invention can make up these well
A difficult problem.
Content of the invention
It is an object of the invention to provide a kind of colouring method of observation of plant leaf portion fungal disease histopathology process, lead to
Cross heretofore described colouring method it can clearly be observed that the funguses structure of blade interior, the technology of successfully solving is asked
Topic.And, also there is good versatility in the leaf diseases of other leaf diseases in willow and other plant.Additionally,
The dyeing course of the present invention is efficient and convenient, and effect quality is good, and can study for related science provides technical support.
For reaching above-mentioned purpose, the technical scheme is that：
A kind of colouring method of observation of plant leaf portion fungal disease histopathology process, methods described includes following steps
Suddenly；
1) preparation of sample blocks：
Select by the plant leaf blade of pathomycete invasion morbidity as process material, and be fabricated to sample blocks；
2) fixing decolouring：
Sample blocks are immersed in fixing destaining solution, after fixing decolouring 4～6h, change fixing destaining solution, fixing decolouring 4～
6h；
3) transparent dyeing：
The sample blocks of fixing decolouring are immersed in transparent stain, 40～60 DEG C of heat treated 4～6h；
4) film-making is observed.
In the present invention, the making of sample blocks adopts 75% blade of alcohol disinfecting or scalpel, is cut into processing material
1cm × 1cm about fritter sample.
In the present invention, described fixing destaining solution is the mixing of 100% solution of trichloroacetic acid/ethanol/chloroform=3/150/50
Solution or ethanol.
In the present invention, described transparent stain is prepared using the method comprising the following steps：
By chloral hydrate solution and dyeing liquor with 6/1～4/1 ratio mix homogeneously, can 60 DEG C of the pre-heat treatment 1min.
Preferably, described chloral hydrate solution is prepared using the method comprising the following steps：20g chloral hydrate adds 4～5ml
Glycerol and 5～6ml water, after fully dissolving, are saved in glass jar.
Preferably, described dyeing liquor is lactophenol cotton blue dyeing liquor or aniline blue dyeing liquor.
The present invention, when film-making is observed, by the sample handled well tabletting, slice, thin piece must not have bubble, using glycerol or thoroughly
Bright stain, as floating supporting agent, is observed in ordinary optical microscope lower sheeting.
Agent prescription according to the present invention：
(1) preparation of fixing destaining solution：It is to be added water 227ml with the trichloroacetic acid of 500g, so that 100% trichloroacetic acid is prepared
Afterwards, with ratio 3/150/50 by 100% solution of trichloroacetic acid, ethanol, chloroform mixing, it is saved in brown bottle；
(2) chloral hydrate saturated solution：20g chloral hydrate adds 4～5ml glycerol and 5～6ml water, after fully dissolving, preserve
In glass jar；
(3) lactophenol cotton blue dyeing liquor：Carbolic acid 10g, lactic acid 10ml, glycerol 20ml, cotton indigo plant 0.02g, distilled water 10ml.
Colouring method in the present invention can be effectively applied to Black spot disease in poplar histopathological study, by host tissue
Internal funguses clear in structure ground coloring display, can significantly distinguish the structure with pathogen for the host by dyeing.
Colouring method in the present invention has extensive versatility, can apply the tissue disease in most of leaf portion fungal disease
In the research work of electrophysiologic procedure, such as using the Staining Protocol in the present invention to willow anthrax, Chinese Rose, Caulis et folium euphorbiae milii tikka
The scab of disease, Paeonia suffruticosa powdery mildew and floral leaf pinta of exposing the wealth has carried out processing to be observed, and all has preferable effect.
The present invention is combined transparent in one step with dyeing, simplifies traditional operating procedure, has saved the time, right
The funguses of blade interior have special Color.
The present invention is directed to the insurmountable Scientific Research Problem of traditional method, and based on conventional reagent, repetition test, by certainly
Chief creating recent studies on, is combined together transparent with dyeing, for successfully completing high definition and the strong dyeing work of contrast effect.This
Invention has the characteristics that convenient high universalizable is strong, provides effective technical support in the research field of plant leaf diseases, has
Application prospect well.
Brief description
Fig. 1 is that coloured differently method applies the Color on Black spot disease in poplar；
Fig. 2 is the impact to Color for the coloured differently time；
Fig. 3 is the impact to Color for the different temperatures；
Fig. 4 is the impact to Color for the coloured differently clarifier configuration proportion；
Fig. 5 is the Color that the present invention applies in other leaf diseases, wherein：A, B are the present invention to willow anthrax
The Color of disease；C is the Color to Chinese Rose for the present invention；D is the Color to Paeonia suffruticosa powdery mildew for the present invention；
E is the Color to Flos Inulae leaf spot for the present invention.
Specific embodiment
For the more preferable practical application effect showing the present invention, below by three examples, in conjunction with accompanying drawing, the present invention is existed
Specific embodiment in practical application is described in detail, and the specific embodiment in embodiment has no effect on this patent
Protection domain.
Embodiment 1：Different transparent colouring methods are applied in Black spot disease in poplar and are compared
From taken outdoors Black spot disease in poplar pathogen poplar Marssonina, carry out separation and Culture.Gather clean poplar leaf, first
Rinsed well with tap water, then carry out surface sterilization 30s, rinsed with sterile water 3 times with 75% ethanol.Poplar Marssonina is mitogenetic
Spore is with 105The concentration of individual/ml is inoculated on blade, cultivates 4～5d in illumination box, and condition of culture is：Humidity
100%, 25 DEG C of temperature, illumination 12h/12h (illumination/dark).With blade or the scalpel of 75% alcohol disinfecting, will inoculate
Blade be cut into 1cm × 1cm about fritter sample.Respectively following process is carried out to material：
Method (1) trichloroacetic acid decolours：
Material is put in the fixing destaining solution of 100% solution of trichloroacetic acid/ethanol/chloroform (3/150/50), changes after 12h
Fixing destaining solution, then entered the fixing decolouring observation of 1d, with aniline blue as staining reagent during observation, with ethanol for floating supporting agent.
Method (2) chloral hydrate transillumination：
Material is put in the ethanol (95%) of equivalent and glacial acetic acid mixed liquor and fixes 24h, be then immersed in the hydration of saturation
In chloral aqueous solution.Take out after transparency of organization and wash, after aniline blue dyeing, observed as floating supporting agent with glycerol.
Method (3) glycerol lactate buffer solution chloral hydrate transillumination：
Material is put into fixing decolouring in ethanol glacial acetic acid mixed liquor, after Sample Fade, moves on on microscope slide, drip two
The glycerol lactate buffer solution dyeing of 1% acid fuchsin, is heated to till being fuming slowly.Then, remove remaining glycerol lactate buffer solution, plus several
Drip the glycerol lactate buffer solution without dyestuff, heating, wash away unnecessary stain, the transparent rear microscopy of saturation chloral hydrate solution.
Method (4) adopts the method for the present invention：
Sample blocks are immersed in the (mixing of 100% solution of trichloroacetic acid/ethanol/chloroform=3/150/50 of fixing destaining solution
Solution) in, after fixing decolouring 6h, change fixing destaining solution, fixing decolouring 6h；The sample blocks of fixing decolouring are immersed transparent dyeing
In agent, 55 DEG C of heat treated 5h；By the sample handled well tabletting, slice, thin piece must not have bubble, using glycerol or transparent stain
As floating supporting agent, observe in ordinary optical microscope lower sheeting.
Described transparent stain is prepared using the method comprising the following steps：
By chloral hydrate solution and lactophenol cotton blue dyeing liquor with 6/1 ratio mix homogeneously, can 60 DEG C of the pre-heat treatment
1min.
Described chloral hydrate solution is prepared using the method comprising the following steps：20g chloral hydrate adds 4～5ml glycerol and 5
～6ml water, after fully dissolving, is saved in glass jar.
A～D in accompanying drawing 1 is followed successively by the result photo of above four kinds of process, can be seen that through side from A, B, C of in figure
Method (1), (2), the process of (3), the funguses structure outline within leaf tissue is unclear, does not have stereovision, is merely able to blade
The conidium on surface effectively colours (as upper left corner area in Figure 1B, 1C), and the structure of funguses does not all effectively catch color, point
Distinguish that the structure being easy to during structure with blade cell is obscured, and, during observing, this several pictures has been best, and
And process of observing is more arduous, looks for such visual field extremely difficult.Figure D is the Color of the present invention, in plant cell
Funguses structure effectively dyeed by lactophenol cotton blue dyeing liquor, with background contrast clearly, can clearly show
Whole funguses architectural feature, picture quality is very high, solves the indeterminable problem of traditional method well.
Embodiment 2：The impact to Color for the different treatment conditions
Early stage is processed with embodiment 1, by the blade of inoculation be cut into 1cm × 1cm about fritter sample after, sample is soaked
In the fixing destaining solution of 100% solution of trichloroacetic acid/ethanol/chloroform (3/150/50), after fixing decolouring 6h, change fixing decolouring
Liquid, continues fixing decolouring 6h, subsequently, carries out in transparent dyeing liquor sample after fixing for the decolouring being put into different proportion preparation
Bright dyeing, specific configuration proportion is as shown in table 1 below.Transparent dye is carried out by different gradient treatment temperatures and process time
Color, specific treatment temperature and process time, as shown in table 1 below.Finally, the sample that each is processed is put on microscope slide, with
Glycerol is that floating supporting agent carries out tabletting observation, and result is respectively as shown in Figure 2, Figure 3 and Figure 4.
Table 1 different disposal time, different disposal temperature and coloured differently liquid proportional
A～F in Fig. 2 is the coloration result of process time 1h, 2h, 3h, 4h, 5h and 6h respectively, as can be seen from the figure
First three hour coloring effect is not so well, after dyeing carries out 4h, starts dark coloured (Fig. 2 D), reaches good dye in 5h
Color effect (Fig. 2 E), after dyeing 6h, coloring does not change significantly, and illustrates that, in 5h, Color has reached optimum state.Attached
The treatment temperature of the A～D in Fig. 3 is respectively 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, when dyeing temperature is spent less than 40 DEG C, sample
Coloring ratio shallower (Fig. 3 A), does not have obvious aberration, but to more than 40 DEG C, the coloring effect of mycelia is just between real silk and host
Start substantially (Fig. 3 B), after 50 DEG C, coloring effect is not strengthening, so 50 DEG C is reasonable selection.A～D in accompanying drawing 4
Use dyeing liquid proportional (chloral hydrate solution/lactophenol cotton blue dyeing liquor) be respectively 7/1,6/1,5/1,4/1, this ratio
During more than 7/1, the coloring effect of mycelia is poor (Fig. 4 A), and when ratio is less than 6/1, Color is very good, and substantially protects
Hold consistent (Fig. 4 B, Fig. 4 C, Fig. 4 D).Consider, the simplicity of operation, time cost and monetary cost, in Black spot disease in poplar
Histopathological study in, several major variables of the present invention, i.e. the preparation ratio of process time, treatment temperature and dyeing liquor
Example, respectively preferably 6h, 50 DEG C and 6/1.
Embodiment 3：Using the colouring method in the present invention, multiple leaf diseases are carried out with dyeing to observe
Gather the sick leaf of some common leaf diseases in Beijing, including willow anthrax, Chinese Rose, Paeonia suffruticosa white lead
Disease and Flos Inulae leaf spot, load in hermetic bag after collection, are saved in 4 degree of refrigerators with to be used.
Rinse the sick leaf of collection with tap water, then dry blade face with filter paper, with blade or the handss of 75% ethanol disinfection
Art knife by blade be cut into 1cm × 1cm about fritter sample, sample is immersed in 100% solution of trichloroacetic acid/ethanol/chloroform
(3/150/50) in fixing destaining solution, after fixing decolouring 6h, change fixing destaining solution, continue fixing decolouring 6h, subsequently, will decolour
Sample after fixation is put in transparent dyeing liquor (chloral hydrate solution/lactophenol cotton blue dyeing liquor=6/1), 50 DEG C dyeing 5～
6h, then the sample of each disease is put on microscope slide, carries out tabletting observation with glycerol for floating supporting agent, result is as shown in Figure 5.
Can be seen that the colouring method of the present invention other leaf diseases willow anthraxs in willow from Fig. 5 A and Fig. 5 B
In application, achieve extraordinary Color, can be very good to observe morphological characteristic in poplar leaf for this pathogen,
Dyeing quality is high, and the aberration in picture is big, by host with apparent the distinguishing of pathogen, the general matter of this colouring method is described
Amount is very high.In the application of Chinese Rose, Chinese Rose pathogen also can posted by Staining Protocol of the present invention
The haustorium structure being formed in main body dyes (Fig. 5 C) well.Equally, for the coloring effect of the haustorium structure of Paeonia suffruticosa Powdery Mildew
Really also very good, can clearly show the morphological characteristic (Fig. 5 D) of these internal structures.It can be seen that Flos Inulae from Fig. 5 E
Inside mycelial structure at leaf spot scab also can be colored liquid and contaminate for blueness, and background colour is slightly miscellaneous, the material due to choosing when possible
Expect empress dowager, or scab has developed into the reason in later stage, but, do not hinder the observation for internal funguses structure.
To sum up state results, it can be seen that the effect of the Staining Protocol practical application of the present invention is very good, the final figure obtaining
Tablet quality is very high, and the versatility of the present invention is very extensive, and generally, any leaf diseases all can be carried out using this method
The dyeing of pathogenic fungi histopathological study is observed, and can provide very big facility for the research work of association area.
The description of the aforementioned specific illustrative embodiment to the present invention illustrate that and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, the present invention, in specific implementation process, can carry out a lot of changing
Become, examples detailed above is to illustrate present invention specific implementation step in actual applications, the staff in help field
The present invention can be preferably applied to carry out scientific research.The scope of the present invention is intended to be limited by claims and its equivalents
Fixed.
Claims (7)
1. a kind of colouring method of observation of plant leaf portion fungal disease histopathology process, methods described comprises the steps；
1) preparation of sample blocks：
Select by the plant leaf blade of pathomycete invasion morbidity as process material, and be fabricated to sample blocks；
2) fixing decolouring：
Sample blocks are immersed in fixing destaining solution, after fixing decolouring 4～6h, change fixing destaining solution, fixing decolouring 4～6h；
3) transparent dyeing：
The sample blocks of fixing decolouring are immersed in transparent stain, 40～60 DEG C of heat treated 4～6h；
4) film-making is observed.
2. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 1, its
It is characterised by, described fungal disease is including Black spot disease in poplar, willow anthrax, Chinese Rose, Paeonia suffruticosa powdery mildew or to expose the wealth
Blade of grass pinta is in interior plant leaf diseases.
3. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 1, its
It is characterised by, described fixing destaining solution is mixed solution or the second of 100% solution of trichloroacetic acid/ethanol/chloroform=3/150/50
Alcohol.
4. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 1, its
It is characterised by, described transparent stain is prepared using the method comprising the following steps：
By chloral hydrate solution and dyeing liquor with 6/1～4/1 ratio mix homogeneously.
5. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 4, its
It is characterised by, described chloral hydrate solution is prepared using the method comprising the following steps：20g chloral hydrate adds 4～5ml glycerol and
5～6ml water, after fully dissolving, is saved in glass jar.
6. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 4, its
It is characterised by, described dyeing liquor is lactophenol cotton blue dyeing liquor or aniline blue dyeing liquor.
7. the colouring method of a kind of observation of plant leaf portion fungal disease histopathology process according to claim 1, its
It is characterised by, when film-making is observed, glycerol or transparent stain are used as floating supporting agent, carry out observation and take pictures.
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| CN113310767A (en) * | 2021-06-01 | 2021-08-27 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Microscopic method for pollen tube and ovule after pollination of water lily and optical microscopic tabletting manufacturing method |
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| CN107446985A (en) * | 2017-08-30 | 2017-12-08 | 李保华 | A set of Apple top layer colonizes and infected the rapid examination method of fungi |
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| CN115824757A (en) * | 2022-12-28 | 2023-03-21 | 九江学院 | Dyeing method of endophytic fungi in oil tea root system |
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