CN107576552B - Paraffin section dyeing method for observing infection process of China rose black spot - Google Patents

Abstract

The invention relates to a paraffin section dyeing method for observing the infection process of China rose black spot, which comprises the steps of firstly dyeing by using a 1% crystal violet water solution after a section is dewaxed to water, then mordanting by using iodine solution, and finally decoloring by using 95% alcohol. The two staining solutions and the two decoloring methods are selected to stain the histiocytes into blue and the hyphae into red, thereby providing a method for observing the disease course development process of the melasma. In addition, the method greatly shortens the dyeing time of observing the process of the Chinese rose black spot infecting the leaves by using the paraffin section through the improved dyeing method, and improves the test efficiency.

Description

Paraffin section dyeing method for observing infection process of China rose black spot

Technical Field

The invention relates to the field of plant microscopy, in particular to a staining method for observing histopathological process of fungal disease of plant leaves

Background

The fungal diseases of plant leaves often cause disease spots, yellowing and early defoliation of the plant leaves, and serious plants directly cause plant death, thereby influencing the normal growth and development of the plants and increasing the cultivation, management and maintenance cost.

The Chinese rose is the first of four big cut flowers, is a perennial woody flower of rosa of rosaceae, and has the advantages of long flowering period, rich flower color, various plant types and the like. The China rose black spot is a fungus disease with extremely high incidence and extremely strong destructiveness in courtyard cultivation, and often causes black spots, yellowing, falling off and other symptoms on the surface of leaves. The disease generally erupts in high-temperature rainy seasons, and the ornamental effect is seriously influenced. The disease is caused by roselle calyx (Marssonina rosae), the fungus can invade the leaves of a host to cause the disease, and the pathogenic fungus can generate a large amount of conidia in the later period of life history and can expand very quickly. In the study of disease resistance of roses, the black spot of roses is often used as a study target to test the disease resistance of varieties. In addition, the research value of the China rose black spot is high in the field of interaction of garden plant pathogenic bacteria and hosts. Although China rose black spot is an important place in the study of diseases of China rose, the histopathology of the disease is not clear, and the study of China rose black spot is restricted. The invention directly provides a reliable method for histopathology research of plant leaf diseases including China rose black spot, and provides technical support for solving the key content of pathology research.

For the research on the histopathological invasion process of the leaf fungal diseases, a plurality of traditional research methods are available, including methods of transparent tape pasting, bleaching observation, tissue overall transparence and the like. However, these methods have certain limitations in the study of the fungal diseases of the leaves of the Chinese rose, and in the study process of the black spot of the Chinese rose, only some pathogenic bacteria structures on the host surface can be obtained by the traditional methods, and the fungi in the plant tissues cannot be clearly shown and stained. The invention can well make up for the defects and solve the problem of scientific research on the technical level.

Disclosure of Invention

The invention aims to solve the problem that fungi and plant cell nuclei cannot be clearly distinguished due to blue coloring of plant cell walls, red coloring of plant cell nuclei and red coloring of pathogenic bacteria in infected leaves prepared by a conventional slicing technology, and provides a staining method for observing the histopathological process of the fungal diseases of plant leaves. The dyeing process is quick and convenient, has good quality, and can provide technical support for related scientific research.

The method comprises the steps of firstly dyeing with 1% crystal violet water solution after the slices are dewaxed to water, then mordanting with iodine solution, and finally decoloring with 95% alcohol.

Preferably, the method of the present invention comprises the steps of:

1) dewaxing the slices to water, and then placing the slices in a 1% crystal violet solution for dyeing for 5-10 min;

2) washing off crystal violet on the surface of the dyed section by using distilled water;

3) treating the washed slices with iodine solution for 30-60 s, and performing mordant dyeing;

4) washing the mordanted slices with 95% alcohol for 15-60 s, and decoloring;

5) dehydrating the mixture for 5 to 10 seconds by using absolute ethyl alcohol.

In the operation, the distilled water is used for flushing away the crystal violet on the surface of the dyed section, so that the continuous dyeing of the crystal violet can be avoided, the proper dyeing can be realized, the section after mordant dyeing is cleaned by 95% alcohol, the dye solution combined with the plant tissue can be removed, and the effects that the fungal hypha is red and the plant tissue is blue can be realized.

Preferably, the specific operation in step 2) is to rapidly wash the slide twice with distilled water for 5s each time.

Preferably, the mordant dyeing time in the step 3) is 30 s.

In the operation process, the scheme of the invention can optimize the dyeing effect that the fungal hyphae are colored red and the plant tissues are blue after the two preferable conditions are selected.

Preferably, after the slices are dewaxed to water in the step 1), the slices are placed in a 1% crystal violet solution for dyeing for 5 min;

preferably, the mordant slices are quickly washed with 95% alcohol for 60s in the step 4), and the washing is carried out for 2 times.

After selecting the above two preferred conditions, the effect of the dyeing can be further optimized.

Preferably, the 1% crystal violet solution is prepared by dissolving 1g of crystal violet in 100ml of distilled water, dissolving the solution sufficiently, and filtering the solution in a filter paper.

Preferably, the iodine solution is prepared by dissolving 1g of iodine and potassium iodide in 100ml of 80% alcohol, and sufficiently dissolving to obtain the iodine solution.

Most preferably, the method of the invention comprises the steps of:

1) dewaxing the slices to water, and placing the slices in a 1% freshly prepared filtered crystal violet solution for dyeing for 5 min;

2) washing the stained section with distilled water for 5s for 2 times;

3) treating the washed slices with iodine solution for 60s, and performing mordant dyeing;

4) quickly cleaning mordant slices with 95% alcohol for 5s for 2 times;

5) dehydrating the mixture for 5 to 10 seconds by using absolute ethyl alcohol.

By adopting the optimal scheme, the formed paraffin section has clear and distinct texture contour, only the fungal hyphae are red, the plant tissues are blue, and the fungal state can be obviously distinguished.

The invention also aims to protect a preparation method of a paraffin section for observing the infection process of the China rose black spot, which comprises the following steps: fixing, dehydrating, transparentizing, waxing, embedding, slicing and unfolding, dewaxing, dyeing and sealing the sample;

the dyeing is carried out by the method described in the application;

the operation of the sealing sheet is as follows:

and (3) the dehydrated slices are subjected to transparency by using a transparent liquid under an optical microscope, and when the fungi and the host tissues are clearly observed, the slices are treated by using dimethylbenzene to carry out mounting.

Preferably, the transparent liquid is prepared from clove oil, absolute ethyl alcohol and xylene according to a volume ratio of 50: 25: 25.

It is a final object of the invention to protect paraffin sections prepared by the methods described herein.

The method of the invention has the following beneficial effects:

(1) the method can ensure that the decoloration of the redundant crystal violet stain attached to the plant tissue is finished by utilizing the optimized alcohol decoloration time, does not influence the dyeing effect on the fungi, realizes the single dyeing of pathogenic bacteria in the leaves infected by the fungi, and provides a method for observing the disease course development process of the black spot.

(2) Greatly shortens the time for observing the process of the Chinese rose black spot infecting the leaves by utilizing the paraffin section, and improves the test efficiency.

Drawings

FIG. 1 is a view of a paraffin section in example 1 of the present invention

FIG. 2 is a view of a paraffin section in example 2 of the present invention

FIG. 3 is a view of a paraffin section in example 3 of the present invention

FIG. 4 is a view of a paraffin section in example 4 of the present invention

FIG. 5 is a view of a paraffin section in example 5 of the present invention

FIG. 6 is a paraffin section view of example 6 of the present invention

Fig. 7 is a view of a paraffin section of comparative example 1.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The experimental method comprises the following steps:

1. samples and sources used for tableting: the sample is 12dpi leaf spot parts after the rosewood-disc-spore bacterium liquid is artificially sprayed. The source is as follows: the plant source is an artificial hybridization progeny of Beijing forestry university, and the pathogenic bacteria source is a single bacterial strain obtained by separating on a black spot disease plant of the small Tangshan base of the national flower engineering center by using a tissue separation method.

2. The fixing method comprises the following steps: the inoculated leaf was cut into small samples of about 10mm in length and about 5mm in width using an alcohol sterilized blade or scalpel. Fixing in FAA fixing solution prepared from 70% alcohol, vacuumizing, fixing for 24 hr, and storing in 4 deg.C refrigerator. The FAA stationary liquid is prepared from 70% alcohol by volume: glacial acetic acid: the formaldehyde volume ratio is 90:5: 5.

3. And (3) dehydrating: taking the material out of the FAA stationary liquid, sucking the excess FAA stationary liquid by filter paper, putting the FAA stationary liquid into a penicillin bottle with a rubber plug, adding the penicillin bottle into 80% alcohol by volume concentration, vacuumizing for 10 minutes, and standing for 10 minutes; then 85%, 90%, 95%, 100% alcohol is replaced, and the same vacuum filtration and standing process are carried out after each replacement.

4. And (3) transparency: replacing xylene/alcohol with anhydrous alcohol of 1/3 volume ratio, and standing for 30 min; changing 2/3 volume ratio xylene/alcohol, standing for 30 min; after pure dimethylbenzene is replaced, standing for 20min, and repeating the process twice to finish the transparency.

5. Wax dipping: placing the transparent sample in 1/2 paraffin which is prepared by heating liquid paraffin and dimethylbenzene in an oven at 60 ℃ in advance and uniformly mixing according to the volume ratio of 1:1, plugging a bottle stopper, and placing the bottle stopper in a thermostat at 45 ℃ overnight; placing the sample in a 60 ℃ oven the next day, opening the bottle stopper, after the xylene is completely volatilized, replacing the melted paraffin with the melting point of 56 ℃, and placing the sample in a 60 ℃ thermostat for 4 hours; then the same paraffin is changed again, and the process is repeated for 3 times; and after paraffin is replaced for the last time, standing for 2 hours, and then embedding.

6. Embedding: and taking the glass vial containing the sample out of the incubator, pouring the paraffin containing the sample into an embedding small box containing paraffin with the same temperature, adjusting the position of the sample by using preheated tweezers, placing the sample in the direction of the section, standing and cooling. And (4) after the surface is solidified, putting the mixture into a basin, after the surface is completely solidified, removing the embedding box, airing the mixture at room temperature, and storing the mixture for later use.

7. Slicing and unfolding: fixing the embedding box on a slicer, adjusting the slice thickness to 8-10 μm, slicing, coating a very small amount of gelatin-coated tablets on a glass slide with a little finger, placing the glass slide on a 42-degree 1-DEG C spreading table, adding 3-5 drops of distilled water on the glass slide, placing a wax tape with the length of 1/5-2/5 of a cover glass on the water surface of the glass slide to rapidly spread the wax tape, absorbing excessive water with absorbent paper, placing the water tape on the spreading table again, marking, and baking in a 42-DEG C oven for a day before carrying out the next step.

8. Dewaxing: the piece to be dyed is placed in 100% xylene twice, each time for 30min, and paraffin is removed.

9. Rehydration: the dewaxed slices are sequentially subjected to 1/2 xylene +1/2 alcohol, 100% alcohol, 95% xylene, 90% xylene, 80% xylene, 70% xylene, 50% xylene and water, each step is 3 min.

10. Dyeing and sealing:

placing the rehydrated slices in a filtered and freshly prepared 1% crystal violet solution for dyeing for 5 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 15 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. And then sealing with balsam, wherein the clearing agent is prepared from clove oil, absolute ethyl alcohol and xylene according to a volume ratio of 50: 25: 25.

Example 2:

in the dyeing and mounting stage of example 1, the wafer which is completed with rehydration is placed in a filtered and freshly prepared 1% crystal violet solution for dyeing for 5 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 30 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. Followed by sealing with balsam.

Example 3:

in the dyeing and mounting stage of example 1, the wafer which is completed with rehydration is placed in a filtered and freshly prepared 1% crystal violet solution for dyeing for 5 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 60 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. Followed by sealing with balsam.

Example 4:

in the dyeing and mounting stage of example 1, the wafer which is completed with rehydration is placed in a filtered and freshly prepared 1% crystal violet solution for dyeing for 10 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 15 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. Followed by sealing with balsam.

Example 5:

in the dyeing and mounting stage of example 1, the wafer which is completed with rehydration is placed in a filtered and freshly prepared 1% crystal violet solution for dyeing for 10 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 30 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. Followed by sealing with balsam.

Example 6:

in the dyeing and mounting stage of example 1, the wafer which is completed with rehydration is placed in a filtered and freshly prepared 1% crystal violet solution for dyeing for 10 min; subsequently, the slide was rapidly rinsed with distilled water for 5 seconds each for a total of two times; mordanting with iodine solution for 30s, and decolorizing with 95% ethanol for 60 s; then the absolute ethyl alcohol is taken to be dehydrated quickly. After dehydration, appropriate clearing was performed with a clearing solution under an optical microscope, and when the fungus and host tissues were clearly shown, treatment with xylene was performed. Followed by sealing with balsam.

Comparative example 1

The staining method in comparative example 1 is a conventional reddish-fast green staining method, specifically, the slice dewaxed to water was placed in a 0.5% safranin solution with 50% ethanol as a solvent for 4-5h, and then dehydrated in 50%, 70%, 80%, 90%, 95% ethanol in sequence, each for 1 min; after subsequent counterstaining in 0.3% fast green in 95% Yichun as solvent for 30s, the solution was further dehydrated in 100% ethanol for 2 times for 30s each. After dehydration was complete, the reaction mixture was mixed with xylene at equal volume ratios: the transparent treatment is carried out in alcohol and pure xylene, each time for 3min, wherein the transparent treatment is carried out 2 times. Transparency is completed. After which a conventional mounting is performed.

In order to illustrate the effects of the present invention, applicants used a conventional paraffin section staining method as a control group, and further took paraffin sections obtained in examples 1, 2, 3, 4, 5, 6 and 1, and photographed the above 7 groups of materials by observation under a leica biomicroscope, to obtain fig. 1 (example 1), 2 (example 2), 3 (example 3), 4 (example 4), 5 (example 5), 6 (example 6) and 7 (control group), respectively. As can be seen from the figure, the plant tissues in the figures 1 and 2 are slightly discolored, and the plant tissues are blue; FIG. 4, FIG. 5 and FIG. 6 are darker in color and the fungal results are partially bluish; FIG. 3 is a clear paraffin section with red hypha and blue plant tissue, which can distinguish the fungus status.

As can be seen from comparison of fig. 7 and 3, only the hyphae in fig. 3 are singly colored, while the hyphae and the plant cell sum in fig. 7 are both red colored, and the plant cell wall is blue colored, so that the fungi and the plant cell nucleus cannot be clearly distinguished.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A paraffin section staining method for observing the infection process of Chinese rose black spot is characterized by comprising the following steps:

1) dewaxing the slices to water, and then placing the slices in a 1% crystal violet solution for dyeing for 5-10 min;

2) washing off crystal violet on the surface of the dyed section by using distilled water;

3) treating the washed slices with iodine solution for 30-40 s, and performing mordant dyeing;

4) washing the mordanted slices with 95% alcohol for 15-60 s, and decoloring;

5) dehydrating the mixture for 5 to 10 seconds by using absolute ethyl alcohol.

2. The method as claimed in claim 1, wherein the specific operation in step 2) is to rapidly wash the slide twice with distilled water for 5s each time.

3. The method according to claim 1 or 2, characterized in that the mordant time in step 3) is 30 s.

4. The method according to claim 1, wherein after the slices are dewaxed to water in the step 1), the slices are stained in a 1% crystal violet solution for 5 min;

and/or in the step 4), quickly washing the mordant slices with 95% alcohol for 60s for 2 times.

5. The method according to any one of claims 1, 2 or 4, wherein the 1% crystal violet solution is prepared by dissolving 1g of crystal violet in 100ml of distilled water, dissolving the solution sufficiently, and filtering the solution in filter paper;

and/or the iodine solution is prepared by dissolving 1g of iodine and potassium iodide in 100ml of 80% alcohol respectively, and fully dissolving to obtain the iodine solution.

6. The method according to claim 3, wherein the 1% crystal violet solution is prepared by dissolving 1g of crystal violet in 100ml of distilled water, dissolving the solution sufficiently, and filtering the solution in a filter paper;

and/or the iodine solution is prepared by dissolving 1g of iodine and potassium iodide in 100ml of 80% alcohol respectively, and fully dissolving to obtain the iodine solution.

7. The method of claim 1, comprising the steps of:

1) dewaxing the slices to water, and placing the slices in a 1% freshly prepared filtered crystal violet solution for dyeing for 5 min;

2) washing the stained section with distilled water for 5s for 2 times;

3) treating the washed slices with iodine solution for 30s, and performing mordant dyeing;

4) quickly cleaning the mordanted slices with 95% alcohol for 60s for 2 times;

5) dehydrating the mixture for 5 to 10 seconds by using absolute ethyl alcohol.

8. A method for preparing a paraffin section for observing the infection process of Chinese rose black spot, which is characterized in that a sample is subjected to fixing, dehydrating, transparentizing, waxing, embedding, slicing and spreading, dewaxing, dyeing and sealing treatment, wherein the dyeing is carried out by the method of any one of claims 1-7, and the sealing operation is as follows:

and (3) the dehydrated slices are subjected to transparency by using a transparent liquid under an optical microscope, and when the fungi and the host tissues are clearly observed, the slices are treated by using dimethylbenzene to carry out mounting.

9. The method according to claim 8, wherein the transparent liquid is prepared from clove oil, absolute ethyl alcohol and xylene in a volume ratio of 50: 25: 25.

10. Paraffin sections prepared by the method of claim 9.

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