阅读说明：本技术 一种新型孢子鞭毛染色液以及染色观察方法 (Novel spore flagellum staining solution and staining observation method ) 是由 史娟 姜粉霞 马新 于 2019-08-27 设计创作，主要内容包括：本发明涉及生物技术领域,尤其涉及一种新型孢子鞭毛染色液以及染色观察方法。所述的染色液为番红或者固绿。一种新型孢子鞭毛染色观察方法,其特征在于,首先材料需要再15度低温下预培养1.5h,室温下进行染色处理后置于20℃培养箱中染色30min,取出冲洗多余的染色液,吸干边缘多余的液体,置于显微镜下观察,可清晰地观察到游动孢子的孢子鞭毛。建立霜霉病菌游动孢子鞭毛的染色技术,可为霜霉病菌游动孢子发育机制、靶向运动等相关研究提供支持。该方法具有操作步骤简单,容易掌握,鞭毛观察明显,且不需要特殊设备即可进行。(The invention relates to the technical field of biology, in particular to a novel spore flagellum staining solution and a staining observation method. The staining solution is safranin or fast green. A novel spore flagellum dyeing observation method is characterized in that firstly, materials need to be pre-cultured for 1.5h at low temperature of 15 ℃, the materials are placed in an incubator at 20 ℃ for dyeing for 30min after being dyed at room temperature, the excess dyeing liquid is taken out and washed, the excess liquid on the edge is sucked dry, and the materials are placed under a microscope for observation, so that the spore flagellum of zoospores can be observed clearly. The establishment of the dyeing technology of the flagella of the downy mildew zoospores can provide support for the relevant researches such as the development mechanism, the targeted movement and the like of the downy mildew zoospores. The method has the advantages of simple operation steps, easy mastering, obvious flagellum observation and no need of special equipment.)

1. A novel spore flagellum staining solution is characterized in that the staining solution is safranin or fast green.

2. A novel spore flagellum staining observation method is characterized in that firstly, materials need to be pre-cultured for 1.5h at low temperature of 15 ℃, the materials are placed in an incubator at 20 ℃ for staining for 0.5h after being stained at room temperature, the excess staining solution is taken out and washed, the excess liquid on the edge is sucked dry, and the materials are placed under a microscope for observation, so that the spore flagellum of zoospores can be observed clearly.

3. The method for observing the flagella of spores as claimed in claim, comprising the steps of,

preparing a dyeing solution: weighing 0.1g of safranin, dissolving in distilled water, dissolving to 100ml, putting into a brown dropping bottle, and labeling for later use; weighing 0.1g of fast green, dissolving in 95% ethanol, dissolving to 100ml, transferring into a brown dropping bottle after all the fast green is dissolved, and labeling for later use;

pre-culture of peronospora parasitica: carrying out moisture-preserving culture on downy mildew leaves collected in the field for 12h in a 15-20 ℃ incubator; and (3) acquisition standard: collecting fresh yellow polygonal scabs on the front of the leaves, and gray-black or black frosty mildew layer on the back of the leaves, namely sporangium peduncles or sporangium of peronospora parasitica, quickly filling into a moisture-preserving bag, and preventing the loss of water of the leaves and influencing the sporangium; or collecting downy mildew leaves before sunset in the afternoon, and quickly filling the collected downy mildew leaves into a moisture-preserving bag to prevent the leaves from losing water; taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with 15cm bottom and filter paper with saturated water for moisture-keeping culture, and placing into a 20 deg.C incubator for 12 hr;

preparation of spore suspension: brushing the pre-cultured downy mildew bacteria with a clean brush to obtain a mildew layer, namely the sporangium of the bacteria, placing the mildew layer in sterilized water on a concave glass slide while avoiding epidermal cells of leaves, repeating the brushing process for 2 to 3 times, covering a cover glass, placing the cover glass into a culture dish of 9cm, and preserving moisture for 0.5h at room temperature of less than or equal to 26 ℃;

dyeing: sucking the staining solution by a dropper, dropping the staining solution on one side of the concave slide, properly inclining the staining solution to enable the staining solution to enter the sterilized water of the concave slide, putting the suspension in the concave slide into a culture dish when the suspension in the concave slide is in uniform red and blue, and placing the concave slide in a 20-DEG incubator for staining for 0.5 h;

and (3) decoloring: slowly dripping sterilizing water along one side of the cover glass to wash off redundant dyeing liquid, and wiping the redundant dyeing liquid around the cover glass with absorbent paper;

and (4) microscopic observation: placing the cleaned glass slide under a low power microscope for observation, and performing high power microscope observation after finding a target; spore flagella can be clearly observed; and a photograph is taken.

4. A novel observation method for dyeing flagella of spores as claimed in claim 3, wherein the moisture-keeping culture in the culture dish of the filter paper with saturated water laid on the bottom of 15cm is carried out by the special design equipment which is the moisture-keeping culture structure comprising the culture dish lower part (1) and the culture dish upper part (2) buckled with each other, the culture dish lower part (1) is heat-sealed with a cube-shaped stainless steel filter screen (5) downwards, the stainless steel filter screen (5) is placed with sponge (9), the culture dish lower part (1) comprises a threaded hole, and the hand-held shaft (6) passing through the threaded hole; the handheld shaft (6) is a threaded shaft, and the sponge (9) can be extruded by downwards moving the extrusion plate (8) below the handheld shaft by rotating the handheld shaft; in the extrusion process, water in the sponge can downwards pass through the stainless steel filter screen (5) and drip on the moisturizing culture structure (3); therefore, water can be added without opening the lower part (1) and the upper part (2) of the culture dish to avoid the entry of the mixed bacteria in the air;

the moisturizing culture structure (3) comprises a coating structure, the middle of the coating structure comprises a clamping space (4), and the clamping space (4) is a water holding material;

the downy mildew leaves can be placed in the clamping space (4) so that both sides of the downy mildew leaves can be contacted with the water holding material.

5. The method for observing the dyeing of the flagella of the novel spores as claimed in claim 4, wherein the water-holding material is filter paper.

Technical Field

The invention relates to the technical field of biology, in particular to a novel spore flagellum staining solution and a staining observation method.

Background

Downy mildew is taxonomically belonging to the species oomycete. Is a kind of eukaryotic microorganism with similar shape and infection characteristics to fungi. Downy mildew can infect many important crops and the resulting downy mildew often causes significant economic losses to farmers.

The zoospore is the most important infection inoculum of the peronospora parasitica, the zoospore moves by means of flagella and targets host plant cells, once the zoospore contacts the host plant, the zoospore rapidly stops and starts to germinate, and a germinating germ tube often forms an adherent cell similar structure so as to be beneficial to further invading the host plant. Therefore, the observation of the zoospore flagella not only is an important content in the biological basic research of the peronospora parasitica, but also provides an important basis for the research of the development mechanism of the zoospores. The flagella is an accessory structure and is slender, and the flagella can not be easily observed under an optical microscope, so that a dyeing method which is simple and easy to operate and has an obvious observation effect is sought, the research on the mechanism of the development of the zoospores of the peronospora parasitica to further form the resting spores is necessary, and an important observation method is provided for the in-depth research on the flagella development mechanism, the ultrastructure and the like. Bacterial flagella are important locomotor organs, and the presence or absence of bacterial flagella is essential for the classification and identification of bacteria, so that technologies for staining bacterial flagella are numerous. However, since oomycetes are not prokaryotic microorganisms and their zoospores have no cell walls, the conventional staining methods for bacterial flagella are not suitable for staining and observing the flagella of the zoospores of peronospora parasitica.

Disclosure of Invention

The purpose of the invention is as follows: in order to provide a novel staining solution for flagella of spores with better effect and a staining observation method, the concrete purpose is to see a plurality of substantial technical effects of the concrete implementation part.

In order to achieve the purpose, the invention adopts the following technical scheme:

a novel spore flagellum staining solution is characterized in that the staining solution is safranin or fast green.

A novel spore flagellum staining observation method is characterized in that firstly, materials need to be pre-cultured for 1.5h at low temperature of 15 ℃, the materials are placed in an incubator at 20 ℃ for staining for 0.5h after being stained at room temperature, the excess staining solution is taken out and washed, the excess liquid on the edge is sucked dry, and the materials are placed under a microscope for observation, so that the spore flagellum of zoospores can be observed clearly.

The invention further adopts the technical scheme that the method comprises the following steps,

preparing a dyeing solution: weighing 0.1g of safranin, dissolving in distilled water, dissolving to 100ml, putting into a brown dropping bottle, and labeling for later use; weighing 0.1g of fast green, dissolving in 95% ethanol, dissolving to 100ml, transferring into a brown dropping bottle after all the fast green is dissolved, and labeling for later use;

pre-culture of peronospora parasitica: carrying out moisture-preserving culture on downy mildew leaves collected in the field for 12h in a 15-20 ℃ incubator; and (3) acquisition standard: collecting the polygonal scab with bright yellow on the front of the leaf and the frosty mildew layer with gray to gray black or black on the back of the leaf, namely the sporangium peduncle or sporangium of the downy mildew, quickly filling the collected sporangium into a moisture-preserving bag to prevent the water loss of the leaf and influence on the sporangium; or collecting downy mildew leaves before sunset in the afternoon, and quickly filling the collected downy mildew leaves into a moisture-preserving bag to prevent the leaves from losing water; taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with 15cm bottom and filter paper with saturated water for moisture-keeping culture, and placing into a 20 deg.C incubator for 12 hr;

preparation of spore suspension: brushing the pre-cultured downy mildew bacteria with a clean brush to obtain a mildew layer, namely the sporangium of the bacteria, placing the mildew layer in sterilized water on a concave glass slide while avoiding epidermal cells of leaves, repeating the brushing process for 2 to 3 times, covering a cover glass, placing the cover glass into a culture dish of 9cm, and preserving moisture for 0.5h at room temperature of less than or equal to 26 ℃;

dyeing: sucking the staining solution by a dropper, dropping the staining solution on one side of the concave slide, properly inclining the staining solution to enable the staining solution to enter the sterilized water of the concave slide, putting the suspension in the concave slide into a culture dish when the suspension in the concave slide is in uniform red and blue, and placing the concave slide in a 20-DEG incubator for staining for 0.5 h;

and (3) decoloring: slowly dripping sterilizing water along one side of the cover glass to wash off redundant dyeing liquid, and wiping the redundant dyeing liquid around the cover glass with absorbent paper;

and (4) microscopic observation: placing the cleaned glass slide under a low power microscope for observation, and performing high power microscope observation after finding a target; spore flagella can be clearly observed; and a photograph is taken.

The technical scheme is that the culture in a moisture-preserving culture dish with the filter paper with the saturated moisture paved at the bottom of 15cm is finished by adopting a special design device which is a moisture-preserving culture structure, the moisture-preserving culture structure comprises a culture dish lower part 1 and a culture dish upper part 2 which are mutually buckled, a cubic stainless steel filter screen 5 is downwards thermally bonded on the culture dish lower part 1, a sponge 9 is placed in the stainless steel filter screen 5, a threaded hole is formed in the culture dish lower part 1, and a handheld shaft 6 penetrating through the threaded hole is further included; the handheld shaft 6 is a threaded shaft, and the sponge 9 can be extruded by downward movement of the extrusion plate 8 below the handheld shaft by rotating the handheld shaft; in the extrusion process, water in the sponge can downwards pass through the stainless steel filter screen 5 and drip on the moisturizing culture structure 3; therefore, water can be added without opening the lower part 1 and the upper part 2 of the culture dish to avoid the entry of the mixed bacteria in the air;

the moisturizing culture structure 3 comprises a coating structure, the middle part of the coating structure comprises a clamping space 4, and the clamping space 4 is a water holding material;

the downy mildew leaves can be placed in the holding space 4 such that both sides of the downy mildew leaves are in contact with the water holding material.

The invention further adopts the technical scheme that the water holding material is filter paper.

Compared with the prior art, the invention adopting the technical scheme has the following beneficial effects: the establishment of the dyeing technology of the flagella of the downy mildew zoospores can provide support for the relevant researches such as the development mechanism, the targeted movement and the like of the downy mildew zoospores. The method has the advantages of simple operation steps, easy mastering, obvious flagellum observation and no need of special equipment. Zoospores, which are vegetative propagules of downy mildew, develop from sporangia and, therefore, are present in nature for a very short period of time. The observation of the zoospore flagella can be realized by the method.

Drawings

To further illustrate the present invention, further description is provided below with reference to the accompanying drawings:

FIG. 1 shows safranin staining; FIG. 2 is fast green staining; FIG. 3 shows unstained zoospore flagella; FIG. 4 is a moisturizing culture structure specifically designed in this patent; wherein: 1. below the culture dish; 2. above the culture dish; 3. a moisture-retaining culture structure; 4. a clamping space; 5. a stainless steel filter screen; 6. a hand-held shaft; 7. a gasket; 8. a pressing plate; 9. a sponge.

Detailed Description

The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention. The patent provides a plurality of parallel schemes, and different expressions belong to an improved scheme based on a basic scheme or a parallel scheme. Each solution has its own unique features.

We refer to relevant documents, and carry out flagellum staining observation on downy mildew zoospores by adopting four staining agents of safranin, aniline blue, methylene blue, fast green and the like in sequence. 2 kinds of coloring agents of safranin and fast green are screened out to be used for dyeing flagella of zoospores of downy mildew, and a relatively ideal observation effect is obtained. The flagella of the downy mildew zoospores can be clearly observed under a microscope through dyeing observation of the 2 coloring agents, and a method is provided for observing the movement law of the zoospores, the flagella change of the zoospores at different periods and the flagella form. Flagella are clearly observed through background contrast by utilizing the principle that four different coloring agents such as safranin, 0.5% aniline blue, 0.1% methylene blue, fast green and the like are combined with zoospore cytoplasm. The most excellent observation effect can be obtained by screening 2 coloring agents of safranin and fast green for dyeing treatment through the definition of flagellum. The key technical points are that firstly, the material needs to be pre-cultured for 1.5h at low temperature of 15 ℃, the material is placed in an incubator at 20 ℃ for dyeing for 0.5h after being dyed at room temperature, the redundant dyeing liquid is taken out and washed, the redundant liquid at the edge is sucked dry, and the material is placed under a microscope for observation, so that the flagella of zoospores can be clearly observed. No other alternatives have been searched for to accomplish the present invention.

1, preparation of a dyeing solution: weighing 0.1g of safranin, dissolving in distilled water, dissolving to 100ml, putting into a brown dropping bottle, and labeling for later use; weighing 0.1g of fast green, dissolving in 95% ethanol, dissolving to 100ml, transferring into a brown dropping bottle after all the fast green is dissolved, and labeling for later use.

2. Pre-culture of peronospora parasitica: carrying out moisture-preserving culture on the downy mildew leaves collected in the field in an incubator at 15-20 ℃ for 12 h. And (3) acquisition standard: collecting fresh yellow polygonal scab on the front of the leaf, gray to gray black or black frost mildew layer (sporangium peduncle or sporangium of peronospora parasitica) on the back of the leaf, and rapidly filling into a moisture-keeping bag to prevent the water loss of the leaf and influence on the sporangium; or collecting downy mildew leaves before sunset in the afternoon, and rapidly packaging into a moisture-keeping bag to prevent water loss of the leaves. Taking back to the laboratory, wrapping petiole with cotton soaked with distilled water, placing into a culture dish with 15cm bottom and filter paper with saturated water for moisture-keeping culture, and placing into a 20 deg.C incubator for 12 hr.

3. Preparation of spore suspension: brushing the pre-cultured downy mildew bacteria with a clean brush pen gently to obtain a mildew layer (sporangium of the bacteria, avoiding epidermal cells of leaves) and placing in sterilized water on a concave glass slide, repeating for 2-3 times, covering with a cover glass, and placing in a 9cm culture dish for moisturizing for 0.5h (room temperature less than or equal to 26 ℃);

4. dyeing: sucking the staining solution by a dropper, dropping the staining solution on one side of the concave slide, properly inclining the staining solution to enable the staining solution to enter the sterilized water of the concave slide, putting the suspension in the concave slide into a culture dish when the suspension in the concave slide is in uniform red and blue, and placing the concave slide in a 20-DEG incubator for staining for 0.5 h;

5. and (3) decoloring: slowly dripping sterilizing water along one side of the cover glass to wash off redundant dyeing liquid, and wiping the redundant dyeing liquid around the cover glass with absorbent paper;

6. and (4) microscopic observation: and (4) placing the cleaned glass slide under an optical microscope for observation under a low power microscope, and carrying out high power microscope observation after a target is found. The flagella were clearly observed. And a photograph is taken.

The technical scheme is that the culture in a moisture-preserving culture dish with the filter paper with the saturated moisture paved at the bottom of 15cm is finished by adopting a special design device which is a moisture-preserving culture structure, the moisture-preserving culture structure comprises a culture dish lower part 1 and a culture dish upper part 2 which are mutually buckled, a cubic stainless steel filter screen 5 is downwards thermally bonded on the culture dish lower part 1, a sponge 9 is placed in the stainless steel filter screen 5, a threaded hole is formed in the culture dish lower part 1, and a handheld shaft 6 penetrating through the threaded hole is further included; the handheld shaft 6 is a threaded shaft, and the sponge 9 can be extruded by downward movement of the extrusion plate 8 below the handheld shaft by rotating the handheld shaft; in the extrusion process, water in the sponge can downwards pass through the stainless steel filter screen 5 and drip on the moisturizing culture structure 3; therefore, water can be added without opening the lower part 1 and the upper part 2 of the culture dish to avoid the entry of the mixed bacteria in the air;

the moisturizing culture structure 3 comprises a coating structure, the middle part of the coating structure comprises a clamping space 4, and the clamping space 4 is a water holding material;

the downy mildew leaves can be placed in the holding space 4 such that both sides of the downy mildew leaves are in contact with the water holding material. The technical scheme of the invention has the following substantial technical effects and the realization process: the defects of the prior art are that when the culture dish is required to be opened continuously and water is required to be added continuously during cultivation, the culture effect is poor if water is forgotten to be added; this patent directly adopts the mode that does not open culture dish below 1 to add water, avoids the miscellaneous fungus in the air to get into.

In addition, the existence of the centre gripping space 4 of this patent makes the leaf both sides can both hold water and cultivate, has avoided the problem that makes a confused which side of leaf has the bacterial of cultivateing sometimes and has caused the cultivation mistake in the time of original cultivation.

The water holding material is filter paper.

It should be noted that the plurality of schemes provided in this patent include their own basic schemes, which are independent of each other and are not restricted to each other, but they may be combined with each other without conflict, so as to achieve a plurality of effects.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to illustrate the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is to be limited to the embodiments described above.