1. A preparation method of a protozoan cyst transmission electron microscope sample is characterized by comprising the following steps:

selecting cells in the protozoan cyst stage or cysts after experimental treatment as samples, fixing the selected cysts by using a fixing solution I, rinsing the fixed cysts by using a rinsing solution after the fixing treatment is finished, carrying out secondary fixing treatment on the rinsed cysts by using a fixing solution II after the rinsing treatment is finished, carrying out secondary rinsing treatment on the cysts after the secondary fixing treatment by using the rinsing solution after the secondary fixing treatment is finished, dewatering the cysts after the secondary rinsing treatment by using an organic solvent after the secondary rinsing treatment is finished, permeating and embedding the dewatered cysts by using an embedding agent after the dewatering treatment is finished, and polymerizing the cysts in turn after the embedding treatment is finished, slicing and dyeing to obtain a protozoan cyst transmission electron microscope sample, and finally verifying and observing the obtained protozoan cyst transmission electron microscope sample.

2. The preparation method according to claim 1, comprising the following steps:

(1) sampling:

selecting a required protozoan cyst stage cell or a cyst after experimental treatment as a sample, wherein the selected cyst is a single cyst of a resting cyst, an infectious cyst or a reproductive cyst, the diameter of the selected single cyst is not too large, but the single cyst is good in development, and before treatment, observing whether the development condition of the cyst is an expected effect under an optical microscope;

(2) fixing treatment:

transferring the single capsule selected in the step (1) to one corner of a glass slide, sucking the redundant culture solution, washing the capsule into a glass fixing cylinder filled with a small amount of fixing solution I by using the fixing solution I (after the capsule is poured into the fixing cylinder, the whole capsule is soaked in the fixing cylinder), and carefully shaking the capsule in the glass fixing cylinder for 5 min; then, carefully transferring the sample to a 2mL centrifuge tube, adding 1mL of stationary liquid I, and standing for 1 hour at room temperature; then transferring the sample into a refrigerator at 4 ℃ to be fixed for 24 hours in a dark place, and keeping the centrifugal tube laid horizontally during the fixing treatment, so that the fixing liquid is in full contact with the sample;

(3) rinsing treatment:

rinsing the sample obtained after the fixing treatment in the step (2) by using sterilized seawater once, and then rinsing the sample by using a rinsing solution for three times, wherein the rinsing is performed for 4 times; putting the sample into an equipartition instrument during each rinsing, adding sterilized seawater or rinsing liquid, and then performing oscillation rinsing, wherein before each immersion rinsing, the sterilized seawater or the rinsing liquid in the previous step needs to be carefully sucked to the greatest extent, and the oscillation rinsing is performed at room temperature;

(4) secondary fixing treatment:

sucking the rinsing liquid in the sample obtained after rinsing treatment in the step (3) until only the last drop is left, then immediately adding a drop of fixing liquid II, and then transferring the sample to a refrigerator at 4 ℃ for secondary fixing treatment; keeping the centrifugal tube in a dark place and keeping the centrifugal tube lying for 1.5 to 2 hours during secondary fixation treatment;

(5) and (3) secondary rinsing treatment:

sucking the fixing liquid II in the sample obtained after the secondary fixing treatment in the step (4) to be dry, and immediately adding a rinsing liquid to rinse the sample for three times; putting the sample into an equipartition instrument during each rinsing, adding a rinsing liquid, and then carrying out oscillation rinsing, wherein the rinsing liquid in the previous step needs to be carefully sucked as dry as possible before each immersion cleaning;

(6) and (3) dehydration treatment:

absorbing the rinsing liquid in the sample obtained after the secondary rinsing treatment in the step (5), dewatering the sample step by utilizing alcohol with different concentrations from low to high, and then continuously dewatering the sample step by utilizing acetone with different concentrations from low to high to replace free water in the sample;

(7) embedding treatment:

carrying out gradual tissue infiltration treatment on the sample obtained after dehydration treatment in the step (6) by using embedding agents with different concentrations;

(8) polymerization treatment:

polymerizing the sample obtained after the embedding treatment in the step (7) at 70 ℃ for 24 hours, and then treating at 60 ℃ for 36 hours;

(9) slicing:

slicing comprises trimming and slicing, taking the sample obtained after the polymerization treatment in the step (8) out of a centrifugal tube, grinding the sample into pieces with the length of 1cm, and then carrying out continuous ultrathin slicing on a slicer;

(10) dyeing treatment:

dyeing the ultrathin slice obtained after the slicing in the step (9) by using a coloring agent, so that heavy metal salt is combined with some structures and components of cells, the scattering capacity of electrons is increased, the purpose of improving contrast is further achieved, and the displayed picture is clear and visible; and after dyeing is finished, placing the obtained sample on a transmission electron microscope, and carrying out sample loading observation to further inspect the sample preparation result.

3. The method according to claim 2, wherein in the step (2), the fixative solution I is a mixture of glutaraldehyde with a mass concentration of 3%, Tritan X-100 with a mass concentration of 0.3%, and sterilized seawater with a salinity of 22%, and the volume ratio of the three is 12:3: 85.

4. The preparation method according to claim 2, wherein in the step (3), the sterilized seawater is sterilized seawater with salinity of 22% o, and the rinsing solution is 0.1mol/L PBS solution; putting the sample into an equipartition instrument during each rinsing, adding 800 mu L of sterilized seawater or rinsing liquid into a centrifugal tube, and then performing shaking rinsing, wherein the time of each shaking rinsing is 15 minutes; when the apparatus is placed evenly for shaking rinsing, the rotating speed is 2400r/min, and the circulation is 30s multiplied by 3.

5. The method according to claim 2, wherein in step (4), the fixative II is osmate solution with a mass concentration of 1%.

6. The preparation method according to claim 2, wherein in the step (5), the rinsing solution is 0.1mol/L PBS solution, the sample is placed in an equipartition instrument during each rinsing, 800 μ L of the rinsing solution is added into a centrifuge tube for shaking rinsing, and the time of each shaking rinsing is 15 minutes; when the apparatus is placed evenly for shaking rinsing, the rotating speed is 2400r/min, and the circulation is 30s multiplied by 3.

7. The method according to claim 2, wherein in the step (6), the dehydration treatment specifically comprises the steps of:

sucking up rinsing liquid in the sample obtained after the secondary rinsing treatment in the step (5), adding 800 mu L of alcohol with the mass concentration of 30% into a centrifugal tube, treating the sample on a shaking instrument for 20 minutes at room temperature, and sucking up the sample; adding 800 microliter of alcohol with the mass concentration of 50% into the centrifuge tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry; continuously adding 800 mu L of alcohol with the mass concentration of 70% into the centrifugal tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry; finally, adding 800 mu L of 90% alcohol into the centrifugal tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry the mixture, thereby completing the step-by-step dehydration treatment of the sample by the alcohol with different concentrations;

transferring the capsules subjected to the step-by-step dehydration treatment by using alcohol with different concentrations into a 1mL centrifuge tube, and continuously dehydrating by using acetone (no damage to samples during transfer): adding 400 mu L of acetone with the mass concentration of 90% into a centrifuge tube, processing the mixture on a shaking instrument for 10 minutes at room temperature, and then sucking the mixture to dry; then adding 400 mu L of anhydrous acetone into the centrifuge tube, processing the mixture on a shaking instrument for 10 minutes at room temperature, and then sucking the mixture to dry; the step of dehydrating the anhydrous acetone is repeated for three times, and the anhydrous acetone is absorbed for the last time until three drops of anhydrous acetone remain, so that the step-by-step dehydration treatment of the sample by the acetone with different concentrations is completed;

in the step, alcohol and acetone with different concentrations are prepared at present.

8. The method of claim 2, wherein the step (7) of performing the step-wise tissue infiltration treatment comprises the steps of:

adding 1.5 drops of embedding agent into a sample obtained after dehydration treatment, wherein the mass ratio of the embedding agent to acetone in a system is 1: 2, covering a cover to perform infiltration treatment for 2 hours;

secondly, adding a drop of half embedding agent into the system in the first step to ensure that the mass ratio of the embedding agent to the acetone in the system is 1: 1, covering a cover to perform infiltration treatment for 2 hours;

carefully sucking out all the liquid added in the system in the step II, adding 500 mu L embedding agent, vacuumizing for 30 minutes and then carrying out polymerization treatment;

the embedding medium in the step I-III is a Spurr embedding medium.

Background

Protozoan cysts are mainly classified into "resting cysts", "infectious cysts" and "reproductive cysts". "resting cysts" are dormant states of protozoa in response to adverse conditions, commonly found in ciliates (cysts, servant, acropolis, etc.); the infection cyst is the transmission stage in the life history of higher animal parasites, and the chitin cyst wall has acid resistance and can prevent the body of the parasite from being digested by the host (endophytic dysentery, giardia lamblia, etc.); unlike the two above, the cysts that undergo normal cell division and produce larvae are "reproductive cysts," a particular allergic stage in the biological life history, which are formed under ex vivo conditions by spontaneous regulation. Cysts of this type are common in fish parasitic protozoa, such as the chaulmoogra, the amylovorin, and the cryptocaryon irritans. The study of protozoan cysts has recently received sufficient attention. Over the years, much research has focused on the structure and formation of cysts, dedifferentiation and redifferentiation of the ciliated organelles of the cell cortex, and the association of various cysts with the evolution of the protozoan system. Especially, the study of the characteristics of the internal microstructure of the cyst is helpful for deeply understanding the development rule of the parasite, provides a theoretical basis for the prevention and treatment of diseases, provides a new thought for the development of new research means and medicines, can effectively explain the regional differences of the parasite and many mechanisms in biological evolution, and provides a scientific basis for the development of biological energy, the promotion of ecological balance of the nature and the like, so that how to research the subtlety of the micro world by means of a modern scientific and technical method is very important.

Transmission Electron Microscopy (TEM) is an important tool for studying biological ultrastructure. The resolution ratio is as high as 0.2nm, the micro structure of most cells can be observed, the observation requirement of unicellular microorganisms is met, and the application range is extremely wide. In the transmission electron microscope imaging, high-speed electron beams emitted by an electron gun are converged by a 1-2-level condenser and then uniformly irradiated on an observation area of a sample, incident electrons collide and do not collide with the sample, most of the electrons penetrate through the sample due to the thinness of the sample, and the intensity distribution of the electrons corresponds to the appearance, the organization and the structure of the observed sample area one by one. The electrons projected out of the sample are magnified and projected on a fluorescent screen for observing the image through a three-level magnetic lens of an objective lens, a middle lens and a projection lens, the fluorescent screen converts the electron intensity distribution into light intensity distribution visible to human eyes, and the image corresponding to the appearance, the organization and the structure of the sample is displayed on the fluorescent screen for the experiment.

Whether a TEM sample is well prepared is an important prerequisite for accurately observing the microstructure in a cell. The sample preparation process of the traditional transmission electron microscope ultrathin section generally comprises the steps of sampling, fixing, soaking, dehydrating, embedding, permeating, polymerizing, slicing, dyeing and the like. Although the sample preparation method has a single property, the diversity of samples causes uneven sample preparation results. Due to the particularities of protozoan encapsulation structures, a ring of "capsule wall" is formed around the periphery of the cell. The encapsulating wall is a derivative of a protozoan cell and is an armor of the cell, and provides a protective microenvironment for the cell, so that the damage of a large amount of chemical drugs and other adverse factors to the protoplast can be prevented. Protozoan cyst walls are generally morphologically diverse and multilayered structures that vary in biological composition. For example, the capsule wall of the cryptocaryon irritans is in a multi-layer structure with the shape of about 4 microns and composed of 3 areas with different electron densities, the middle part is compact, the thickness is about half of the capsule wall, and the structures on the inner side and the outer side are loose.

Therefore, to observe the internal microstructure of the capsule by transmission electron microscopy, the sample preparation method and technique become the key to the success of sample preparation. To prepare a good sample and obtain a clearly visible image, the following difficulties need to be overcome: 1. ensuring that the size of the sample is the optimum size; 2. the stationary liquid can quickly penetrate through the capsule wall to the interior of the capsule; 3. the dehydrating agent can enter the capsule and replace the free water in the cell in a short time; 4. the embedding agent can sufficiently permeate into the capsule and the like. As long as one of the steps is not well treated, the sample preparation fails, and the obtained internal structure is degraded to different degrees. However, in terms of the current experimental method and operation technology, the quality of the prepared protozoan cyst electron microscope sample is mixed, cavities with different degrees often appear in the cyst, the organelle structure in the cyst is difficult to accurately observe in a large visual field, the ultrastructure of the cell is difficult to clearly present, and the failure rate is high. And a set of more standard sample preparation method is not available, and sample preparation is usually based on personal experience, and the obtained result has the characteristics of instability, non-repeatability and the like.

Disclosure of Invention

The invention aims to solve the technical problems of high degradation and failure rate in the capsule, difficulty in clear presentation of the ultrastructure of cells, instability and unrepeatability of sample preparation results and the like in the preparation of a protozoan capsule TEM sample in the prior art, and provides a preparation method of a protozoan capsule transmission electron microscope sample. According to the invention, through multiple experimental tests, combined with experimental experiences, according to the characteristics of protozoan cysts, on the basis of the traditional sample preparation method, the method for fixing the cysts and the selection of embedding agents are optimized, the time for immersion, dehydration and embedding is improved, the skills in part of sample preparation processes are increased, the ultrastructure of the cysts can be completely and clearly presented, the result is stable, the repeatability is strong, and the technical problems of large degradation of cytoplasm and the like in the previous sample preparation are solved.

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

a preparation method of a protozoan cyst transmission electron microscope sample comprises the following steps:

selecting cells in the protozoan cyst stage or cysts after experimental treatment as samples, fixing the selected cysts by using a fixing solution I, rinsing the fixed cysts by using a rinsing solution after the fixing treatment is finished, carrying out secondary fixing treatment on the rinsed cysts by using a fixing solution II after the rinsing treatment is finished, carrying out secondary rinsing treatment on the cysts after the secondary fixing treatment by using the rinsing solution after the secondary fixing treatment is finished, dewatering the cysts after the secondary rinsing treatment by using an organic solvent after the secondary rinsing treatment is finished, permeating and embedding the dewatered cysts by using an embedding agent after the dewatering treatment is finished, and polymerizing the cysts in turn after the embedding treatment is finished, slicing and dyeing to obtain a protozoan cyst transmission electron microscope sample, and finally verifying and observing the obtained protozoan cyst transmission electron microscope sample.

In the above technical scheme, the preparation method specifically comprises the following steps:

(1) sampling:

selecting a required protozoan cyst stage cell or a cyst after experimental treatment as a sample, wherein the selected cyst is a single cyst of a resting cyst, an infectious cyst or a reproductive cyst, the diameter of the selected single cyst is not too large, but the single cyst is good in development, and before treatment, observing whether the development condition of the cyst is an expected effect under an optical microscope;

(2) fixing treatment:

transferring the single capsule selected in the step (1) to one corner of a glass slide, sucking the redundant culture solution, washing the capsule into a glass fixing cylinder filled with a small amount of fixing solution I by using the fixing solution I (after the capsule is poured into the fixing cylinder, the whole capsule is soaked in the fixing cylinder), and carefully shaking the capsule in the glass fixing cylinder for 5 min; then, carefully transferring the sample to a 2mL centrifuge tube, adding 1mL of stationary liquid I, and standing for 1 hour at room temperature; then transferring the sample into a refrigerator at 4 ℃ to be fixed for 24 hours in a dark place, and keeping the centrifugal tube laid horizontally during the fixing treatment, so that the fixing liquid is in full contact with the sample;

(3) rinsing treatment:

rinsing the sample obtained after the fixing treatment in the step (2) by using sterilized seawater once, and then rinsing the sample by using a rinsing solution for three times, wherein the rinsing is performed for 4 times; putting the sample into an equipartition instrument during each rinsing, adding sterilized seawater or rinsing liquid, and then performing oscillation rinsing, wherein before each immersion rinsing, the sterilized seawater or the rinsing liquid in the previous step needs to be carefully sucked to the greatest extent, and the oscillation rinsing is performed at room temperature;

(4) secondary fixing treatment:

sucking the rinsing liquid in the sample obtained after rinsing treatment in the step (3) until only the last drop is left, then immediately adding a drop of fixing liquid II, and then transferring the sample to a refrigerator at 4 ℃ for secondary fixing treatment; keeping the centrifugal tube in a dark place and keeping the centrifugal tube lying for 1.5 to 2 hours during secondary fixation treatment;

(5) and (3) secondary rinsing treatment:

sucking the fixing liquid II in the sample obtained after the secondary fixing treatment in the step (4) to be dry, and immediately adding a rinsing liquid to rinse the sample for three times; putting the sample into an equipartition instrument during each rinsing, adding a rinsing liquid, and then carrying out oscillation rinsing, wherein the rinsing liquid in the previous step needs to be carefully sucked as dry as possible before each immersion cleaning;

(6) and (3) dehydration treatment:

absorbing the rinsing liquid in the sample obtained after the secondary rinsing treatment in the step (5), dewatering the sample step by utilizing alcohol with different concentrations from low to high, and then continuously dewatering the sample step by utilizing acetone with different concentrations from low to high to replace free water in the sample;

(7) embedding treatment:

carrying out gradual tissue infiltration treatment on the sample obtained after dehydration treatment in the step (6) by using embedding agents with different concentrations;

(8) polymerization treatment:

polymerizing the sample obtained after the embedding treatment in the step (7) at 70 ℃ for 24 hours, and then treating at 60 ℃ for 36 hours;

(9) slicing:

slicing comprises trimming and slicing, taking the sample obtained after the polymerization treatment in the step (8) out of a centrifugal tube, grinding the sample into pieces with the length of 1cm, and then carrying out continuous ultrathin slicing on a slicer;

(10) dyeing treatment:

dyeing the ultrathin slice obtained after the slicing in the step (9) by using a coloring agent, so that heavy metal salt is combined with some structures and components of cells, the scattering capacity of electrons is increased, the purpose of improving contrast is further achieved, and the displayed picture is clear and visible; and after dyeing is finished, placing the obtained sample on a transmission electron microscope, and carrying out sample loading observation to further inspect the sample preparation result.

In the above technical scheme, in the step (2), the fixing solution I is a solution obtained by mixing 3% by mass of Glutaraldehyde (Glutaraldehyde 25% EM grade), 0.3% by mass of Tritan X-100, and 22% by mass of sterilized seawater, and the volume ratio of the three is 12:3: 85.

In the technical scheme, in the step (3), the sterilized seawater is sterilized seawater with salinity of 22 per mill, and the rinsing solution is 0.1mol/L PBS solution; putting the sample into an equipartition instrument during each rinsing, adding 800 mu L of sterilized seawater or rinsing liquid into a centrifugal tube, and then performing shaking rinsing, wherein the time of each shaking rinsing is 15 minutes; when the apparatus is placed evenly for shaking rinsing, the rotating speed is 2400r/min, and the circulation is 30s multiplied by 3.

In the above technical scheme, in step (4), the fixing solution II is osmate solution (SPI-Chem) with a mass concentration of 1%^TM)。

In the technical scheme, in the step (5), the rinsing solution is 0.1mol/L PBS solution, the sample is placed in an equipartition instrument during each rinsing, 800 mu L of the rinsing solution is added into a centrifugal tube for shaking rinsing, and the time of each shaking rinsing is 15 minutes; when the apparatus is placed evenly for shaking rinsing, the rotating speed is 2400r/min, and the circulation is 30s multiplied by 3.

In the above technical solution, in the step (6), the dehydration treatment specifically includes the following steps:

sucking up rinsing liquid in the sample obtained after the secondary rinsing treatment in the step (5), adding 800 mu L of alcohol with the mass concentration of 30% into a centrifugal tube, treating the sample on a shaking instrument for 20 minutes at room temperature, and sucking up the sample; adding 800 microliter of alcohol with the mass concentration of 50% into the centrifuge tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry; continuously adding 800 mu L of alcohol with the mass concentration of 70% into the centrifugal tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry; finally, adding 800 mu L of 90% alcohol into the centrifugal tube, processing the mixture on a shaking instrument for 20 minutes at room temperature, and then sucking the mixture to dry the mixture, thereby completing the step-by-step dehydration treatment of the sample by the alcohol with different concentrations;

transferring the capsules subjected to the step-by-step dehydration treatment by using alcohol with different concentrations into a 1mL centrifuge tube, and continuously dehydrating by using acetone (no damage to samples during transfer): adding 400 mu L of acetone with the mass concentration of 90% into a centrifuge tube, processing the mixture on a shaking instrument for 10 minutes at room temperature, and then sucking the mixture to dry; then adding 400 mu L of anhydrous acetone into the centrifuge tube, processing the mixture on a shaking instrument for 10 minutes at room temperature, and then sucking the mixture to dry; the step of dehydrating the anhydrous acetone is repeated for three times, and the anhydrous acetone is absorbed for the last time until three drops of anhydrous acetone remain, so that the step-by-step dehydration treatment of the sample by the acetone with different concentrations is completed;

in the step, alcohol and acetone with different concentrations are prepared at present.

In the above technical solution, in the step (7), the step-by-step tissue infiltration treatment includes the following steps:

adding 1.5 drops of embedding agent into a sample obtained after dehydration treatment, wherein the mass ratio of the embedding agent to acetone in a system is 1: 2, covering a cover to perform infiltration treatment for 2 hours;

secondly, adding a drop of half embedding agent into the system in the first step to ensure that the mass ratio of the embedding agent to the acetone in the system is 1: 1, covering a cover to perform infiltration treatment for 2 hours;

carefully sucking out all the liquid added in the system in the step II, adding 500 mu L embedding agent, vacuumizing for 30 minutes and then carrying out polymerization treatment;

the embedding medium in the step I-III is a Spurr embedding medium.

In the above technical scheme, in the step (10), the staining agents are uranyl acetate (TED PELLA) and lead citrate (Merck KGaA), and the uranyl acetate staining and the lead citrate staining are performed according to a traditional two-color staining method.

In the technical scheme, the use purpose of the averaging instrument and the oscillator is the same, the averaging instrument is used for short-time quick oscillation, the oscillation of the oscillator is continuous and slow, and the oscillation is performed fully.

In the prior art, for the preparation of a protozoan electron microscope sample, the whole cell is generally used as a sample for preparation; although the capsule is a part of cells, the capsule has a special structure, namely a capsule wall, which hinders the penetration of chemicals such as stationary liquid, rinsing liquid or embedding medium and brings great difficulty to the preparation of samples; in order to overcome the technical problems, the invention improves the traditional method, and has the following technical effects compared with the prior art:

(1) the invention provides a set of relatively standard preparation method of protozoan cyst transmission electron microscope samples, which enables the cyst cell ultrastructure to be clearly and completely presented, and the method has certain stability and repeatability.

(2) The traditional fixing method is improved, the capsule is treated by using glutaraldehyde and Tritan X-100 under the condition of not damaging the capsule, the glutaraldehyde and the Tritan X-100 play a role in fixing, and the Tritan X-100 have the characteristic of increasing the permeability of the capsule wall and do not kill protozoa, so that the fixing liquid can be promoted to permeate the capsule wall, the sample is quickly fixed, and the difficulty of insufficient sample fixing in the past is solved.

(3) The invention utilizes plant cell embedding medium (Spurr embedding medium) to replace animal cell embedding medium (Epon812 epoxy resin embedding medium) in the traditional sample preparation method, so that the embedding medium can better and completely penetrate into the capsule.

(4) In the past sample preparation method, the processing time in each step is optimized and improved according to the characteristics of the capsule, and an equipartition instrument and a vibration instrument are used in some processing, so that each step can be fully processed, the effectiveness of a sample preparation result is improved, and a foundation is provided for clear presentation of a sample.

(5) In the sample preparation process, the invention summarizes the reasons of the prior scholars that the sample preparation fails, systematically improves the sample preparation method, combines the exploration of the individual preliminary sample preparation, summarizes some operation skills in the sample preparation, and leads the sample preparation of the special protozoan capsule to have the characteristics of stability and repeatability.

(6) The invention can be applied to the preparation of most protozoan cyst TEM samples, such as Cryptocaryon irritans, Torulopsis multifida, and cyst-derived weevils.

Drawings

FIG. 1 is a TEM image of Cryptocaryon irritans cysts obtained by conventional sampling method (3% glutaraldehyde fixation + Epon812 epoxy resin embedding medium), with a smaller magnification and a larger field of view;

FIG. 2 is a TEM image of Cryptocaryon irritans cysts obtained by conventional sampling method (3% glutaraldehyde fixation + Epon812 epoxy resin embedding medium), with larger magnification and smaller field of view;

FIG. 3 is a TEM image of Cryptocaryon irritans cysts obtained by conventional sampling (3% glutaraldehyde fixation + Epon812 epoxy resin embedding medium), with a larger magnification and a smaller field of view (different from the area taken in FIG. 2);

FIG. 4 is a TEM image (3% glutaraldehyde fixation + Spurr embedding medium) of the Cryptocaryon irritans cysts modified by conventional methods in example 1, with a smaller magnification and a larger field of view;

FIG. 5 is a TEM image (3% glutaraldehyde fixation + Spurr embedding medium) of the Cryptocaryon irritans cysts of example 1, with greater magnification and smaller field of view, obtained after a modification of the conventional method;

FIG. 6 is a TEM image (3% glutaraldehyde fixation + Spurr embedding medium) of the Cryptocaryon irritans cysts of example 1, obtained after a modification of the conventional method, with a larger magnification and a smaller field of view (different from the area of the image of FIG. 5);

FIG. 7 is a TEM image of the Cryptocaryon irritans encysted in example 2, obtained after a modification of the conventional method (3% glutaraldehyde + 0.3% Tritan X-100 mixed solution fixation + Spurr embedding medium), with a smaller magnification and a larger field of view;

FIG. 8 is a TEM image of the Cryptocaryon irritans encysted in example 2, obtained after a modification of the conventional method (3% glutaraldehyde + 0.3% Tritan X-100 mixed solution fixation + Spurr embedding medium), with a larger magnification and a smaller field of view;

FIG. 9 is a TEM image of the Cryptocaryon irritans encysted in example 2, obtained after modification of the conventional method (3% glutaraldehyde + 0.3% Tritan X-100 mixed solution fixation + Spurr embedding medium), with a larger magnification and a smaller field of view (different from the area of FIG. 8).

Detailed Description

The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to the following descriptions:

the invention will now be illustrated with reference to specific examples:

example 1:

after the traditional method is optimized, glutaraldehyde with the concentration of 3% which is prepared by diluting sterilized seawater is used as a stationary liquid, and a plant tissue embedding medium (Spurr embedding medium) is used for replacing an animal tissue cell embedding medium (Epon812 epoxy resin embedding medium) to prepare the cryptocaryon irritans cyst transmission electron microscope ultrathin section sample:

(1) selecting a single cryptocaryon volvatus cyst, wherein the diameter of the single cryptocaryon volvatus cyst is not suitable for being too large, but the cryptocaryon volvatus cyst grows well, and before fixation, observing whether the growth condition of the single cryptocaryon volvatus cyst is an expected effect under an optical microscope;

(2) front fixing: transferring the capsules selected in the step (1) to one corner of a glass slide, sucking off excessive culture solution, washing the capsules into a glass fixing cylinder filled with a small amount of mixed fixing solution by using prepared 3% Glutaraldehyde (Glutaraldehyde 25% EM grade), carefully shaking the samples in the fixing cylinder for 5min, then carefully transferring the samples into a 2mL centrifuge tube and adding 1mL of mixed fixing solution, standing at room temperature for 1 hour, and then transferring the samples to 4 ℃ for storage for 24 hours for next sample preparation. The whole treatment process keeps light-resistant and the centrifuge tube is laid flat, which is beneficial to the full contact of the fixing liquid and the sample.

(3) Pre-immersion washing: the capsules in (2) were rinsed with two solutions (22% saline sterile seawater and 0.1mol PBS solution), carefully sucking off the solution from the previous step (as dry as possible) before each rinse, then immediately adding 800 μ L of the next rinse for a total of 4 rinses (1 st with sterile seawater, 2-4 th with PBS solution), 15 minutes each rinse, and shaking with a homogenizer after each rinse addition (2400r/min, 30s × 3 cycles). The whole experiment was carried out at room temperature and the centrifuge tube was kept flat.

(4) Post-fixing: pipetting the rinse solution in step (3) to the last drop only, and immediately adding an equal volume of 1% osmic acid (SPI-Chem)^TM) And then transferred to a refrigerator at 4 ℃ to be fixed for 2.5 hours in a dark place.

(5) Post-immersion cleaning: the fixative in step (4) was blotted dry and immediately added with 800 μ L of leachant (0.1mol PBS solution) for a total of 3 leachates, 15 minutes each, and shaken with a homogenizer after each addition of leachant (2400r/min, 30s x 3 cycles). The entire experiment was performed at room temperature, and the centrifuge tube was kept flat.

(6) And (3) dehydrating: the capsules in step (5) were treated with two organic solutions (different concentrations of alcohol and acetone, now ready for use) to replace the free water therein. The method mainly comprises the following steps:

a. sucking the immersion cleaning liquid in the step (5) to dryness, adding 800 mu L of 30% alcohol, treating on a shaking instrument for 20 minutes at room temperature, and sucking to dryness;

b. adding 800 μ L of 50% ethanol, treating on a shaking instrument at room temperature for 20 min, and sucking to dry;

c. adding 800 μ L of 70% ethanol, treating on a shaking instrument at room temperature for 20 min, and sucking to dry;

d. adding 800 μ L of 90% ethanol, treating at room temperature for 20 min on a shaking apparatus, and sucking to one drop;

e. transferring the capsule into a 1mL centrifuge tube, and continuously dehydrating with acetone (the sample is not damaged when transferring);

f. adding 400 mu L of 90% acetone, treating the mixture on a shaking instrument for 10 minutes at room temperature, and then sucking the mixture to dry;

g. add 400. mu.L of anhydrous acetone, treat on a shaker for 10 minutes at room temperature and then suck dry, repeat the procedure three times, last suction until the last three drops of anhydrous acetone.

(7) Embedding: the encapsulation was gradually infiltrated with embedding medium (Spurr embedding medium). The method comprises the following specific steps:

a. adding 1.5 drops of embedding medium into a, and allowing the embedding medium/acetone to 1/2 for 2 hours;

b. adding a drop of semi-embedding medium into a, and allowing the embedding medium/acetone to 1/1 for 2 hours;

c. carefully aspirate all the fluid previously added in b, add 500. mu.L of embedding medium, evacuate 30 minutes before removing the sample for further processing.

(8) Polymerization: polymerization was carried out at 70 ℃ for 24 hours.

(9) Slicing: including trimming and slicing, the polymerized sample is removed from the centrifuge tube and ground to a length (about 1cm) suitable for slicing, followed by successive ultra-thin sectioning on a microtome.

(10) Dyeing: for double staining, uranium dioxyacetate (TED PELLA) and lead citrate (Merck KGaA) are utilized to combine heavy metal salt with certain structures and components of cells, so that the scattering capacity of electrons is increased, the purpose of improving contrast is further achieved, and the displayed picture is clear and visible.

(11) And (3) sample loading and observation: on a transmission electron microscope (Hitachi H-7650), sample preparation results are further examined by sample loading observation, and an electron microscope image is shown in fig. 4-6, and is a sample result obtained by using 3% glutaraldehyde as a fixing solution and treating the fixing solution by using a Spurr embedding agent, compared with the traditional method (fig. 1-3), observation under the electron microscope shows that the internal substructure of cells is clear and identifiable, the cell structure is complete, the integral transparency of the cells is improved, and the size and the shape are distinct; the phenomenon that cytoplasm is dissolved and a large number of cavities and the like do not appear from the internal structure of the cells in the graph; the observation result is still very stable after repeated operation, and the experimental result is reliable.

Example 2:

after the traditional method is optimized, fixing the mixed solution of 3 percent glutaraldehyde and 0.3 percent Tritan X-100 as a fixing solution, and replacing an animal tissue cell embedding agent (Epon812 epoxy resin embedding agent) with a plant tissue embedding agent (Spurr embedding agent) to prepare the cryptocaryon irritans cyst transmission electron microscope ultrathin section sample by the specific steps:

in this example, the treatment is substantially the same as that in example 1 except that different fixative solutions (3% glutaraldehyde and 0.3% Tritan X-100 mixed solution) are used, and the electron microscope images are shown in fig. 7-9, which is the result of the sample treated by using 3% glutaraldehyde + 0.3% Tritan X-100 mixed solution as the fixative solution and using the sprur embedding medium, compared with the conventional method (fig. 1-3), the observation under the electron microscope shows that the internal substructure of the cell is clear and distinguishable, the cell structure is complete, the overall transparency of the cell is improved, and the size and the shape are clear; the phenomenon that cytoplasm is dissolved and a large number of cavities and the like do not appear from the internal structure of the cells in the graph; the observation result is still very stable after repeated operation, and the experimental result is reliable.

The above examples are only for illustrating the technical concept and features of the present invention, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.