Method for directly dyeing and fixing cystorotifer sample by eosin Y

Abstract

The invention discloses a method for directly dyeing and fixing a cystorotifer sample by eosin Y. The method specifically comprises the following steps: collecting a fresh cystorotifer sample by using a filter screen with the mesh aperture of 0.0750 mm (200 meshes); the method comprises the following steps: collecting a fresh sample of the cystorotifer, putting the fresh sample into a beaker, removing impurities by using a suction tube, controlling the collected sample to be about 1mL, maintaining the number of the cystorotifer to be 40-80, then directly adding 3-5mL of 0.2-1.0% eosin Y alcohol-soluble solution into the sample, dyeing the cystorotifer for 20-30 minutes, and fixing the cystorotifer at the same time. According to the invention, not only is the cystorotifer sample perfectly fixed, but also the cystorotifer is stained with purple or pink, so that various tissues and organs of the cystorotifer can be conveniently observed. Under the observation of a microscope, the tissue structures of the cyst rotifer are clearer and are convenient to observe.

Description

Technical Field

[0001] The invention belongs to the field of rotifer cultivation in aquaculture, and in particular relates to a method for directly staining and fixing a crystal capsule rotifer sample with eosin Y. Background Art

[0002] Rotifers are an important group of aquatic plankton. They are small, generally 100-1000µm, with a short life cycle and generation time, and a fast reproduction rate. In production, rotifers in pond water are often used as the starter bait for fry, or as bait for early development of fish such as fry and juveniles, and are also bait for many shrimps and crabs. If there are more rotifers in the water, the bait for small fish, shrimps and crabs will be abundant, and the growth rate will be faster.

[0003] In the process of studying rotifers, especially crystal cyst rotifers, it is often necessary to fix the crystal cyst rotifer samples, observe and analyze them. However, using the existing methods of fixing crystal cyst rotifers, such as formalin, Bonn's solution, pure ethanol and other commonly used fixatives for fixation, crystal cyst rotifers often have distorted appearance, curled body, deformed tissues and organs, blurred structure and other phenomena, which brings many inconveniences to the biological research of crystal cyst rotifer morphology and structure. In the process of our research, we changed the original practice of fixing with formalin, Bonn's solution, pure ethanol and other fixatives, and then staining or microscopic observation. We directly added eosin solution to the fresh crystal cyst rotifer samples for staining, and then dehydrated and preserved them with ethanol. This can fix and preserve the crystal cyst rotifer samples well. At the same time, the external morphology of the crystal cyst rotifer is not distorted, and the microscopic structure of the tissues and organs will not be deformed, and it is clear. Summary of the invention

[0004] The main purpose of the present invention is to propose a method for directly staining and fixing a crystal cyst rotifer sample with eosin Y, aiming to eliminate the phenomenon that the crystal cyst rotifer appears distorted in appearance, curled in worm body, deformed in tissues and organs, blurred in structure, etc. after being fixed with commonly used fixatives such as formalin, Bonn's solution, and pure ethanol, which brings many inconveniences to the research on biological aspects such as the morphological structure of rotifers.

[0005] To achieve the above object, the present invention provides a method for directly staining and fixing a crystal rotifer sample with eosin Y, comprising the following steps.

[0006] S1. Use a filter with a mesh size of 0.0750 mm (200 mesh) to collect fresh crystal rotifer samples; after collecting the fresh crystal rotifer samples, put them into a beaker, let them stand for 20-30 minutes, and then use a pipette to carefully and slowly suck out the upper clear liquid, and control the biomass to 40-80 crystal rotifers in 1 mL of water.

[0007] S2, staining, directly staining the crystal cyst rotifer with eosin Y ethanol solution, and also fixing the crystal cyst rotifer; Furthermore, the eosin Y solution has a mass concentration of 0.2-1.0% ethanol solution and contains glacial acetic acid with a mass concentration of 0.05-0.20%; Furthermore, the dissolution method of the eosin Y solution is to weigh 2-10g of eosin Y, crush it into powder, dissolve it with 95% ethanol, transfer it to a 1000mL volumetric flask, then add glacial acetic acid to control its mass concentration to 0.05-0.20%, and dilute it to 1000mL with 95% ethanol solution, shake well, and store it at room temperature away from light for later use.

[0008] S3, aeration water washing, add 10-20mL aeration water to the eosin solution of crystal cyst rotifer, let it stand for 10-15min, carefully remove the supernatant with a pipette, and wash the dye solution with aeration water 3-5 times; Then use different concentrations of ethanol solution for multiple dehydration: In the first step, the sample was washed with aerated water for 3-5 times, and then 20-50 mL of 25% ethanol solution was added for dehydration. Each time, it was allowed to stand for 20-30 min. The upper clear liquid was carefully removed with a pipette, and the lower sample liquid was retained for further processing; Furthermore, add 20-50 mL of 50% ethanol solution to the lower sample solution, let it stand for 20-30 min, carefully remove the upper clear liquid with a pipette, and keep the lower sample solution for further processing; Further, add 20-50 mL of 75% ethanol solution to the above lower layer sample solution, let it stand for 20-30 min, carefully remove the upper layer clear liquid with a pipette, and keep the lower layer sample solution for further processing; Furthermore, 20-50 mL of anhydrous ethanol was added to the lower layer sample solution, and the solution was allowed to stand for 20-30 min. The upper layer clear liquid was carefully removed with a pipette, and the lower layer sample solution was retained for further processing.

[0009] S4. Take the lower layer sample prepared above, add anhydrous ethanol, and observe it under a microscope. It can be seen that the shape of the crystal cyst rotifer is not distorted or wrinkled, and the structure of tissues and organs is clear. Store it in anhydrous ethanol and make sure it is sealed.

[0010] Through the above careful operation, under the microscope, the crystal cyst rotifer is purple or pink, with a good outline, no shrinkage, no distortion, and the structure of the tissues and organs is intact and clear. The crystal cyst rotifer samples fixed by this method can be stored in anhydrous ethanol solution for a long time and can be observed and analyzed at any time.

[0011] Beneficial effects: In the technical solution provided by the present invention, eosin Y solution is directly added to the fresh crystal cyst rotifer sample for dyeing, and then dehydrated and preserved with ethanol. This can well fix and preserve the crystal cyst rotifer sample. At the same time, the external contour morphological structure of the crystal cyst rotifer will not be deformed or distorted, and the sample with intact tissue and organ structure can be preserved and can be observed and analyzed at any time. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. It is obvious that the drawings described here are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The crystal rotifer directly stained and fixed by the eosin group of the present invention (4×10 times).

[0014] Figure 2 The crystal cyst rotifer (4×10 times) fixed in the formalin control group of the present invention.

[0015] Figure 3 The crystal rotifer directly stained and fixed by the eosin group of the present invention (4×10 times).

[0016] Figure 4 The crystal rotifer fixed in the Bonn control group of the present invention (4×10 times).

[0017] Figure 5 The crystal rotifer directly stained and fixed by the eosin group of the present invention (4×10 times).

[0018] Figure 6 The crystal rotifer (4×10 times) after fixation in the anhydrous ethanol control group of the present invention. DETAILED DESCRIPTION

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work. The chemical reagents used in the embodiments are analytically pure or ultra-pure.

[0020] The present invention is further described below in conjunction with specific embodiments. Example 1

[0021] S1. Use a filter with a mesh size of 0.0750 mm (200 mesh) to collect a fresh sample of crystal rotifer, put it into a beaker, let it stand for 20 minutes, then discard part of the supernatant and control the biomass to 40 crystal rotifers / mL of the sample.

[0022] S2. The above 40 crystal cyst rotifers / mL samples were divided into two treatment groups. One group was directly added with 3 mL of 0.2% eosin Y ethanol solution to the sample to stain the crystal cyst rotifers and fixed for 20 minutes, which was recorded as the eosin group; the other group was directly added with 3 mL of 0.2% formalin solution to the sample to fix the crystal cyst rotifers for 20 minutes, which was recorded as the formalin control group.

[0023] S3, respectively add 5 mL of water to the above fixed rotifer solution for aeration and washing, let it stand for 10 min, carefully remove the supernatant with a pipette, wash the fixed solution with aeration water 3 times, then add 20 mL of 25% ethanol solution for dehydration, let it stand for dehydration for 10 min, and carefully remove the supernatant with a pipette; Further, add 20 mL of 50% ethanol solution, let it stand for dehydration for 10 min, and carefully remove the supernatant with a pipette; Furthermore, add 20 mL of 75% ethanol, let it stand for dehydration for 10 min, and carefully remove the supernatant with a pipette; Furthermore, 20 mL of anhydrous ethanol was added, and the mixture was allowed to stand for dehydration for 10 min, and the supernatant was carefully removed with a pipette.

[0024] S4. Add anhydrous ethanol to the above dehydrated samples and observe them under a microscope.

[0025] The samples of the eosin group were observed under a 40x microscope. Figure 1 As shown in the figure, after fixation with eosin Y staining, the tissue and organ structure of the crystal cyst rotifer sample was intact, the structure was clearly visible, and it would not be deformed or twisted, and it could be observed and analyzed at any time. Figure 2 As shown, it was found that although the tissue and organ structures of the fixed crystal rotifer samples were intact, the structure was unclear and there were deformation and distortion.

[0026] Example 2 S1. Use a filter with a mesh size of 0.0750 mm (200 mesh) to collect a fresh sample of crystal rotifer, put it into a beaker, let it stand for 25 minutes, discard part of the supernatant, control the biomass to 60 crystal rotifers / mL water, and divide it into two treatment groups.

[0027] S2. In the two sample groups treated above, 4 mL of 0.6% eosin Y ethanol solution was added to one group of samples to stain and fix the crystal cyst rotifer for 25 minutes, which was recorded as the eosin group; 30 mL of 0.6% Bonn's solution was added to the other group of samples to fix the crystal cyst rotifer for 8 minutes, which was recorded as the Bonn's solution control group.

[0028] S3, respectively add 15mL of aerated water to the above fixed rotifer solution for washing, let it stand for 15min, carefully remove the supernatant with a pipette, wash the dye solution with aerated water 4 times, then add 40mL of 25% ethanol for dehydration, let it stand for dehydration for 25min, and carefully remove the supernatant with a pipette; Furthermore, 40 mL of 50% ethanol was added and the mixture was allowed to stand for dehydration for 25 min, and the supernatant was carefully removed with a pipette; Further, add 40 mL of 75% ethanol, let stand for dehydration for 25 min, and carefully remove the supernatant with a pipette; Furthermore, 40 mL of anhydrous ethanol was added and the mixture was allowed to stand for dehydration for 25 min, and the supernatant was carefully removed with a pipette.

[0029] S4. Finally, add anhydrous ethanol to the above lower layer sample and observe it under a microscope.

[0030] The structure was observed by 40 times microscope. Figure 3 As shown, it was found that after fixation with eosin staining, the tissue and organ structure of the crystal cyst rotifer sample was intact, the structure was clearly visible, without deformation or distortion, and could be observed and analyzed at any time. Figure 4 This is the control group of Bonn. When observed under a 40x microscope, the structure of the fixed crystal rotifer sample was blurred and appeared deformed and distorted.

[0031] Example 3 S1. Collect fresh samples of crystal cyst rotifers, put them into a beaker, let them stand for 30 minutes, then discard part of the supernatant. The biomass was controlled to 80 crystal cyst rotifers / mL water and divided into two treatment groups.

[0032] S2. 5 mL of 1.0% eosin Y ethanol solution was added to one group of direct samples to stain and fix the crystal cyst rotifer for 30 minutes, which was recorded as the eosin group; 5 mL of 1.0% anhydrous ethanol was added to another group of direct samples to fix the crystal cyst rotifer for 30 minutes, which was recorded as the anhydrous ethanol control group.

[0033] S3, add 20mL of aerated water to the fixed rotifer solution for washing, let it stand for 5 minutes, carefully remove the supernatant with a pipette, wash the dye solution with aerated water for 5 times, then add 50mL of 25% ethanol for dehydration, let it stand for dehydration for 20 minutes, and carefully remove the supernatant with a pipette; Further, add 50 mL of 50% ethanol, let it stand for dehydration for 20 min, and carefully remove the supernatant with a pipette; Furthermore, add 50 mL of 75% ethanol and let it stand for dehydration for 20 min, and carefully remove the supernatant with a pipette; Furthermore, 50 mL of anhydrous ethanol was added, and the mixture was allowed to stand for dehydration for 20 min, and the supernatant was carefully removed with a pipette.

[0034] S4. Finally, add anhydrous ethanol and observe under a microscope. Observe the structure of the crystal cyst rotifer with a 40x microscope.

[0035] The structure of eosin group Figure 5 As shown, from Figure 5 It can be observed that after fixation with eosin Y staining, the tissue and organ structure of the crystal cyst rotifer sample is intact, the structure is clearly visible, and it will not be deformed or twisted. It can be observed and analyzed at any time and has a long storage time. In the anhydrous ethanol control group, the crystal cyst rotifer sample structure is fixed with anhydrous ethanol and observed under a microscope. The crystal cyst rotifer appears severely blurred, deformed, and twisted. Figure 6 shown.

[0036] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for directly staining and fixing a crystal rotifer sample with eosin Y, characterized in that: Fresh crystal cyst rotifer samples were collected for sampling and processing, the processed biological sample liquid was aspirated into a beaker, eosin Y ethanol solution was added to stain the crystal cyst rotifer, and then the samples were washed with aerated water and dehydrated with ethanol solutions of different concentrations, and the fixed crystal cyst rotifers were observed under a microscope.

2. A method for directly staining and fixing a crystal rotifer sample with eosin Y as claimed in claim 1, characterized in that , Use a filter with a mesh size of 0.0750mm (200 mesh) to collect fresh crystal rotifer samples and put them into a beaker. After standing for 20-30 minutes, use a pipette to carefully and slowly suck out the upper clear liquid, so that the collected sample is controlled at about 1mL, and the number of crystal rotifers is maintained at 40-80.

3. A method for directly staining and fixing a crystal rotifer sample with eosin Y as claimed in claim 1, characterized in that: In the fresh crystal cyst rotifer sample, soak it with eosin Y ethanol solution for 20-30 minutes, then add 5-20mL of aerated water for washing, let it stand for 10-20 minutes, and use a pipette to carefully and slowly suck off the upper aqueous solution, leaving the lower purple crystal cyst rotifer sample solution. Repeat this operation 3-5 times.

4. A method for directly staining and fixing a crystal rotifer sample with eosin Y as claimed in claim 1, characterized in that: After washing the samples with aerated water for 3 to 5 times, they were then cleaned with ethanol solutions of different concentrations: Add 20-50 mL of 25% ethanol solution, let it stand for 10-20 minutes, carefully and slowly remove the upper clear liquid with a pipette, and keep the purple crystal rotifer sample liquid in the lower layer; Further, add 20-50 mL of 50% ethanol, let it stand for 10-20 min, and carefully remove the upper clear liquid with a pipette; retain the purple crystal rotifer sample liquid in the lower layer; Further, add 20-50 mL of 75% ethanol, let stand for 20-30 min, and carefully remove the supernatant with a pipette; Further, add 20-50 mL of anhydrous ethanol, let stand for 20-30 min, and carefully remove the supernatant with a pipette; Furthermore, anhydrous ethanol was added, and the sample was transferred into a glass bottle with a lid and sealed for storage.

5. A method for directly staining and fixing a crystal rotifer sample with eosin Y as described in claim 1, characterized in that: The mass concentration of the ethanol-soluble solution of Eosin Y is 0.2-1.0%. The dissolution method is to weigh 2-10g of Eosin Y in a mortar, grind it into powder, transfer it to a 100mL beaker, dissolve it in 95% ethanol and make it up to 1000mL, add 0.5-2.0mL of glacial acetic acid, shake it well and store it at room temperature for later use.

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