Method for preparing freshwater fish otolith sample

Abstract

The invention relates to the technical field of otolith sample preparation, in particular to a freshwater fish otolith sample preparation method which comprises the following steps: 1, sampling otolith; 2, otolith cleaning; 3, weighing the otolith; 4, fixing the otolith; 5, vacuumizing and hardening; 6, cutting the otolith; 7, fixing and cutting for the second time; 8, otolith polishing; 9, polishing the otolith; 10, otolith dyeing. According to the method, the freshwater fish otolith sample can be well prepared.

Description

Technical Field

[0001] The invention relates to the technical field of otolith sample preparation, in particular to a method for preparing otolith samples of freshwater fish. Background Art

[0002] Fish otoliths are hard structures deposited in the inner ear of true bony fish. They are divided into three pairs, including stellate otoliths, micro-otoliths, and sagittal otoliths. They are mainly composed of calcium carbonate, and also contain various trace chemical elements and organic matter. They play an important role in regulating balance and hearing.

[0003] Since otoliths grow with daily and annual rhythms and are inert and stable structures, the trace chemical elements deposited in otoliths have good stability, so they are powerful tools for fish age identification and habitat inversion. Currently, there are many research directions through the combination of otoliths and fish ecology, such as the determination of spawning and nursery grounds, migration routes, habitat utilization, etc.

[0004] In the process of using otoliths for the above research directions, researchers generally need to prepare specific otolith samples to obtain clear and flat otolith sun or annual ring sections for subsequent analysis. However, the otolith sample preparation process is relatively complicated. Different otoliths of freshwater fish have different characteristics, such as length, width and thickness, so the methods of preparing otolith samples are also different. Currently, the commonly used method is to embed otoliths with curing materials such as nail polish, hardened resin, hot melt adhesive, and then grind them. The whole process is relatively complicated, time-consuming, and the effect is not ideal. The above methods often have the following problems: (1) The method of using otolith slices for preparation has the problems of long curing time, difficult to grasp during grinding, inconvenient operation, and easy to cause wear and damage; (2) Curing materials such as hardened resin and hot melt adhesive are very easy to generate bubbles during the preparation process, which affects sample observation and measurement after solidification; (3) Most of the above methods use curing and direct grinding, which is time-consuming and labor-intensive, and prolongs the preparation time. (4) At the same time, the previous methods themselves are limited and cannot be widely used for the preparation of otoliths of most freshwater fish, and are not suitable for large-scale promotion. Summary of the invention

[0005] The present invention provides a method for preparing otolith samples of freshwater fish, which can solve the problems that the existing otolith sample preparation method is single, time-consuming, difficult to operate and has a small application range.

[0006] A method for preparing otolith samples of freshwater fish according to the present invention comprises the following steps:

[0007] 1. Otolith sampling;

[0008] 2. Otolith cleaning;

[0009] 3. Weighing of otoliths;

[0010] 4. Otolith fixation;

[0011] First, prepare the resin liquid. The curing agent and the resin are configured in a ratio of 10:3. The total amount of the prepared solution is the number of otoliths prepared multiplied by 2 grams. After the solution is prepared, use tweezers to pick up the dried otoliths and place them in a square silicone mold with the convex side facing down and the concave side facing up. Then, pour the prepared resin into each mold according to 2 grams of solution.

[0012] 5. Evacuate and harden;

[0013] Place the embedded otolith mold into a vacuum pump, and evacuate the solution for 1-2 minutes at an atmospheric pressure of -0.15 to 0.1 MPa to remove the bubbles in the solution; then take out the mold, carefully adjust the otolith to its original position with tweezers, place it on a constant temperature heating table and heat it for about 30 minutes to further remove bubbles and accelerate solidification, and then place it at room temperature for 2-4 hours to wait for hardening;

[0014] 6. Otolith cutting;

[0015] The hardened otolith is taken out from the square plastic mold, fixed on a low-speed high-density cutting machine, and the hardened resin is cut horizontally or vertically at the 2MM vertical edge near the tip of the otolith. After the cutting is completed, the sample is taken out and polished on a polishing machine. It is observed under a dark field microscope every 5-10 seconds until the core of the otolith is observed; then the polished otolith is fixed on the cutting machine again and cut along the unpolished two sides to obtain small slices;

[0016] 7. Secondary fixation and cutting;

[0017] Specifically:

[0018] A. Continue to prepare the solution according to step 4;

[0019] B. Place the obtained otolith slice vertically into a cylindrical silicone mold with the otolith at the top and the polished surface facing downward, so that the otolith slice remains in the center of the mold;

[0020] C. Print the serial number label according to the otolith number, and place it in the center of the bottom of the silicone mold with the reverse side facing up;

[0021] D. Perform vacuum extraction according to step 5 to remove bubbles in the solution, and place it to harden to obtain a cylindrical resin;

[0022] E. Take out the hardened otolith sample from the mold, fix it on the cutter again, and cut it at the 2mm vertical edge of the unpolished otolith end to obtain a cylindrical otolith resin sample with a height smaller than the original resin;

[0023] 8. Otolith polishing;

[0024] 9. Otolith polishing;

[0025] 10. Otolith staining.

[0026] Preferably, in step one, surgical scissors and tweezers are used to remove the otoliths in the fish's head, the surface of the fish's otoliths is preliminarily cleaned to remove blood stains, capsules, mucus and other dirt, and the otoliths are placed in an otolith tube for storage.

[0027] Preferably, in step 2, the otoliths are taken out of the tube, put into an ultrasonic cleaner for cleaning for 3-5 minutes, taken out with tweezers, the moisture on the surface of the otoliths is absorbed with absorbent paper, and placed at room temperature for drying to obtain dry and clean fish otoliths.

[0028] Preferably, in step three, the cleaned and dried otoliths are placed in left-right order on an electronic balance for weight measurement, and the data are recorded and saved for subsequent use in age regression.

[0029] Preferably, in step eight, the cut columnar otolith sample is first polished with 1000# sandpaper and observed under a dark field microscope until the edge and core of the otolith are clear but the core is not exposed, and then polished with 3000-5000# sandpaper until the core is completely exposed, the overall growth rings of the otolith are clear, and the core is obvious, and then the polishing is stopped; during the polishing, it is observed by an electron microscope every 3-5 seconds to obtain an otolith sample with an overall height of 1 cm and an otolith thickness of about 0.06 mm.

[0030] Preferably, in step nine, the surface of the cylindrical otolith sample is wiped clean, and then polished on a metallographic polishing machine using a diamond suspension polishing liquid until the upper end surface of the otolith sample presents a mirror reflection, thereby obtaining a fish otolith sample that can be preserved for a long time and placed in a sample box.

[0031] Preferably, in step ten, firstly, etching preparation is performed, and 1% hydrochloric acid or 3-5% EDTA is prepared as an etchant to remove impurities on the surface of the otoliths and prepare for dyeing; after the etching is completed, the otoliths are immediately taken out from the etchant and washed with sufficient water to remove residual chemical reagents; after washing, the otoliths are dried using a dryer, and wiping with paper towels or other materials is avoided to prevent contamination or damage; after the surface of the otoliths is dried, 1% toluidine blue is prepared as a dye to dye the otoliths so as to observe their internal structure; after dyeing is completed, the otoliths are taken out from the dye and rinsed with water to remove excess dye; finally, the dyed otoliths are dried using the dryer again, and wiping with paper towels or other materials is also avoided.

[0032] Preferably, if 1% hydrochloric acid etching is selected, the otoliths are immersed in hydrochloric acid for 1 minute, and in the subsequent staining process, the otoliths are immersed in toluidine blue for 30-120 minutes; if 3-5% EDTA is selected, the otoliths are immersed in EDTA for 3 minutes, and in the subsequent staining process, the otoliths are immersed in toluidine blue for 5-30 minutes.

[0033] The beneficial effects of the present invention are as follows:

[0034] (1) The present invention uses a fixed resin material and a heating device to shorten the hardening time and the curing time. In addition, the polishing is easy to grasp and operate, and it is not easy to cause wear and damage.

[0035] (2) The curing materials such as the hardened resin and hot melt adhesive of the present invention are not prone to generate bubbles during the preparation process, and the bubbles in the resin can be quickly removed by vacuum extraction. The vacuum extraction time is short, which is convenient for subsequent observation.

[0036] (3) The present invention uses cutting and secondary cutting methods to increase the preparation speed and reduce the workload; adding paper labels to the samples prevents sample confusion and ensures long-term preservation of the labels. The cylindrical samples formed by secondary fixation are easy to operate, and the alternate pasting method makes it difficult for the resin in the mold to overflow and be lost; the samples are not easily damaged and have a very long shelf life.

[0037] (4) The method provided by the present invention is widely applicable to the preparation of otoliths of most freshwater fish and is suitable for large-scale promotion.

[0038] (5) The present invention etches and dyes the otoliths after the original otolith samples are tested, which can clarify the internal structure of the otoliths and accurately distinguish the otolith growth rings, thereby providing further data reference for subsequent age identification and related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention is a flow chart of a method for preparing otolith samples of freshwater fish in an embodiment.

[0040] Figure 2 Schematic diagram of otolith fixation in the embodiment.

[0041] Figure 3 Schematic diagram of the cross section and longitudinal section of otolith in the embodiment.

[0042] Figure 4 This is a schematic diagram of a cross-section sample of the otolith of the full-lipped schizothorax in the embodiment.

[0043] Figure 5 It is a schematic diagram of a longitudinal section of a full-lipped split-bellied fish otolith sample in the embodiment. DETAILED DESCRIPTION

[0044] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the embodiments are only for explaining the present invention and are not intended to limit it.

[0045] Example

[0046] like Figure 1 As shown, this embodiment provides a method for preparing otolith samples of freshwater fish, which comprises the following steps:

[0047] 1. Otolith sampling;

[0048] Use surgical scissors and tweezers to remove the otoliths from the fish's head, perform preliminary cleaning on the surface of the fish's otoliths to remove blood stains, capsules, mucus and other dirt, and store them in an otolith tube.

[0049] 2. Otolith cleaning;

[0050] The otoliths were taken out of the tube, placed in an ultrasonic cleaner (model AK-009SD) for cleaning for 3-5 minutes, then taken out with tweezers, the moisture on the surface of the otoliths was absorbed with absorbent paper, and placed at room temperature to dry to obtain dry and clean fish otoliths.

[0051] 3. Weighing of otoliths;

[0052] The cleaned and dried otoliths were placed in left-right order on an electronic balance (model FA1004) for weight measurement, and the data was recorded and saved for subsequent age calculation.

[0053] 4. Otolith fixation;

[0054] First, prepare the resin liquid. The curing agent (model JCM4) and the resin (model JCM4) are configured in a ratio of 10:3. The total amount of the prepared solution is the number of otoliths to be prepared multiplied by 2 grams. After the solution is prepared, use tweezers to pick up the dried otoliths with the convex side facing down and the concave side facing up and place them in a square silicone mold (2.5*2.5CM [outside]; 2*2CM [inside]). Then, pour the prepared resin into each mold at a concentration of 2 grams (that is, completely embed the otoliths. The pouring solution volume can be adjusted according to the size of the otoliths to meet the preparation requirements of otoliths of different fish). Figure 2 shown.

[0055] 5. Evacuate and harden;

[0056] Place the embedded otolith mold in a vacuum pump (model TWH; RS-4), and evacuate the solution for 1-2 minutes at an atmospheric pressure of -0.15 to 0.1 MPa to remove the bubbles in the solution; then remove the mold, carefully adjust the otolith to its original position with tweezers, place it on a constant temperature heating table (model X1030T) for about 30 minutes to further remove bubbles and accelerate solidification, and then place it at room temperature for 2-4 hours to wait for hardening. This can shorten the solidification time of the prepared sample.

[0057] 6. Otolith cutting;

[0058] The hardened otoliths were taken out from the square plastic mold and fixed on a low-speed high-density cutting machine (model TechCut4). The hardened resin was cut horizontally or vertically at the 2MM vertical edge near the tip of the otolith (the horizontal cut [perpendicular to the long axis of the otolith] or the vertical cut [perpendicular to the short axis of the otolith] can be made according to the morphological characteristics of the otoliths and the experimental requirements). After the cutting is completed, the sample was removed and polished with 500-600# sandpaper on a metallographic polishing machine (model P-2T). The sample was observed under a dark field microscope (model OLYMPUS; BX51) every 5-10 seconds until the core of the otolith was observed (but not exposed). The polished otoliths were then fixed on the cutting machine again and cut along the unpolished sides (i.e., the polished opposite sides and any one side) to obtain small slices of approximately square size (1*1cm).

[0059] Schematic diagram of transverse and longitudinal sections of otoliths Figure 3 As shown. According to the different cutting methods of otoliths, the otolith area can be maximized to meet the experimental requirements. The cross-section of otoliths is generally prepared for the determination of microchemical elements, and the longitudinal section of otoliths can make the otolith annual rings clear to the greatest extent, which is often used for age identification. Figure 4 The following is a schematic diagram of a cross-section of a complete-lipped schizothorax otolith sample. Figure 5 Shown is a schematic diagram of a longitudinal section of a sample of the otolith of the complete-lipped schizothorax.

[0060] 7. Secondary fixation and cutting;

[0061] Specifically:

[0062] A. Continue to prepare the solution according to step 4 (Note: this time, multiply the number of otoliths by 5 grams);

[0063] B. Place the obtained otolith slice vertically into a cylindrical silicone mold (2.5*2.5 cm [outer side]; 2*2 cm [inner side]) with the otolith placed at the top and the polished surface facing downward, so that the otolith slice remains in the center of the mold;

[0064] C. Print a 1*0.5cm serial number label according to the otolith number, and place it in the center of the bottom of the silicone mold with the reverse side facing up;

[0065] D. Perform vacuum extraction according to step 5 to remove bubbles in the solution (Note: Use transparent tape to stick along the upper end of the mold before vacuum extraction to form a transparent cylinder on the upper end of the mold to prevent resin from overflowing when vacuum extraction is performed), and let it harden to obtain a cylindrical resin; the second vacuum extraction step is to wrap the mold with transparent tape to ensure the vacuum extraction speed and prevent the resin from overflowing and causing insufficient solution in the mold;

[0066] E. Take out the hardened otolith sample from the mold, fix it on the cutter again, and cut it at the 2MM vertical edge of the unpolished otolith end to obtain a cylindrical otolith resin sample that is smaller in height than the original resin.

[0067] The two cuts used in this method are both to increase the grinding speed of the otolith samples, and after the second cut, the otolith samples can be directly hand-held and ground without being fixed on a glass slide. Glass products are fragile and can easily cause harm to experimenters.

[0068] 8. Otolith polishing;

[0069] The cut columnar otolith samples were first polished with 1000# sandpaper and observed under a dark field microscope until the edge and core of the otolith were clear but the core was not exposed. Then they were polished with 3000-5000# sandpaper until the core was completely exposed, the overall growth rings of the otolith were clear, and the core was obvious. Then the polishing was stopped. During the polishing process, the samples were observed under an electron microscope every 3-5 seconds. The overall height of the otolith samples was 1 cm and the thickness of the otoliths was about 0.06 mm.

[0070] 9. Otolith polishing;

[0071] Wipe the surface of the cylindrical otolith sample clean, then use diamond suspension polishing liquid to polish it on a metallographic polishing machine (model PG-1A) until the upper end of the otolith sample shows a mirror reflection. You can get a fish otolith sample that can be preserved for a long time and put it into a sample box (length: 2.5CM; width: 2CM; height: 2CM).

[0072] 10. Otolith staining;

[0073] First, prepare for etching and prepare 1% hydrochloric acid or 3-5% EDTA as an etchant to remove impurities on the surface of the otoliths in preparation for dyeing; after etching is completed, immediately remove the otoliths from the etchant and wash them with enough water to remove residual chemical reagents; after washing, use a dryer to dry the otoliths, and avoid wiping them with paper towels or other materials to prevent contamination or damage; after the surface of the otoliths is dry, prepare 1% toluidine blue as a dye to dye the otoliths to facilitate observation of their internal structure; after dyeing is completed, remove the otoliths from the dye and rinse them with water to remove excess dye; finally, use a dryer again to dry the dyed otoliths, and also avoid wiping them with paper towels or other materials.

[0074] If 1% hydrochloric acid etching is selected, immerse the otoliths in hydrochloric acid for 1 minute, and in the subsequent staining process, immerse the otoliths in toluidine blue for 30-120 minutes; if 3-5% EDTA is selected, immerse the otoliths in EDTA for 3 minutes, and in the subsequent staining process, immerse the otoliths in toluidine blue for 5-30 minutes.

[0075] Through this series of steps, the otoliths will be etched and dyed to make their internal structure more clearly visible, facilitating subsequent observation and analysis.

[0076] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing otolith samples of freshwater fish, characterized in that: The following steps are involved:

1. Otolith sampling; 2. Otolith cleaning; 3. Weighing of otoliths; 4. Otolith fixation; First, prepare the resin liquid. The curing agent and the resin are configured in a ratio of 10:

3. The total amount of the prepared solution is the number of otoliths prepared multiplied by 2 grams. After the solution is prepared, use tweezers to pick up the dried otoliths and place them in a square silicone mold with the convex side facing down and the concave side facing up. Then, pour the prepared resin into each mold according to 2 grams of solution.

5. Evacuate and harden; Place the embedded otolith mold into a vacuum pump, and evacuate the solution for 1-2 minutes at an atmospheric pressure of -0.15 to 0.1 MPa to remove the bubbles in the solution; then take out the mold, carefully adjust the otolith to its original position with tweezers, place it on a constant temperature heating table and heat it for about 30 minutes to further remove bubbles and accelerate solidification, and then place it at room temperature for 2-4 hours to wait for hardening; 6. Otolith cutting; The hardened otolith is taken out from the square plastic mold, fixed on a low-speed high-density cutting machine, and the hardened resin is cut horizontally or vertically at the 2MM vertical edge near the tip of the otolith. After the cutting is completed, the sample is taken out and polished on a polishing machine. It is observed under a dark field microscope every 5-10 seconds until the core of the otolith is observed; then the polished otolith is fixed on the cutting machine again and cut along the unpolished two sides to obtain small slices; 7. Secondary fixation and cutting; Specifically: A. Continue to prepare the solution according to step 4; B. Place the obtained otolith slice vertically into a cylindrical silicone mold with the otolith at the top and the polished surface facing downward, so that the otolith slice remains in the center of the mold; C. Print the serial number label according to the otolith number, and place it in the center of the bottom of the silicone mold with the reverse side facing up; D. Perform vacuum extraction according to step 5 to remove bubbles in the solution, and place it to harden to obtain a cylindrical resin; E. Take out the hardened otolith sample from the mold, fix it on the cutter again, and cut it at the 2mm vertical edge of the unpolished otolith end to obtain a cylindrical otolith resin sample with a height smaller than the original resin; 8. Otolith polishing; 9. Otolith polishing; 10. Otolith staining.

2. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step one, surgical scissors and tweezers are used to remove the otoliths from the fish's head, and the surface of the fish's otoliths is preliminarily cleaned to remove blood stains, capsules, mucus and other dirt, and then placed in an otolith tube for storage.

3. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step 2, the otolith is taken out from the tube, put into an ultrasonic cleaning machine for cleaning for 3-5 minutes, then taken out with tweezers, the moisture on the surface of the otolith is absorbed with absorbent paper, and placed at room temperature to dry to obtain dry and clean fish otoliths.

4. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step three, the cleaned and dried otoliths are placed in left-right order on an electronic balance for weight measurement, and the data is recorded and saved for subsequent age calculation.

5. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step eight, the cut columnar otolith sample is first polished with 1000# sandpaper and observed under a dark field microscope until the edge and core of the otolith are clear but the core is not exposed, and then polished with 3000-5000# sandpaper until the core is completely exposed, the overall growth rings of the otolith are clear, and the core is obvious, then the polishing is stopped; during the polishing process, it is observed through an electron microscope every 3-5 seconds, and the otolith sample is 1 cm high and 0.06 mm thick.

6. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step nine, the surface of the cylindrical otolith sample is wiped clean, and then polished on a metallographic polishing machine using a diamond suspension polishing liquid until the upper end surface of the otolith sample presents a mirror reflection, thereby obtaining a fish otolith sample that can be preserved for a long time and placed in a sample box.

7. The method for preparing otolith samples of freshwater fish according to claim 1, characterized in that: In step 10, firstly, etching preparation is performed, and 1% hydrochloric acid or 3-5% EDTA is prepared as an etchant to remove impurities on the surface of the otoliths in preparation for dyeing; After the etching is completed, the otoliths are immediately removed from the etchant and washed with sufficient water to remove the residual chemical reagents; after washing, the otoliths are dried in a dryer, and paper towels or other materials are avoided to prevent contamination or damage; after the surface of the otoliths is dry, 1% toluidine blue is prepared as a dye to dye the otoliths for the purpose of observing their internal structure; After dyeing is completed, remove the otoliths from the dye and rinse them with water to remove excess dye; finally, use the dryer to dry the dyed otoliths again, and avoid wiping them with paper towels or other materials.

8. The method for preparing otolith samples of freshwater fish according to claim 7, characterized in that: If 1% hydrochloric acid etching is selected, immerse the otoliths in hydrochloric acid for 1 minute, and in the subsequent staining process, immerse the otoliths in toluidine blue for 30-120 minutes; if 3-5% EDTA is selected, immerse the otoliths in EDTA for 3 minutes, and in the subsequent staining process, immerse the otoliths in toluidine blue for 5-30 minutes.

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