Manufacturing method of transparent fish specimen and transparent fish specimen

Abstract

The invention discloses a manufacturing method of a transparent fish specimen and the transparent fish specimen, and belongs to the field of specimen manufacturing. The method comprises the following steps: dehydrating, fixing, degreasing and pre-transparentizing a fish body; soaking the fish body in an alcian blue coloring agent for 1-2 days until the cartilage of the fish turns blue; soaking the fish body in a decolorizing agent for 2-24 hours; soaking the fish body in an alizarin red coloring agent for 2-3 days until the hard bone of the fish becomes red; soaking the fish body in an alcian blue coloring agent for 1-2 hours until the outline of the fish body is light green; and performing transparency treatment on the fish body to obtain the transparent fish specimen. The transparent fish specimen prepared by the method can be comprehensively presented from inside to outside, is simple and convenient to manufacture, short in period, low in cost, beneficial to long-term storage, convenient to display and gorgeous and attractive in color, and can be widely applied to the fields of scientific research and teaching, species identification, science popularization, biological safety and the like.

Description

Technical Field

[0001] This application belongs to the field of specimen production, and specifically relates to a method for producing a transparent fish specimen and a transparent fish specimen. Background Art

[0002] The production of fish specimens is a process of preserving the original state of tissues such as the internal organs, bones, scales, and fur of fish through various production methods. According to the different production materials and methods, it is mainly divided into types such as immersion, skinning and shaping, drying, bone making, casting, and slide making. Some of the above types of specimens only show the outline of the fish, some can only see the internal characteristics of the fish, and some can only observe local details, which have certain limitations.

[0003] In order to more completely display the characteristics of fish, transparent fish specimens are usually used. The production of transparent fish specimens generally makes the fish meat transparent through chemical methods and stains the fish bones. However, when the fish is relatively large in size (for example, the body length is greater than 10 cm), it is difficult to stain the spine while preserving the fish meat. The existing transparentization methods will also cause fish with more cartilage to show the outline, while fish with more hard bones cannot be seen clearly. In the prior art, there is a solution of using trypsin solution to digest part of the fish meat, but this solution will result in incomplete preservation of the fish meat. In addition, there are differences between fish meat and internal organ tissues, and it is difficult to obtain a transparent fish specimen that simultaneously preserves the complete fish meat and internal organs using the existing methods. Summary of the Invention

[0004] The purpose of this application is to provide a method for producing a transparent fish specimen and a transparent fish specimen, which can at least solve one problem in the prior art, so as to obtain a transparent fish specimen that can preserve the complete fish meat and internal organs and present the characteristics of fish comprehensively from the inside to the outside.

[0005] In order to solve the above technical problems, this application is implemented as follows: In the first aspect, an embodiment of this application provides a method for producing a transparent fish specimen, which includes: Performing dehydration fixation, degreasing, and pre-transparency treatment on the fish body; Soaking the fish body in Alcian blue stain for 1 - 2 days until the fish cartilage turns blue; Soaking the fish body in a decolorizing agent for 2 - 24 hours; Soaking the fish body in alizarin red stain for 2 - 3 days until the fish hard bones turn red; Soaking the fish body in Alcian blue stain for 1 - 2 hours until the outline of the fish body shows light green; Performing transparency treatment on the fish body to obtain a transparent fish specimen.

[0006] Further, the steps of dehydrating, fixing, defatting, and pre-clearing the fish body specifically include: soaking the fish body in absolute ethanol for 7 to 14 days for dehydration and fixation, then putting it into acetone for 2 to 4 days for defatting; soaking the fish body in a pre-clearing solution for 1 to 3 days until the fish muscle becomes semi-transparent; the pre-clearing solution is a potassium hydroxide solution with a mass fraction of 1% to 5%; the semi-transparent state means that the bone morphology inside the fish can be clearly observed.

[0007] Further, after the step of soaking the fish body in Alcian blue stain for 1 to 2 days until the fish cartilage turns blue, the following steps are also included: rinsing the excess dye on the surface of the fish body with pure water or distilled water, and continuously soaking the fish body, changing the water once an hour until the water no longer changes color.

[0008] Further, the step of soaking the fish body in a decolorizing agent for 2 to 24 hours specifically includes: soaking the fish body in the decolorizing agent for 2 to 24 hours, and puncturing or extracting larger bubbles during the soaking process until bubbles are generated in the fish spine, and then the fish body is soaked in pure water or distilled water for 1 day.

[0009] Further, the decolorizing agent is a mixture of a hydrogen peroxide solution with a mass fraction of 3% to 5% and glycerol in a volume ratio of 2 to 4:1.

[0010] Further, the steps of clearing the fish body to obtain a transparent fish specimen specifically include: soaking the fish body in a first-gradient clearing solution, a second-gradient clearing solution, and a third-gradient clearing solution for 2 to 3 days each to obtain a transparent fish specimen; Among them, the first-gradient clearing solution, the second-gradient clearing solution, and the third-gradient clearing solution are all mixtures of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2%, and the content of glycerol in the first-gradient clearing solution, the second-gradient clearing solution, and the third-gradient clearing solution increases in turn.

[0011] Further, the first-gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed in a volume ratio of 0.5 to 1.5:3, the second-gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed in a volume ratio of 0.5 to 1.5:1, and the third-gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed in a volume ratio of 2.5 to 3.5:1.

[0012] Further, the preparation method of the Alcian blue stain is: dissolving 90 mg to 110 mg of Alcian blue in 700 ml of absolute ethanol, adding 300 ml of glacial acetic acid, and mixing well.

[0013] Further, the preparation method of the alizarin red staining agent is as follows: Dissolve 600 mg - 700 mg of alizarin red powder in 100 ml of absolute ethanol until saturated, add 900 ml of a 1% potassium hydroxide solution by mass, and mix well.

[0014] In a second aspect, the present application provides a transparent fish specimen, which is prepared according to the above - mentioned method for making a transparent fish specimen.

[0015] The present application has at least the following beneficial effects: (1) By using physical and chemical methods for pretreatment, the hard bones, soft bones, gill arches, muscles, and soft tissues of fish are colored respectively. It can not only retain the fish meat, but also retain soft tissues such as eyeballs, gill arches, internal organs, swim bladders, and even ossified ctenoid scales, enabling the fish specimen to be presented comprehensively from the inside to the outside. (2) It can be used to make transparent specimens of small - sized fish (body length less than 15 cm), which only takes 15 - 20 days; it can also be used to make transparent specimens of medium - sized fish (body length of 15 - 30 cm), which only takes 30 - 35 days; and it can even be used for large - sized fish.

[0016] (3) The production is simple, the cycle is short, and the cost is low. The specimen is conducive to long - term preservation, convenient for display, and has gorgeous colors. It can be widely applied in many fields such as scientific research and teaching, species identification, science popularization, and biological safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a photo of the transparent specimen of Hypomesus olidus in Example 1 of the present application.

[0018] Figure 2 It is a photo of the transparent specimen of Scatophagus argus in Example 2 of the present application.

[0019] Figure 3 It is a photo of the transparent specimen of Terapon jarbua in Example 3 of the present application.

[0020] Figure 4 It is a photo of the transparent specimen of Monacanthus chinensis in Example 3 of the present application.

[0021] Figure 5 It is a photo of the Scatophagus argus specimen in Comparative Example 1.

[0022] Figure 6 It is a photo of the Siganus fuscescens specimen in Comparative Example 2.

[0023] Figure 7 It is a photo of the Siganus fuscescens specimen in Comparative Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] Next, in combination with the accompanying drawings in the embodiments of the present application, the technical solutions and the resulting technical effects in the embodiments of the present application will be clearly and completely described to fully elaborate the purpose, solution, and effect of the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0025] In an embodiment of the present application, a method for making a transparent fish specimen is provided, and the method includes: Dehydrating, fixing, degreasing, and pre-transparenting the fish body; Soaking the fish body in Alcian blue stain for 1 - 2 days until the fish cartilage turns blue; Soaking the fish body in a decolorizing agent for 2 - 24 hours; Soaking the fish body in alizarin red stain for 2 - 3 days until the fish hard bones turn red; Soaking the fish body in Alcian blue stain for 1 - 2 hours until the outline of the fish body shows light green; Performing a transparent treatment on the fish body to obtain a transparent fish specimen.

[0026] In some embodiments of the present application, the step of dehydrating, fixing, degreasing, and pre-transparenting the fish body specifically includes: soaking the fish body in absolute ethanol for 7 - 14 days for dehydration and fixation, and then putting it into acetone for 2 - 4 days for degreasing; soaking the fish body in a pre-transparent solution for 1 - 3 days until the fish muscle becomes semi-transparent; the pre-transparent solution is a potassium hydroxide solution with a mass fraction of 1% - 5%; the semi-transparent state means that the internal bone morphology of the fish can be clearly observed.

[0027] In this embodiment, the pre-transparent solution is a potassium hydroxide solution with a mass fraction of 1% - 5%. Soaking the fish body in the pre-transparent solution until the fish meat becomes semi-transparent is a key step. Pre-transparency can cause protein denaturation, destroy the muscle fiber structure, and at the same time is conducive to observing the immediate effects presented by the fish body at any time during the subsequent blue staining, red staining, and decoloring steps, accurately controlling the processing time. Pre-transparency before staining can observe the staining degree of internal tissues such as the internal organs and gills of the fish, which is conducive to retaining internal features such as internal organs.

[0028] Among them, semi-transparent means that the internal bone morphology of the fish can be clearly observed. Among them, the concentration of the potassium hydroxide solution has an obvious impact on the final effect. Too high a concentration will quickly corrode parts such as the fish fins, causing them to fall off; too low a concentration will make it difficult for the later staining solution to fully penetrate into the fish vertebrae, resulting in staining failure.

[0029] In some embodiments of the present application, the fish body is soaked in Alcian blue stain for 1 to 2 days until the fish cartilage turns blue. The excess dye on the surface of the fish body is rinsed with pure water or distilled water, and the fish body is continuously soaked, with the water changed every hour until the water no longer changes color. Alcian blue is a type of copper phthalocyanine cationic conjugated dye, which easily combines with acidic groups to form insoluble complexes. When the pH value is 1.0 to 2.5, sulfate radicals in the tissue muscle ionize a negative charge and form a salt bond with the cations in Alcian blue, dyeing the tissue muscle blue. Alcian blue can also dye the main components such as chondroitin sulfate and keratan sulfate in cartilage blue.

[0030] In some embodiments of the present application, the fish body is soaked in a decolorizing agent for 2 to 24 hours, and large bubbles are punctured or extracted during the soaking process until bubbles are generated in the fish body spine. After that, the fish body is soaked in pure water or distilled water for 1 day. The decolorizing agent is a mixture of a hydrogen peroxide solution with a mass fraction of 3% to 5% and glycerol mixed at a volume ratio of 2 to 4:1. The decolorizing agent has various functions. Among them, hydrogen peroxide not only removes the floating color but also generates a large number of bubbles in the fish muscle, destroying the muscle compactness and making it easier for the later dye to reach the spine directly. Glycerol can soften the muscle and ensure that the muscle bundle fibers do not break. The ratio of the hydrogen peroxide solution with a mass fraction of 3% to 5% and glycerol is adjusted according to the size of the fish. Small fish (body length less than 15 cm) are soaked in a mixture of a hydrogen peroxide solution with a mass fraction of 3% and glycerol mixed at a volume ratio of 2 to 4:1, and medium-sized fish (body length greater than 15 cm) are soaked in a mixture of a hydrogen peroxide solution with a mass fraction of 5% and glycerol mixed at a volume ratio of 2 to 4:1. The soaking time is based on observing bubbles generated in the muscles on both sides of the fish body spine, and then it can be fished out and washed with water immediately.

[0031] In some embodiments of the present application, the fish body is soaked in alizarin red stain for 2 to 3 days until the fish hard bones turn red. Alizarin red is an anionic dye that easily complexes with various metal ions to form red complexes. When alizarin red forms a complex with calcium ions in completely ossified bones (hard bones) in a chelating manner, an orange-red deposit (i.e., calcium nodules) will be produced on the bone surface, thus achieving the color display effect. Alizarin red is effective in staining mature bone tissues.

[0032] In some embodiments of the present application, the fish body is soaked in Alcian blue stain for 1 to 2 hours until the outline of the fish body shows a light green color. Re-staining the fish body in Alcian blue stain is beneficial for presenting the complete outline of the fish, and can solve the problem that in some transparent specimens of fish, only the bones can be seen and the outline cannot be seen.

[0033] In some embodiments of the present application, the fish body is soaked in the first-gradient transparent solution, the second-gradient transparent solution, and the third-gradient transparent solution for 2 to 3 days in sequence to obtain a transparent fish specimen. Among them, the first gradient transparent solution, the second gradient transparent solution, and the third gradient transparent solution are all mixtures of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2%. The content of glycerol in the first gradient transparent solution, the second gradient transparent solution, and the third gradient transparent solution increases in sequence. The role of the gradient transparent solution is to gradually make the muscles and tissues transparent and remove the excess pigments on the fish body surface. The basic transparency of the fish meat means that the fish meat is translucent, and the color systems of the fish cartilage, hard bone, joints, and ligaments can be clearly observed.

[0034] Further, the first gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed at a volume ratio of 0.5 to 1.5:3, the second gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed at a volume ratio of 0.5 to 1.5:1, and the third gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% mixed at a volume ratio of 2.5 to 3.5:1.

[0035] In some embodiments of the present application, the preparation method of the alcian blue staining agent is: dissolving 90 mg to 110 mg of alcian blue in 700 ml of absolute ethanol, and adding 300 ml of glacial acetic acid, and mixing evenly.

[0036] In some embodiments of the present application, the preparation method of the alizarin red staining agent is: dissolving 600 mg to 700 mg of alizarin red powder in 100 ml of absolute ethanol until saturated, and adding 900 ml of a potassium hydroxide solution with a mass fraction of 1%, and mixing evenly.

[0037] In the embodiments of the present application, absolute ethanol is used to fix the fish body, acetone is used for degreasing, a potassium hydroxide solution with a mass fraction of 1% to 5% is also used for pre-transparency, the alcian blue staining agent is used to stain the fish cartilage, a decolorizing agent is used to remove the alcian blue staining agent that is not bound to the cartilage and destroy the compactness of the fish meat, the alizarin red staining agent is used to stain the fish hard bone, and after the hard bone staining, the alcian blue staining agent is used for re-staining, and the gradient transparent solution is used for transparency; in this way, not only can the fish meat be retained, but also soft tissues such as the eyeball, gill arch, internal organs, swim bladder, and even ossified ctenoid scales can be retained, so that the fish specimen can present the fish characteristics in all directions from the inside to the outside.

[0038] Some typical examples and comparative examples are introduced below.

[0039] In the following examples and comparative examples, the fish samples are fresh individuals or individuals that have died for no more than 12 hours, with no damage on the surface, normal body, intact fins and rays, and complete characteristic tissues such as whiskers, teeth, spines, and scutes. Clean the surface and gills of the fish and remove mucus; remove all scales, especially the front ends of the pectoral and pelvic fins and the scales on the head; make a small cut on the ventral side to facilitate the entry of liquid into the abdominal cavity. The preparation method of Alcian blue dye is: dissolve 100 mg of Alcian blue in 700 ml of anhydrous ethanol, add 300 ml of glacial acetic acid, and mix well. The preparation method of alizarin red dye is: dissolve alizarin red powder in 100 ml of anhydrous ethanol to prepare a saturated solution, add 900 ml of 1% potassium hydroxide solution by mass, and mix well. Among them, the Alizarin Red powder is calculated based on the maximum amount of powder that can be dissolved in ethanol, but the solubility of Alizarin Red powder in ethanol is affected by temperature. When the water temperature is 15°C, 100 ml of anhydrous ethanol can dissolve about 610 mg of Alizarin Red powder (purity ≥ 99%). When the water temperature is 30°C, 100 ml of anhydrous ethanol can dissolve about 680 mg of Alizarin Red powder, so about 610mg~680mg of Alizarin Red powder is dissolved.

[0040] Example 1 Fish sample parameters: small male fish, body length (excluding tail fin) 10cm, long strip, thin abdominal muscle. Therefore, only the scales were removed before dehydration, retaining all muscles and tissues including the fish skin, and the abdomen was not opened.

[0041] Follow these steps to prepare transparent fish specimens: (1) Dehydration, fixation and fat removal: Place the fish flat in a container, add anhydrous ethanol until the fish body is completely covered, soak for 7 days, replace the anhydrous ethanol once during the period, and then soak in acetone for 3 days; (2) Pre-transparency: remove the dissolved substances on the fish body, absorb the organic reagents on the surface with absorbent paper, and soak them in a pre-transparency solution for 2 days; the pre-transparency solution is a 1% by mass potassium hydroxide solution; (3) Blue dyeing: Use a scoop net to gently lift the fish and transfer it to a container filled with Alcian blue dye. Soak the fish until the cartilage turns blue. Rinse the fish body with purified water and continue soaking in purified water. Change the purified water every hour until the purified water no longer changes color. (4) Decolorization: Soak the fish in a decolorizer (a large number of bubbles will be generated during the soaking process. Use a needle syringe to puncture or extract larger bubbles in time to prevent excessive expansion that may cause the eyeball to fall off or the body to deform). When bubbles are found near the spine, remove the fish and soak it in pure water for 1 day. The decolorizer is a mixture of 3% by mass hydrogen peroxide solution and propylene glycol in a ratio of 3:1. (5) Red dyeing: Soak the fish in alizarin red dye and remove it when the spine above the fish belly turns red. (6) Counterstaining: After the red staining is completed, repeat the blue staining in step (3), but change the blue staining time to 1 hour; (7) Gradient clearing: Immerse the fish in the first gradient clearing solution for 2 days, immerse the fish in the second gradient clearing solution for 2 days, and immerse the fish in the third gradient clearing solution for 2 days (during this period, large air bubbles in the fish body should be pumped out or removed by strong light irradiation to avoid affecting the display of the fish's posture); among them, the first gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% in a ratio of 1:3, the second gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% in a ratio of 1:1, and the third gradient clearing solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% in a ratio of 3:1; (8) Specimen preservation: Transfer the prepared transparent fish specimen to a container filled with glycerol, and add 1 small grain of thymol to preserve it against corrosion for a long time.

[0042] The transparent specimen of Anchoviella commersonii in this example is as Figure 1 shown. It can be seen that the specimen has the following advantages: 1) The details are completely retained, the overall outline of the fish is clearly visible, the color system is distinct, among which the muscles and soft fin rays are blue; the internal and external bones are red, and the internal organs and eyeballs are yellowish-green; the internal structure is clearly visible, the visibility and distinguishability of the bone structure are strong, it can be distinguished that the degree of hardening of the dorsal fin and anal fin is not high, the hard bones are covered in the muscles, the number of fin rays can be counted, and the degree of hardening of the head, caudal fin and ventral fin is relatively high; the internal organs are easy to identify, and internal organs such as the intestine, swim bladder, ovary, stomach, and kidney can be easily identified.

[0043] Example 2 Fish sample parameters: Scatophagus argus, body length (excluding caudal fin) 11 cm, body height 7 cm, strongly laterally compressed, high degree of hardening of internal and external bones, tough fish skin and large muscle density. Therefore, before rehydration (rehydration is carried out after dehydration and fixation with absolute ethanol, and the purpose of this operation is to make the dehydrated floating fish body sink to the bottom), the fish skin is peeled off, a small opening is made on the ventral side, and a potassium hydroxide solution with a relatively high mass fraction can be used as the pre-clearing solution for treatment.

[0044] This example is basically the same as Example 1, the only difference is that: (2) Pre-clearing: Remove the dissolved substances on the fish surface, dry the surface organic reagents with absorbent paper, and immerse it in the pre-clearing solution for 2 days; among them, the pre-clearing solution is a potassium hydroxide solution with a mass fraction of 5%.

[0045] The transparent specimen of Scatophagus argus in this example is as Figure 2As shown, it can be seen that this specimen has the following advantages: the details are well preserved, the overall outline of the fish is clearly visible and the color system is distinct, with the muscles being light blue and the internal and external skeletons being red; the internal structure is clearly visible, the internal organs and eyeballs are yellow-green, and the intestines can be clearly observed to be a circular coiled structure in its abdomen, and the stomach contents are vaguely visible, which helps researchers to infer the fish's feeding habits through the types of stomach contents; the skeletal structure is highly visible and distinguishable, and it can be seen that the specimen has a high degree of ossification and it is easy to distinguish the fin rays and fin spines by the depth of color concentration.

[0046] Example 3 Fish sample parameters: fine-scaled carp, body length (excluding tail fin) 12cm, fusiform, high degree of ossification of internal and external skeletons, thick muscle groups on both sides of the back, tough fish skin and high muscle density. Therefore, after rehydration, the fish skin was peeled off, a small incision was made on the ventral side, and the decolorizer was treated with a high mass fraction of hydrogen peroxide solution.

[0047] This embodiment is basically the same as Embodiment 1, except that: (4) Decolorization: Soak the fish body in a decolorizer (a large number of bubbles will be generated during the soaking process. Use a needle syringe to puncture or extract larger bubbles in time to prevent excessive expansion that may cause the eyeball to fall off or the body to deform). When bubbles are found near the spine, remove the fish and soak it in pure water for one day. The decolorizer is a mixture of 5% by mass hydrogen peroxide solution and propylene glycol in a ratio of 1:1.

[0048] The transparent fish specimen of this embodiment is as follows Figure 3 As shown, it can be seen that this specimen has the following advantages: the details are well preserved, the overall outline of the fish is clearly visible and the color system is distinct, the fusiform state of the fish is very complete, the muscles and eyes are light blue, the exoskeleton and exoskeleton are red, and the internal organs are white; the skeletal structure is highly visible and distinguishable, and it can be seen that the specimen has a high degree of ossification and it is easy to distinguish the fin rays and fin spines by the depth of color concentration; the internal structure is clearly visible, the frontoquadrate bone, hyoidofrontal bone and the ligaments connecting it to the skull are clearly visible, the teeth are serrated, a pair of otoliths can be seen on the lower side of the ventricle, the edge of the otolith outline is visible, the swim bladder is well developed, the intestine is short and less curved, and the number of gill arches is small.

[0049] Example 4 Fish sample parameters: Chinese pufferfish, body length (excluding tail fin) 10cm, body height 6cm, laterally flattened, head and spine bones are highly ossified, back and abdomen fins are less ossified, fish skin is tough and rough, body side muscles are thick. Therefore, before rehydration (rehydration is performed after dehydration and fixation with anhydrous ethanol, the purpose of this operation is to allow the dehydrated and floating fish body to sink to the bottom), most of the fish skin is peeled off, only the iconic skin flap that can be opened / closed in the lower abdomen is retained, and the ventral side is not opened.

[0050] This embodiment is basically the same as Embodiment 1.

[0051] The transparent specimen of Thamnaconus sinensis in this embodiment is as Figure 4 shown. It can be seen that this specimen has the following advantages: the details are well preserved, the overall contour of the fish is clearly visible, the color system is rich and distinct. Among them, the muscles are light pink, the hard bones transition from purple to red according to their calcification degree, the gills are blue, the soft fin rays are light green, the internal organs are orange, and the intestinal contents are brown; the internal structure is clearly visible; the visibility and distinguishability of the bone structure are relatively strong. The head and body of the fish are connected by the spine, and there are no ribs to protect the abdomen. Only one extended apical spine plays a role in fixing and supporting. A long strip of hard air sac can be seen along the lower edge of the spine. The liver is huge, accounting for nearly half of the abdominal cavity volume; the opening and closing of the skin flap are controlled by two anteriorly fused hard bones. A large number of red dots can be seen on the skin flap, indicating that there is a certain degree of ossification on its surface.

[0052] Comparative Example 1 Fish sample parameters: Scatophagus argus, body length (excluding caudal fin) 11 cm, body height 7 cm, strongly laterally compressed. The fish specimen is made according to the following steps: 1) Dehydration, fixation, and degreasing: Place the fish flat in a container, add absolute ethanol until the fish body is completely submerged, soak for 7 days, change the absolute ethanol once during this period, and then soak in acetone for 3 days; (2) Pre-transparency: Remove the dissolved substances on the fish surface, dry the surface organic reagents with absorbent paper, and soak in the pre-transparency solution for 2 days; among them, the pre-transparency solution is a potassium hydroxide solution with a mass fraction of 5%; (3) Blue staining: Gently lift the fish with a fishing net and transfer it to a container filled with Alcian blue staining agent, soak until the cartilage turns blue, rinse the body surface with pure water, and continuously soak with pure water, changing the pure water once per hour until the pure water no longer changes color; (4) Red staining: Soak the fish body with alizarin red staining agent, and take out the fish when the spine above the fish abdomen turns red; (5) Decolorization: Soak the fish body with the decolorizing agent (a large number of bubbles will be generated during the soaking process of the fish body. Use a needle syringe to pierce or extract the larger bubbles in time to prevent excessive swelling resulting in the eyeball falling off or the body deforming). Take out the fish when bubbles are generated beside the spine and soak in pure water for 1 day; among them, the decolorizing agent is a mixture of a hydrogen peroxide solution with a mass fraction of 3% and glycerol mixed in a ratio of 3:1; (6) Re-staining: After the red staining is completed, repeat the blue staining in step (3), but change the blue staining time to 1 hour; (7)Gradient transparency: Immerse the fish in the first gradient transparency solution for 2 days, then in the second gradient transparency solution for 2 days, and then in the third gradient transparency solution for 2 days (during this period, large air bubbles in the fish body should be pumped out or removed by strong light irradiation to avoid affecting the display of the fish's posture); among them, the first gradient transparency solution is a mixture of glycerol and a 1% potassium hydroxide solution in a ratio of 1:3, the second gradient transparency solution is a mixture of glycerol and a 1% potassium hydroxide solution in a ratio of 1:1, and the third gradient transparency solution is a mixture of glycerol and a 1% potassium hydroxide solution in a ratio of 3:1; (8)Specimen preservation: Transfer the prepared transparent fish specimen to a container filled with glycerol, and add 1 small grain of thymol to enable long-term anti-corrosion storage.

[0053] The Scatophagus argus specimen of this comparative example is as Figure 5 shown. Compared with Example 2, it can be seen that after blue staining, red staining and then decolorization, the fish belly has partially dissolved, the dye only stays on the surface layer of the muscle, the exposed fins are successfully stained, but the spine and internal bones are not colored, and the internal organs are not successfully stained either, and the internal structure is not clearly visible; it can be seen that the method disclosed in this application can make the spine and internal bones successfully colored and clearly identify the internal organs and gastric contents by destroying the muscle compactness before staining, which is convenient for researchers to conduct subsequent research.

[0054] Comparative Example 2 Fish sample parameters: Siganus fuscescens, body length (excluding caudal fin) 12 cm, laterally compressed.

[0055] Prepare the fish specimen according to the following steps: (1)Dehydration fixation and degreasing: Place the fish flat in a container, add absolute ethanol to completely cover the fish body, soak for 7 days, change the absolute ethanol once during this period, and then soak in acetone for 3 days; (2)Pre-transparency: Remove the dissolved substances on the fish surface, dry the surface organic reagents with absorbent paper, and soak in the pre-transparency solution for 2 days; among them, the pre-transparency solution is a 1% potassium hydroxide solution; (3)Blue staining: Gently lift the fish with a fishing net and transfer it to a container filled with Alcian blue staining agent, soak until the cartilage turns blue, rinse the body surface with pure water, and continuously soak with pure water, changing the pure water once per hour until the pure water no longer changes color; (4)Decolorization: Soak the fish body with the decolorizing agent (a large number of air bubbles will be generated during the soaking process of the fish body, and the larger air bubbles should be punctured or pumped out in time with a syringe to avoid excessive expansion causing the eyeballs to fall off or the body to deform), fish out the fish when bubbles are found beside the spine, and soak in pure water for 1 day; among them, the decolorizing agent is a mixture of a 3% hydrogen peroxide solution and glycerol in a ratio of 3:1; (5) Red staining: Immerse the fish body in alizarin red staining agent. Take out the fish when the vertebra above the fish belly turns red. (6) Gradient clearing: Immerse the fish in the first gradient clearing solution for 2 days, then immerse it in the second gradient clearing solution for 2 days, and then immerse it in the third gradient clearing solution for 2 days (during this period, the larger air bubbles in the fish body should be pumped out or removed by strong light irradiation to avoid affecting the display of the fish's posture); among them, the first gradient clearing solution is a mixture of glycerol and 1% potassium hydroxide solution by a ratio of 1:3, the second gradient clearing solution is a mixture of glycerol and 1% potassium hydroxide solution by a ratio of 1:1, and the third gradient clearing solution is a mixture of glycerol and 1% potassium hydroxide solution by a ratio of 3:1. (7) Specimen preservation: Transfer the prepared transparent fish specimen to a container filled with glycerol, and put in 1 small grain of thymol, then it can be preserved against corrosion for a long time.

[0056] The Siganus fuscescens specimen of this comparative example is as Figure 6 shown. It can be seen that after the specimen prepared in this example does not go through the re-staining step, the muscle morphology and eyes cannot be presented.

[0057] Comparative Example 3 Fish sample parameters: Siganus fuscescens, body length (excluding caudal fin) 12 cm, laterally compressed.

[0058] This example is basically the same as Comparative Example 2, and the only difference is that: (6) Re-staining: After the red staining is completed, repeat the blue staining in step (3), but change the blue staining time to 1 hour. The Siganus fuscescens specimen of this comparative example is as Figure 7 shown. Compared with Comparative Example 2, it can be seen that after the re-blue staining, the muscles and eyes are blue, and the internal and external bones are red; the visibility and distinguishability of the bone structure are stronger, and it can be seen that the overall outline of the fish after re-staining is more complete.

[0059] The present application discloses a method for making a transparent fish specimen, which includes: dehydrating and fixing, degreasing and pre-clearing the fish body; immersing the fish body in alcian blue staining agent for 1 - 2 days until the fish cartilage turns blue; immersing the fish body in a decolorizing agent for 2 - 24 hours; immersing the fish body in alizarin red staining agent for 2 - 3 days until the fish hard bones turn red; immersing the fish body in alcian blue staining agent for 1 - 2 hours until the fish body outline shows light green; and performing a clearing treatment on the fish body to obtain a transparent fish specimen.

[0060] As can be seen from the above embodiments, the present application solves the problem that the vertebrates and bilateral bones of medium-sized fish cannot be stained due to muscle obstruction, and can even be applied to the production of large fish specimens. Moreover, the details of the finally obtained specimens are well preserved. It can not only retain the fish meat, but also retain soft tissues such as eyeballs, gill arches, internal organs, swim bladders, and even ossified ctenoid scales. Moreover, the overall outline of the fish is clearly visible and the color system is distinct, which helps researchers infer the feeding habits of fish through the types of stomach contents; the visibility and distinguishability of the bone structure are relatively strong, enabling the fish specimens to be presented comprehensively from the inside to the outside.

[0061] The embodiments of the present application have been described above in conjunction with the accompanying drawings. However, the present application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them belong to the protection scope of the present application.

Claims

1. A method for preparing a transparent fish specimen, characterized in that: The method comprises the following steps: The fish bodies are dehydrated, fixed, defatted and pre-transparentized; Soaking the fish body in Alcian blue dye for 1 to 2 days until the fish cartilage turns blue; Soaking the fish body in a decolorizing agent for 2 to 24 hours; Soaking the fish body in an alizarin red dye for 2 to 3 days until the fish bones turn red; Soak the fish body in Alcian blue dye for 1 to 2 hours until the outline of the fish body turns light green; The fish body is subjected to a transparent treatment to obtain a transparent fish specimen.

2. The method for preparing a transparent fish specimen according to claim 1, characterized in that: The steps of dehydrating, fixing, defatting and pre-transparentizing the fish body specifically include: The fish body was immersed in anhydrous ethanol for 7 to 14 days for dehydration and fixation, and then immersed in acetone for 2 to 4 days for degreasing; Soaking the fish body in a pre-transparent solution for 1 to 3 days until the fish body muscles become translucent; The pre-transparent solution is a potassium hydroxide solution with a mass fraction of 1% to 5%; The semi-transparent state allows the skeleton morphology in the fish body to be clearly observed.

3. The method for preparing transparent fish specimens according to claim 1, characterized in that: The step of soaking the fish body in Alcian blue dye for 1 to 2 days until the fish cartilage turns blue further comprises: Rinse the excess dye on the fish body surface with purified water or distilled water, and continue to immerse the fish body, changing the water once every hour until the water no longer changes color.

4. The method for preparing a transparent fish specimen according to claim 1, characterized in that: The step of soaking the fish body in the decolorizing agent for 2 to 24 hours specifically comprises: The fish body is soaked in a decolorizing agent for 2 to 24 hours, and larger bubbles are punctured or extracted during the soaking process until bubbles are generated in the spine of the fish body. The fish body is then soaked in purified water or distilled water for 1 day.

5. The method for preparing a transparent fish specimen according to claim 4, characterized in that: The decolorizing agent is a mixture of a 3% to 5% by mass hydrogen peroxide solution and glycerol in a volume ratio of 2 to 4:

1.

6. The method for preparing a transparent fish specimen according to claim 1, characterized in that: The step of subjecting the fish body to transparent treatment to obtain a transparent fish specimen specifically comprises: The fish body is sequentially immersed in the first gradient transparent solution, the second gradient transparent solution, and the third gradient transparent solution for 2 to 3 days to obtain a transparent fish specimen; Among them, the first gradient transparent solution, the second gradient transparent solution and the third gradient transparent solution are all mixtures of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2%, wherein the content of glycerol in the first gradient transparent solution, the second gradient transparent solution and the third gradient transparent solution increases successively.

7. The method for preparing a transparent fish specimen according to claim 5, characterized in that: The first gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% at a volume ratio of 0.5 to 1.5:3, the second gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% at a volume ratio of 0.5 to 1.5:1, and the third gradient transparent solution is a mixture of glycerol and a potassium hydroxide solution with a mass fraction of 1% to 2% at a volume ratio of 2.5 to 3.5:

1.

8. The method for preparing a transparent fish specimen according to claim 1, characterized in that: The preparation method of the Alcian blue dye is as follows: 90 mg to 110 mg of Alcian blue is dissolved in 700 ml of anhydrous ethanol, 300 ml of glacial acetic acid is added, and the mixture is mixed.

9. The method for preparing a transparent fish specimen according to claim 1, characterized in that: The preparation method of the alizarin red dye is as follows: dissolving 600 mg to 700 mg of alizarin red powder in 100 ml of anhydrous ethanol until saturated, adding 900 ml of 1% by mass potassium hydroxide solution, and mixing well.

10. A transparent fish specimen, characterized in that: The transparent fish specimen is prepared according to the method for preparing the transparent fish specimen according to any one of claims 1 to 9.

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