Method for producing transparent stained specimen of medium and large adult fish individuals

Abstract

The present invention provides a method for making a transparent stained specimen of medium and large adult fish individuals, belonging to the field of specimen making. The method includes sample pretreatment, specimen making, and specimen preservation; the specimen making includes scaling and skinning, fixing with absolute ethanol, bone staining, pretreatment for clearing, and clearing; the present invention adopts the method of precise targeted intramuscular injection of trypsin digestion solution, which can accelerate the digestion process and thus shorten the production process; the present invention immerses the digested specimen successively in two potassium hydroxide-water-glycerol-diethylene glycol systems, and then transfers it to diethylene glycol-glycerol to complete the clearing, which can not only make the clearing faster and more complete, but also exclude the interference of histamine, pigments, etc., so that the spatial structure of the internal bones of the fish body can be observed through the cleared soft tissues without removing the soft tissues, and a transparent bone stained specimen of adult fish individuals with a body length of 10-50 cm and a body weight of 0-1 kg is successfully made.

Description

Technical Field

[0001] The present invention belongs to the field of specimen production, and particularly relates to a method for producing a transparent stained specimen of medium and large adult fish individuals. Background Art

[0002] In general popular science, scientific research and teaching activities, common types of fish specimens include dry specimens, immersion specimens, and skeletal specimens, etc. Transparent skeletal stained specimens are a type of characteristic specimen, which are not only necessary materials for studying fish skeletal anatomy, but also relatively rare and beautiful specimens for exhibition. In the research of fish development, morphology, anatomy, and even genetic evolution, this type of specimen is an important research object. However, the commonly used production methods at present have great defects and cannot completely transparentize the soft tissues of medium and large fish. Generally, only fish larvae or adult fish individuals with a body length less than 10 cm and not exceeding 50 g can be made into transparent skeletal stained specimens, which greatly limits the application scope of this type of specimen. Currently, major domestic and foreign fish-related scientific research institutions, museums, and specimen museums are all actively researching and developing the production process of transparent skeletal stained specimens of medium and large adult fish individuals, but there are only a few successful cases. Known adult individuals include flatfish, catfish, and scorpionfish, with a body length generally not exceeding 20 cm and a weight not exceeding 100 g. The transparentization effects of soft tissues of fish in different families often vary due to differences in composition and structure. Factors such as fat content, histamine content, amino acid preference, location of connective soft tissues, and bone structure may all lead to the failure of specimen production. The failure is divided into two types: incomplete transparentization and excessive transparentization. Incomplete transparentization will result in the inability to clearly observe the internal bones, while excessive transparentization will lead to specimen damage, such as the shedding of fins, jaws, etc. or the rupture of the abdominal cavity. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for producing a transparent stained specimen of medium and large adult fish individuals. The specimens produced by the method of the present invention are not limited to fish larvae or sizes not exceeding 10 cm in body length. Through an improved transparentization process flow, adult fish individuals with a body length of 10 cm - 50 cm can be made into transparent skeletal stained specimens.

[0004] The present invention is realized by the following technical solutions:

[0005] A method for producing a transparent stained specimen of medium and large adult fish individuals, the method comprising sample pretreatment, specimen production, and specimen preservation;

[0006] The specimen production includes scale and skin removal, fixation with absolute ethanol, cartilage staining, neutralization, hard bone staining, transparentization pretreatment, and transparentization;

[0007] The transparency pre-treatment: For individuals with a large amount of pigment deposition inside, decolorize with 3% hydrogen peroxide by mass percentage; for fish rich in oil content, soak in a lipase solution to decompose excess fat;

[0008] The transparency: Digest with a trypsin solution. For specimens with thick or dense soft tissues, refer to X-ray or CT photos, and use a multi-hole syringe to inject 1% trypsin solution by mass percentage into large muscle groups, taking care to avoid important connective tissues between bones and muscle bundles. The injection positions are in the area between the second myomere and the penultimate fifth myomere of the epaxial muscle and between the second myomere and the penultimate fifth myomere of the hypaxial muscle posterior to the abdominal vertebra, as well as large muscle bundles including the first sub-muscle of the adductor mandibulae. Soak at a constant temperature of 37°C in 0.5% trypsin solution by mass percentage for digestion for 2 - 5 days; Transfer the digested specimens to two potassium hydroxide - water - glycerol - diethylene glycol systems for soaking successively. The first reagent is composed of an aqueous solution of 1.5% potassium hydroxide by mass percentage, pure glycerol, and diethylene glycol prepared in a volume ratio of 7:2:1, and soak for 3 - 7 days; The second reagent is composed of an aqueous solution of 1.5% potassium hydroxide by mass percentage, pure glycerol, and diethylene glycol prepared in a volume ratio of 4:4:2, and soak for 5 - 10 days; Then transfer to diethylene glycol - glycerol and let stand for 1 - 3 months to complete transparency, with the volume ratio of diethylene glycol to glycerol being 1:1.

[0009] Preferably, for the absolute ethanol fixation: Put the fish body into absolute ethanol for fixation. For fish with a body length of more than 30 cm or a body weight of more than 500 g, inject absolute ethanol into the abdominal cavity to fix the internal organs before soaking.

[0010] Preferably, for the cartilage staining: Transfer the fish body fixed with absolute ethanol to an Alcian blue staining solution and soak for 1 - 4 days, then transfer to clean water for washing to remove the excess staining solution.

[0011] Preferably, for the neutralization: Transfer the fish body after washing with water to a saturated sodium borate solution and soak for 2 - 5 days.

[0012] Preferably, for the bone staining: Transfer the fish body after neutralization to an alizarin red staining solution and soak for 2 - 4 days, then transfer to clean water for washing for 1 - 2 hours to remove the excess staining solution.

[0013] Preferably, for fish individuals with a body length exceeding 20 cm, after the diethylene glycol - glycerol treatment, transfer them to a short-term preservation solution. The short-term preservation solution is a diethylene glycol - glycerol system containing 0.1% thymol by volume ratio, with the volume ratio of diethylene glycol to glycerol being 1:2, until transparency is complete.

[0014] The beneficial effects of the present invention compared with the prior art:

[0015] (1) The present invention breaks through the limitations of the volume and fish age of the transparent bone stained specimens of fish. The original method can only produce transparent bone stained specimens of fish larvae and fish with a body length not exceeding 10 cm and a body weight not exceeding 50 g. The present invention can produce transparent bone stained specimens of adult fish individuals with a body length of 10 - 50 cm and a body weight of 0 - 1 kg.

[0016] (2) Compared with other methods for producing transparent bone stained specimens of fish, the present invention has a shorter production cycle when dealing with fish of the same volume and the same species.

[0017] (3) The present invention uses the method of lipase decomposing fat to remove the excess grease in the fish body and avoid the influence of grease on transparency.

[0018] (4) In the process of transparency, the present invention applies the anatomical knowledge theory to accurately inject the trypsin digestive solution at fixed points. Using the trypsin digestive solution can eliminate the interference of amino acid preference. Due to the different thicknesses of the soft tissues of the fish body, the digestion degrees of different parts are different under the same digestion conditions. Injecting at fixed points while avoiding important connecting parts can prevent the fish body from being damaged due to over-digestion. Injecting the high-concentration protease digestive solution into large muscle groups can accelerate the digestion process, thus shortening the production process.

[0019] (5) The present invention immerses the digested specimens successively in two potassium hydroxide - water - glycerol - diethylene glycol systems, and then transfers them to diethylene glycol - glycerol to complete transparency. Compared with the previous potassium hydroxide - water - glycerol system, it can make the transparency faster and more complete, and can also eliminate the interference of histamine, pigments, etc. The experimental results show that the transparency effect of the present invention is significantly better than other methods.

[0020] (6) When accurately injecting the trypsin digestive solution at fixed points by the method of the present invention, a soft X-ray photography system or an animal CT imaging system is used to take pictures of the bones as a reference, which greatly ensures the accuracy of the injection position.

[0021] (7) The formula of the immersion liquid and preservation liquid of the specimens prepared by the present invention has strong stability, no volatility, no toxicity, is not flammable, and is not prone to chemical reactions with contact substances. The preservation effect of the specimens is long-lasting and is suitable for exhibition. Description of the Drawings

[0022] Figure 1 The porous syringe used in Example 1, 1. Syringe barrel, 2. Piston rod, 3. Rubber stopper, 4. Fixed body, 5. Needle, 6. External thread;

[0023] Figure 2 The transparent bone stained specimen of medium and large adult fish successfully prepared by the method of the present invention;

[0024] Figure 3This is a comparison chart of the results of the method of the present invention and the prior art for making fish specimens transparent; where a is the second group of transparency treatment, b is the first group of transparency treatment, and c is the third group of transparency treatment. Detailed implementation mode

[0025] The technical solution of the present invention will be further explained below through embodiments, but the protection scope of the present invention is not limited by any form of the embodiments.

[0026] Embodiment 1

[0027] A method for making a transparent stained specimen of a medium or large adult fish individual, the method includes sample pretreatment, specimen production and specimen preservation, and the specific steps of the method are as follows:

[0028] The sample pretreatment: Select a fish that is intact without injury, has undamaged fins, intact internal organs, and no signs of decay, preferably a fish that has just died and is very fresh. Wash away the dirt and mucus adhering to the fish body, and also clean the dirt in the fish's mouth and gills without damaging the fins. Place the washed fish body on a stainless steel tray, place it upright, use specimen needles to spread and fix the dorsal fin, pectoral fin, ventral fin, anal fin and caudal fin, brush absolute ethanol on the base of each fin and let it stay for 3 - 5 minutes, then remove the specimen needles. At this time, the fins are basically shaped and in a stretched state. Use a soft X-ray photography system or an animal CT imaging system to take pictures of the bones as a reference.

[0029] The specific steps of the specimen production are as follows: (1) Scale and skin removal: Remove the fish scales and skin under a microscope or magnifying glass, and pay attention not to overly damage the inner muscle tissue and internal organs.

[0030] (2) Fixation with absolute ethanol: Place the fish body in absolute ethanol for fixation (note that the posture of the fish body should be flat to ensure that the solution submerges the fish body and avoid any part being exposed to the air). Depending on factors such as the size and muscle thickness of the individual, the soaking time is slightly different. Fish of 10 - 20 cm are soaked for 3 - 4 days, fish of 20 - 30 cm are soaked for 5 days, and fish of 30 - 50 cm are soaked for one week. In addition, for larger individual fish, before soaking, it is necessary to inject absolute ethanol into the abdominal cavity with a syringe to fix the internal organs, and the injection volume should be such that the abdomen becomes slightly hard.

[0031] (3) Cartilage staining: Transfer the fish body fixed with absolute ethanol to a bucket or tank filled with Alcian blue staining solution and soak it for 1 - 4 days, then transfer it to clean water and wash it for 1 - 2 hours to wash away the excess staining solution.

[0032] (4) Neutralization: Transfer the fish body after water washing to a bucket or tank filled with saturated sodium borate and soak it for 2 - 5 days.

[0033] (5) Bone staining: Move the neutralized fish body to a bucket or tank filled with alizarin red staining solution and soak it for 2-4 days. Then transfer it to clean water for 1-2 hours to wash off the excess staining solution.

[0034] (6) Transparent pretreatment: For individuals with more pigmentation inside, soak them in 3% hydrogen peroxide for 1-3 days for decolorization. For fish with rich oil content, soak them in 2% lipase solution at 38°C for 2 days to decompose excess fat. Soak them in clean water for 1 hour to wash away excess reagents.

[0035] (7) Transparentization: Remove excess water from the fish body and digest it with trypsin solution. For specimens with thick or dense soft tissues, based on knowledge of fish anatomy and referring to X-ray or CT images, use a multi-hole syringe to inject 1% by mass trypsin solution into large muscle groups. To prevent specimen disintegration and destruction caused by over-digestion, pay attention to avoid important bone-to-bone connective tissue and muscle bundles, including the intermandibular ligament, dense connective tissue between the lingual arch and the branchial arch, connective tissue between the suspensor and the operculum, connective tissue between the shoulder girdle and the girdle, genioglossus, adductor palatine arch, adductor lingual mandibularis, levator operculum, intermandibularis, abductor girdle, adductor girdle, tilting fin muscle and depressor girdle muscle. The injection position focuses on the area between the second and fifth last myometrium of the axial muscle and the second and fifth last myometrium of the posterior subaxial muscle of the abdominal vertebra, as well as the first divisor of the mandibular adductor muscle and other large muscle bundles. Digestion is carried out in a 0.5% trypsin solution at a constant temperature of 37°C for 2-5 days. The digested specimens are successively transferred to two potassium hydroxide-water-glycerol-diethylene glycol systems for immersion. The first reagent component is a 1.5% potassium hydroxide aqueous solution, pure glycerol and diethylene glycol in a volume ratio of 7:2:1, and the immersion time is 3-7 days; the second reagent component is a 1.5% potassium hydroxide aqueous solution, pure glycerol and diethylene glycol in a volume ratio of 4:4:2, and the immersion time is 5-10 days. Then transfer to diethylene glycol-glycerol and let it stand for 1-3 months to complete transparency. The volume ratio of diethylene glycol and glycerol is 1:1.

[0036] For fish individuals over 20cm in length, an additional step is required. After the above method is completed, they are transferred into a short-term preservation solution. The short-term preservation solution is a diethylene glycol-glycerol system containing 0.1% musmol by volume, wherein the volume ratio of diethylene glycol to glycerol is 1:2, until complete transparency is achieved.

[0037] The porous syringe used in this embodiment is as Figure 1As shown in the figure, it includes a multi-needle injection head and a syringe body. The multi-needle injection head is composed of a fixing body 4 and more than one needle 5. The fixing body 4 and the needle 5 are of an integral structure, made of stainless steel material. The fixing body 4 is cylindrical, and the inner wall of the fixing body 4 is provided with internal threads. The needle 5 is a tubular body with both ends open, and the free end of the needle 5 is sharp. The syringe body includes a syringe barrel 1, a plunger rod 2 located inside the syringe barrel 1, and a rubber stopper 3 located at the top of the plunger rod 2. The rubber stopper 3 is made of rubber material, and the diameter of the rubber stopper 3 is equal to the inner diameter of the syringe barrel 1, which is used to push the liquid. The outer wall of one end of the syringe barrel 1 is provided with external threads 6, and the external threads 6 match the internal threads on the inner wall of the fixing body 4 to seal the liquid in the syringe barrel 1.

[0038] For the preservation of the specimen: Transfer the specimen from the previous step into a specimen bottle of appropriate size, add the preservation liquid, and cover the specimen bottle cap. The preservation liquid consists of glycerol containing 0.1% thymol by volume. Stick a label on the bottle body with information such as the Chinese scientific name, number, Latin name, family, collection date, collection location, collector, and remarks (bottle number) of the specimen.

[0039] The transparent stained bone specimens successfully prepared by the method described in this embodiment are as Figure 2 shown. They are flathead catfish with a body length of 29.8 cm, silver pomfret with a body length of 20.6 cm, and spotted tail goby with a body length of 26 cm respectively.

[0040] Example 2

[0041] Select 12-cm-long anchovies and perform different clearing treatments. The first group is the anchovy transparent bone stained specimens made by the method of the example, and the second and third groups are the anchovy transparent bone stained specimens made by the existing production methods, that is, the control groups. The cleaning, fixation, and bone staining steps of the three control experiments are the same, as follows:

[0042] Select 12-cm-long anchovies that are intact without injury, have undamaged fins, intact internal organs, and no signs of decay. Wash the dirt and mucus adhering to the fish body, in the fish mouth and in the gills, spread out and fix each fin with specimen needles, brush with absolute ethanol and let it stay for 3 minutes, then remove the specimen needles. At this time, the fins are basically shaped and in a stretched state. Use a soft X-ray photography system to take pictures of the bones. Remove the fish scales and fish skin under a magnifying glass, put the fish body into absolute ethanol for fixation, soak for 3 days, then transfer the fish body to Alcian blue staining solution and soak for 1 day, transfer to clean water and wash for 1 - 2 hours, then transfer to a bucket or tank filled with saturated sodium borate and soak for 2 days, transfer it to alizarin red staining solution and soak for 2 days, and then transfer to clean water and wash for 1 - 2 hours to wash away the excess staining solution.

[0043] The clearing steps of the three groups of anchovies are different, as follows:

[0044] Group 1: Absorb the excess moisture from the fish body and digest it using trypsin solution. Refer to the X-ray or CT photos, and use a multi-hole syringe to inject a 1% protease solution by mass into the large muscle groups. The injection locations focus on the area between the second to the penultimate fifth myotomes of the epaxial muscle and the area between the second to the penultimate fifth myotomes of the hypaxial muscle posterior to the abdominal vertebra. Soak it at a constant temperature of 37°C in a 0.5% trypsin solution by mass for 3 days for digestion. Transfer the digested specimen to two potassium hydroxide-water-glycerol-diethylene glycol systems for soaking successively. The first reagent is composed of a 1.5% potassium hydroxide aqueous solution, pure glycerol, and diethylene glycol prepared in a volume ratio of 7:2:1, and the soaking time is 3 days; the second reagent is composed of a 1.5% potassium hydroxide aqueous solution, pure glycerol, and diethylene glycol prepared in a volume ratio of 4:4:2, and the soaking time is 5 days. Then transfer it to diethylene glycol-glycerol and let it stand for 1 month to complete the clearing, and the volume ratio of diethylene glycol to glycerol is 1:1. Put the completely cleared specimen into the preservation solution, the composition of which is glycerol containing 0.1% thymol by volume.

[0045] Group 2: Absorb the excess moisture from the fish body and digest it using trypsin solution. Soak it at a constant temperature of 37°C in a 0.5% trypsin solution by mass for 3 days for digestion. Transfer the specimen to two potassium hydroxide-water-glycerol systems for soaking successively. The first reagent is composed of a 1.5% potassium hydroxide aqueous solution and pure glycerol prepared in a volume ratio of 7:3, and the soaking time is 3 days; the second reagent is composed of a 1.5% potassium hydroxide aqueous solution and pure glycerol prepared in a volume ratio of 4:6, and the soaking time is 5 days. Then transfer it to glycerol and let it stand for 1 month to complete the clearing. Put the cleared specimen into the preservation solution, the composition of which is glycerol containing 0.1% thymol by volume.

[0046] Group 3: Absorb the excess moisture from the fish body and transfer the specimen to two potassium hydroxide-water-glycerol systems for soaking successively. The first reagent is composed of a 1.5% potassium hydroxide aqueous solution and pure glycerol prepared in a volume ratio of 7:3, and the soaking time is 7 days; the second reagent is composed of a 1.5% potassium hydroxide aqueous solution and pure glycerol prepared in a volume ratio of 4:6, and the soaking time is 10 days. Then transfer it to glycerol and let it stand for 1 month to complete the clearing. Put the cleared specimen into the preservation solution, the composition of which is glycerol containing 0.1% thymol by volume.

[0047] Judging from the result comparison of the three groups of experiments, the soft tissues of Group 1 are completely cleared and the fish body is intact, such as Figure 3 the b specimen in; the soft tissues of Group 2 are not completely cleared and the fish body is intact, such as Figure 3 the a specimen in; the soft tissues of Group 3 are relatively completely cleared, but the dorsal fin, ventral fin, part of the skull, and anal fin are detached and the fish body is incomplete, such as Figure 3Specimen c therein. Thus, by using the anatomical theoretical knowledge to inject the trypsin digestive solution at fixed points and adopting the clearing reagent system of the first group, it is possible to achieve complete clearing of soft tissues while ensuring the integrity of the fish body.

Claims

1. A method for preparing transparent stained specimens of medium-to-large adult fish, the method comprising sample pretreatment, specimen preparation and specimen preservation; The specimen preparation includes descaling and skinning, fixation with absolute ethanol, cartilage staining, neutralization, hard bone staining, pretreatment for clearing and clearing, and is characterized in that: The transparent pretreatment: for individuals with more pigmentation inside, 3% by mass hydrogen peroxide is used for decolorization; for fish with rich oil content, lipase solution is used for soaking to decompose excess fat; The transparentization is as follows: firstly digest with trypsin solution, for specimens with thick or dense soft tissue, refer to X-ray or CT photos, use a multi-hole syringe to inject 1% by weight trypsin solution into large muscle groups, pay attention to avoid important bone-to-bone connective tissue and muscle bundles, the injection position is between the second myometrium to the fifth last myometrium of the axial muscle and between the second myometrium to the fifth last myometrium of the posterior subaxial muscle of the abdominal vertebra, and the large muscle bundles including the first divisor of the mandibular adductor muscle, and digest in 0.5% by weight trypsin solution at a constant temperature of 37°C for 2-5 days; the digested specimens are successively transferred to two potassium hydroxide-water-glycerol-diglycol systems for immersion, and the reagent component of the first system is 1.5% by weight The first system is prepared by mixing potassium hydroxide aqueous solution, pure glycerol and diethylene glycol in a volume ratio of 7:2:1, and soaking for 3-7 days; the second system is prepared by mixing potassium hydroxide aqueous solution with a mass percentage of 1.5%, pure glycerol and diethylene glycol in a volume ratio of 4:4:2, and soaking for 5-10 days; then transferred to diethylene glycol-glycerol and allowed to stand for 1-3 months to complete transparency, and the volume ratio of diethylene glycol to glycerol is 1:1; The multi-hole syringe comprises a multi-needle injection head and a syringe body. The multi-needle injection head is composed of a fixed body and more than one needle. The needle is a tubular body with two ends open.

2. The method for making a transparent stained specimen of medium and large adult fish individuals according to claim 1, characterized in that, The anhydrous ethanol fixation is to fix the fish body in anhydrous ethanol. Before the fish with a body length of more than 30 cm or a weight of more than 500 g is immersed, anhydrous ethanol needs to be injected into the abdominal cavity to fix the internal organs.

3. The method for making a transparent stained specimen of medium and large adult fish individuals according to claim 1, characterized in that, The cartilage staining is as follows: the fish body fixed with anhydrous ethanol is transferred to an Alcian blue staining solution and soaked for 1-4 days, and then transferred to clean water for washing to remove excess staining solution.

4. The method for producing a transparent stained specimen of a medium or large-sized adult fish individual according to claim 1, characterized in that, Neutralization: The cartilage-stained fish body is moved to a saturated sodium borate solution and immersed for 2-5 days.

5. The manufacturing method of a transparent stained specimen of medium and large adult fish individuals according to claim 1, characterized in that, The bone staining is as follows: the neutralized fish body is transferred to an Alizarin Red staining solution and soaked for 2-4 days, and then transferred to clean water for washing for 1-2 hours to wash away the excess staining solution.

6. The method for making a transparent stained specimen of a medium or large-sized adult fish individual according to claim 1, characterized in that, For fish with a body length of more than 20 cm, they need to be transferred into a short-term preservation solution after the diethylene glycol-glycerol treatment. The short-term preservation solution is a diethylene glycol-glycerol system containing 0.1% musmol by volume, and the volume ratio of diethylene glycol to glycerol is 1:2, until complete transparency.

Images