GB/T 18654.12-2026Inspection of germplasm for fishes - Part 12: Test method for the karyotype (English PDF)

5.2 Solution Preparation

5.2.1 KMnO4 solution Weigh out KMnO4 (5.1.1) according to the volume of the disinfected water and prepare a 1mg/L~2mg/L KMnO4 solution. 5.2.2 75% ethanol solution Take 100 mL of 95% ethanol (5.1.2), add

26.67 mL of deionized water or distilled water, and mix well.

5.2.3 Physiological saline Prepare a 0.7%~0.9% NaCl solution by weighing 7.0g~9.0g NaCl (5.1.3), adding 1L of sterile water, mixing well, and incubating at 121°C. Sterilize at 103.4 kPa for 30 min in an autoclave (6.2). 5.2.4

0.1 mol/L NaHCO3 solution Weigh 8.4g NaHCO3 (5.1.4), add 1L of sterile water, mix well and set aside. Filter using a bacterial filter (5.3.6).

5.2.5 Culture medium A culture medium was prepared by mixing calf serum (5.1.5) or fetal bovine serum (5.1.6) with cell culture medium (5.1.7) at a volume ratio of 1.4.Each milliliter of culture medium... The solution contained penicillin (5.1.8), streptomycin (5.1.9), and kanamycin (5.1.10) at concentrations of 100 IU, 100 mg, and 50 IU, respectively, and was prepared using a

0.1 mol/L solution. Adjust the pH of the NaHCO3 solution (5.2.4) to 7.2.

5.2.6 PHA solution According to the fish size and the dosage used for injection or tissue culture, weigh PHA (5.1.11) and mix with sterile physiological saline (5.2.3) and deionized water. Prepare a PHA solution of appropriate concentration using water or distilled water. Filter the solution using a bacterial filter (5.3.6) during cell culture.

5.2.7 Colchicine solution Depending on the culture method, temperature, and fish size, weigh out colchicine (5.1.12) and prepare a solution with a mass concentration of 5 µg/mL. A 10 mg/mL colchicine solution. Filter the solution using a bacterial filter (5.3.6) during cell culture. 5.2.8

0.2 mol/L Na2HPO4 solution Weigh 28.4g of anhydrous Na2HPO4 (5.1.13), add 1L of sterile water, mix well and set aside. 5.2.9

0.2 mol/L NaH2PO4 solution Weigh 24.0g of anhydrous NaH2PO4 (5.1.14), add 1L of sterile water, mix well and set aside.

5.2.11 Hypotonic solution Weigh out

5.59 g of KCl from 0.0375 mol/L,

0.05 mol/L, or

0.075 mol/L KCl solutions, respectively. (5.1.16) Add 1L of sterile water, mix well, and store at 4°C for later use.

5.2.12 Carnoy's fixative Methanol (5.1.17) or anhydrous ethanol (5.1.18) and glacial acetic acid (5.1.19) are mixed at a volume ratio of

3.1 and used immediately.

5.2.13 Chromic acid cleaning solution Mix potassium dichromate (5.1.20). water. concentrated sulfuric acid (5.1.21) in a ratio of 1 (g). 2 (mL). 18 (mL).

5.2.14 Giemsa stock solution 0.5g of Giemsa powder (5.1.22) and 33mL of glycerin (5.1.23) were mixed with a small amount of glycerin in a mortar (5.3.10) and ground. When there are no particles, pour in the remaining glycerin, keep warm at 56°C for 2 hours, add 33mL of methanol (5.1.13), and store away from light.

5.3 Materials

5.3.1 White porcelain plate.

5.3.2 Disinfect gauze or disinfectant paper towels.

5.3.3 Petri dish. 5.3.4 10mL beaker. 5.3.5 25mL culture flask.

5.3.6 Bacterial filter (membrane pore size 0.22µm).

5.3.7 Centrifuge tubes (10mL, 15mL).

5.3.8 Glass slides. Glass slides are immersed in chromic acid cleaning solution (5.2.13) for at least 12 hours, rinsed with purified water, and then soaked in anhydrous ethanol (5.1.18) for 1 hour. Store at -20°C until needed. Alternatively, purchase clean, ready-to-use glass slides.

5.3.9 Alcohol lamp.

5.3.10 Mortar and pestle.

5.3.11 Silk sieve [aperture 60 mesh (

0.250 mm) ~ 100 mesh (

0.150 mm)].

7.2 PBS (5.2.10) and Giemsa stock solution (5.2.14) at a volume ratio of

9.1 and use immediately. Alternatively, use commercial Giemsa. The concentrate and diluent are mixed at a volume ratio of

1.9 and prepared immediately before use.

5.2.16 Heparin solution Under aseptic conditions, prepare a heparin solution with a concentration of 500 IU/mL using sterile water and heparin sodium (5.1.24). The specific concentration can be determined according to... Adjustments to different fish species and tissue cells. 5.2.17 50% glacial acetic acid solution Add 100 mL of glacial acetic acid (5.1.19) to 100 mL of deionized water or distilled water, mix well, and use immediately.

8.1.1 Somatic cell in vitro culture method

8.1.1.1 In vitro culture of kidney and spleen tissue cells 8.1.1.1.1 Sampling Disinfect the sample fish with 1 mg/L~2 mg/L KMnO4 solution (5.2.1) for 30 min, then cut the gill vessels or disconnect the tail to bleed them. Dissolve in 75% ethanol. Disinfect the fish with liquid (5.2.2) and place it on a white porcelain dish (5.3.1) in a sterile room or ultra-clean workbench (6.3). The white porcelain dish should be pre-placed with sterilized gauze. Cloth or sterile wipes (5.3.2). Take head kidney, whole kidney, or spleen tissue, and wash 2-3 times in sterile saline (5.2.3). In a container... Place the sample tissue into a petri dish (5.3.3) containing 3-5 mL of culture medium (5.2.5) or a 10 mL beaker (5.3.4), and gently stir. Let it stand for 3-5 minutes. Transfer the upper cell suspension to a 25mL culture flask (5.3.5), then add an appropriate amount of culture medium. (5.2.5) Prepare each bottle to contain 4 mL or 5 mL. Add PHA solution (5.2.6) using a pipette, generally

0.1 mL per 5 mL of culture medium. Add

0.2 mL to bring the PHA (5.1.11) concentration in the culture flask to 5 µg/mL~10 µg/mL. Shake gently to mix. 8.1.1.1.2 Tissue Culture Place the inoculated culture flasks (5.3.5) flat in a constant temperature incubator or carbon dioxide incubator (6.4). The incubation temperature is generally 18°C~ The optimal incubation temperature is 28°C, and the incubation period is 1 to 5 days. Choose the appropriate incubation temperature and time based on specific circumstances. Gently shake the culture flask daily. 1 to 2 times. 8.1.1.1.3 Cell Collection Add colchicine solution (5.2.7) using a pipette

1.5 to 5 hours in advance (this may be earlier or later for some fish species) to the colchicine solution. (5.1.12) The final concentration is 0.1 µg/mL to 10 µg/mL. When collecting cells, first shake the culture flask until the cells are suspended, then pour it into a centrifuge. For the heart tube (5.3.7), wash out the residual cells in the culture flask (5.3.5) with a small amount of physiological saline (5.2.3) or PBS (5.2.10). Adherent cells can be removed with a small amount of saline. Dissolve the contents in 0.25% trypsin digestion solution (5.1.15), then pour into a centrifuge tube (5.3.7). Place in a centrifuge (6.5) at 700 rpm. Centrifuge at 1000 rpm for 5 to 8 minutes, remove the supernatant, and keep about

0.5 mL of cell pellet. 8.1.1.1.4 Hypoosmolarity Add 4 mL of hypotonic solution (5.2.11) to centrifuge tube 8.1.1.1.3 (5.3.7), mix well with a pipette, and incubate at room temperature or 37°C. Let stand for 20-50 minutes, then add 0.5-

1.0 mL of Carnoy's fixative (5.2.12), mix well by pipetting, and let stand for 2-3 minutes. Centrifuge at 700 rpm to 1000 rpm for 5 to 8 minutes. 8.1.1.1.5 Fixed Remove the supernatant, add a small amount of Carnoy's fixative (5.2.12), mix well by pipetting, and then add 2-3 mL of Carnoy's fixative. (5.2.12) Let stand for 20 minutes, then centrifuge according to the procedure in 8.1.1.1.3.Repeat the fixation process 2 to 3 times. 8.1.1.1.6 Droplets After the final fixation and centrifugation, aspirate the supernatant, add an appropriate amount of freshly prepared Carnoy's fixative (5.2.12), and mix thoroughly by pipetting. Aspirate the cell suspension. For the liquid, place 3 to 5 drops onto a frozen slide (5.3.8) tilted at 30° to 45°, ensuring each drop is dispersed and does not cover the other. The slide height should be higher than 50 cm. Gently blow on the slide to spread the cells. Place the slide at an angle and allow it to air dry naturally, or place the slide sample-side up over an alcohol lamp (5.3.9) flame. Dry the slide by passing it through a flame 4-5 times quickly. Alternatively, a spreading method can be used. place the frozen slide (5.3.8) on a horizontal surface and absorb the fine... For cell suspension, disperse 3 to 5 drops at a height of 5 to 10 cm from the glass slide, spread evenly, and allow to air dry or flame dry. 8.1.1.1.7 Staining Place a clean piece of glass on a horizontal surface. Use clean glass slides as a support, spaced slightly shorter than the length of the glass slides. The slides to be stained should be finely... Place the slide with the cell side down on the rack. Add the staining solution (5.2.15) dropwise from under the slide (avoiding air bubbles). Stain for 20-30 minutes, then rinse with tap water. Rinse the back of the slide to remove the stain, and after drying, it can be used for observation and analysis.

8.1.1.2 In vitro culture of blood cells 8.1.1.2.1 Sampling Aseptic blood collection is acceptable; blood can be drawn from the tail vein (artery) or the heart. 8.1.1.2.2 Lymphocyte Culture 8.1.1.2.2.1 Using a syringe, add heparin solution (5.2.16) dropwise along the wall of the centrifuge tube (5.3.7) to moisten the tube wall. Add to each centrifuge tube (5.3.7) 0.1mL~0.3mL. 8.1.1.2.2.2 Remove the syringe needle containing sterile blood, discard the first 2-3 drops of blood, and drip the remaining blood into the centrifuge tube des...