Procedure
1. Take a healthy fry or fish and condition them. 2. The conditioned fishes were injected intramuscularly with 0.01-0.05% colchicine at a dosage of 0.5 ml to 1.0 ml/100 g body weight of the fish. If the fishes are small fry, add required quantity (0.1% to 0.001%) of colchicine in the aquarium. 3. The fishes should be kept in well aerated aquaria for 1-2 hr after colchicine treatment. 4. Dissect the fish and isolate the kidney (or/and spleen) for larger fish and gill arches for smaller fish avoiding the upper membrane and blood clots. 5. Immediately put the tissue in 6-8 ml hypotonic solution (0.56% KCI or 0.08% sodium citrate in sterile water or distilled water) in 15 ml centrifuge tubes. 6 By using tissue homogenizer make the tissues to cell suspension (for hatchlings and early fry whole body tissues can be taken). 7. Carefully decant the upper liquid cell suspension into a 15 ml clinical centrifuge tube and try to avoid any particulate piece to come into that. If any such piece of tissue comes then remove it from the cell suspension by carefully decanting into another tube. Gently with Pasteur pipette mix it. Incubate it for 20-25 min at room temperature for swelling. 8. Gently add 1.0 ml freshly prepared chilled Carnoy's fixative through the sides of the tube. Mix it gently with Pasteur pipette. 9. Spin at 1500 rpm in clinical centrifuge for 10 min at room temperature with proper balancing to pellet down the cells from the suspension. 10. Drain the supernatant with a pipette and slowly overlay 6-8 ml freshly prepared chilled fixative. 11. Keep the tubes in refrigerator (at 4 ° C) for half an hour for thorough fixation (the duration of fixation may be prolonged further). 12. Mix the contents and centrifuge cell suspension at 1200-1500 rpm for 10 min at room temperature. Remove the supernatant without disturbing cell pellet at the bottom and add fresh fixative (5-6 ml). 13. Repeat the above step for 3-4 times till clear transparent cell suspension is obtained. 14. Take a clean glass slides (treated with 50% ethanol). A drop of the cell suspension with fixative can be dropped from a Pasteur pipette held at a height of 1-1.5 feet to the slide. 15. Allow the slide to air or flame dry. 16. Keep the slide for ageing for 1-3 days in dust free place. 17. Stain the slide in 5% Giemsa solution (pH 6.8) for 20 min by keeping the slides back to back into the staining jar. 18. Wash the slide with distilled water thoroughly and store the slides in a slide box. Make the slides permanent by mounting in DPX mountant. 19. Observe the slide under Nikon microscope under oil immersion objective (100X). |