Principle
The basic principle of getting metaphase chromosomes is to arrest a mitotically dividing cell at the subsequent stage. Here mitosis is induced in some heamopoetic tissues: viz., kidney, spleen etc., in the living system of a fish with a mitogen like concanavaline A (or may be with phytohaemagglutinin). This induced mitosis is allowed to continue in vivo for some specific time interval and then the cells are treated with colchicine. Colchicine is a potent inhibitor of tubulin polymerization and it dismantles the microtubule spindles. Thus the separation of the chromosomes is stopped. But the concentration and time of this treatment must be very critically maintained. Otherwise the chromosomes will be excessively contracted. The tissues are isolated from the system after the colchicine treatment and minced to get a single cell suspension in hypotonic solution which also helps to swell the cell very much. They are fixed with Carnoy's solution (3:1 methanol, acetic acid ) which helps to clear up the cytoplasmic content of the cells and the nucleolar proteins. Then the fixed cells are burst open on a cleaned/warmed slide by falling a drop of cell suspension from sufficient height (1-2 feet is sufficient). |