Taxonomy of Finfish (1+2)

6.1.5.1 Chromosome Preparation

Chromosomes can be prepared from two sources: i) in vivo- from the actively dividing tissues like kidney, spleen or gill and ii) in vitro- from the cultured cells. The in vivo method requires colchicines treatment of the fishes 2-3 hours before chromosome preparation. Colchicine prevents the polymerization of tubuline that form microtubules. As a result, cell division is arrested at metaphase stage. The number of metaphase plates obtained depends up on mitotic activity, which is affected by the season, health and age of the individual fish. Prior treatment of a mitogen such as phytohaemaglutanin, cobalt chloride, phenyl hydrazine, which stimulates cell diviosns, is useful in obtaining greater number of metaphase plates.

A good number of metaphase plates can be obtained by short term in vitro culture of blood cells, abdominal fluid, fin tip or scale epithelium. The cultured cells are treated with colchicines to three hours before harvest. Long term culture not recommended for karyotypes study because aneuploid and polyploid cells are often produced.

The cells obtained from in vivo or in vitro sources are treated with a hypotonic solution, which is 0.56% potassium chloride or 1% sodium citrate. As a result the cells swell in size. The cells are sedimented by low speed centrifugation and fixed in methanol: acetic acid (3:1 V/V) with two to three changes. The chromosomes slides are prepared by flame drying or air drying method.