ABSTRACT

The protocol for fi broblast culture from fi sh fi n presented in this chapter is based on the method by Ojima (1978; 1982) later modifi ed by Amemiya et al. (1984). Briefl y, pieces of fi ns are sterilized, and placed as aseptically as possible into culture fl asks using a suitable medium with adjusted pH, antibiotic/ antimycotic agents and fetal calf serum. The cell culture is maintained at the temperature appropriate to the species under study, monitored daily for checking the explant outgrowth and possible contamination. When a monolayer of fi broblast cells covers the fl ask bottom, attaining around 70-80% of confl uence, the cells are treated with colchicine and detached from the bottom surface by trypsin application. The cells are harvested and then hypotonized and fi xed according to conventional procedure for chromosome preparations. The fi rst protocols were based on the use of bicarbonate-buffered media (Eagel’s Medium 199), which require frequent changes of the medium or culture in a CO2-enriched atmosphere. Our modifi cation applies L-15

medium, which is self-buffered by its amino acid composition and does not imply frequent changes, thus signifi cantly reducing the manipulations and the consequent risk of contamination.