Antibiotic and antimitotic solution

Asian seabass larvae of 1–2 days post-hatching (dph) age from the Marine Aquaculture Center (St John's Island, Singapore) were sacrificed by placing on ice and they were seeded into cell culture dishes in L15 or RPMI medium supplemented with 20% fetal calf serum and antibiotic and antimitotic solution (Sigma). The primary culture was incubated at 29°C with 5% CO. After 1–2 weeks, the cells were incubated with 0.01% of colchicine for 5–6 h. Chromosome spreads were prepared using the method outlined by Pradeep et al. (2011 (link)). Chromomycin A3 (CMA3) and DAPI fluorochrome stainings followed the methodologies of Schmid (1980 (link)) and Schweizer (1980 (link)), respectively.

The fin clips and scales with a small piece of tissue from the scale pocket were seeded into cell culture dishes and plated in RPMI Medium 1640 (Life Technologies) supplemented with 20% fetal calf serum containing antibiotic and antimitotic solution (Sigma-Aldrich, USA). The primary cultures were incubated at 29 °C with 5% CO₂. After 1–2 weeks, the cells were incubated with 0.01% of colchicine for 5–6 hours. Chromosome spreads were prepared using the method outlined earlier89 . Images were acquired using Zeiss/MetaMorph epifluorescence microscope equipped with a (CCD) camera.