Phagocytic activity of leucocytes
was determined according to Puangkaew et al.
(2004). Briefly, a 300-Al volume of the leucocyte
suspension in L-15 medium (Sigma-Aldrich) containing
2106 cells was seeded into a chamber slide (Lab-
Tek Nalge Nunc International, IL, USA) and incu-
244 A. Panigrahi et al. / Aquaculture 243 (2005) 241–254
bated at 15 8C for 2 h after which the non-adherent
cells were removed. They were further incubated for 1
h after adding opsonized fluorescent latex beads (2
Am; Sigma) to each chamber to maintain the cells to
beads ratio of 1:10. The latex beads had been
opsonized earlier by incubating 2107 beads/ml with
the serum of very same fish from which the cell
sample originated at 15 8C for 1 h. After washing and
fixation, the slides were stained with Diff Quick
solution (Kokusai Shiyaku Hyougo, Japan). The
phagocytic activity (PA) was expressed as the
percentage of phagocytic cells quantified from 300
adherent cells under microscope. The phagocytic
index (PI) was expressed as the average number of
particle beads ingested by each phagocytic cell.