2.7. Phagocytic activity
The phagocytic activity was determined following the in vivo
method described by Dias [23] and Silva [24] with slight modi-
fications. The fish were anesthetized, and 3.0 mL of a Saccharomyces
cerevisiae (Baker's yeast, Type II, Sigma, USA) yeast
solution with 8000 cells mm3 was injected into the coelomic
cavity. After 4 h of incubation (time required for the migration of
phagocytic cells to the coelomic cavity), the animals were killed
by a high dosage of anesthetic and spinal cord dissection; the
coelomic cavity of each fish was washed with 3.0 mL of PBS. The
liquid was collected using a Pasteur pipette and centrifuged at
251.5 g for 5 min, and the supernatant was discarded. Aliquots
of the sediments were examined between a slide and coverslip
under a contrast phase microscope (400) with a green filter,
and 100 phagocytes were counted to determine the phagocytic
capacity (PC) and phagocytic index (PI): PC ¼ (number of
phagocytes that phagocyted yeast/number of phagocytes
observed) and IP ¼ (number of yeasts ingested/number of
phagocytes observed).