A challenge test was performed by immersing the surviving larvae
from the growth experiment in a Streptococcus agalac
the Ministry of Fisheries and Marine Affairs, Indonesia. About 10 μL of
stock culture was inoculated into Brain Heart Infusion broth (Criterion
C5141, Hardy Diagnostics, California, USA) and incubated for 24 h at
30 °C under constant agitation (140 rpm). After culture, the cells were
washed twice in sterile saline. Twelve units of 2 L plastic tanks were
filled with 1.5 L of chlorinated and aerated freshwater and added with
S. agalactiae culture at a density of 107 CFU/mL. From three out of four
growth test tanks per treatment, ten healthy fish were selected and
transferred to an immersion tank.