2.4.2. Serum bactericidal activity
Bactericidal activity was determined following the method of Kampen et al. [17] with modifications for plasma. In each well of a round-bottom 96-well microtiter plate, 20 μL of sample plasma or Hank's balanced salt solution was added for positive control and incubated for 2.5 h with aliquots of a 24 h culture of A. hydrophila. To each well, 25 μL of 3-(4, 5 dimethyl-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT, 2.5 mg/mL) was added and incubated for 10 min to allow the formation of formazan. Plates were centrifuged at 2000 × gfor 10 min and the precipitate was dissolved in 200 μL of dimethyl sulfoxide. The absorbance of the dissolved formazan was recorded at 560 nm. Bactericidal activity was calculated as the decrease in the number of viable A. hydrophila cells by subtracting the absorbance of sample from that of control and expressed as absorbance units.