Microbroth dilution assay

The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined as previously described [12,30] following standard methods recommended by the National Committee of Laboratory Safety and Standards for MIC₉₀ and MBC determinations of clinical isolates with some modifications. Overnight bacterial cultures were diluted to 80–88% transmittance in their appropriate medium. The cultures were then diluted again to 1:200 and 100 μL was pipetted into each of 3 wells in 11 columns of a 96-well plate (Corning, Inc., Tewksbury, MA), the 12^th column loaded with media without bacteria as a sterility control and blank.

The synthetic piscidin peptides (piscidins 1, 4, and 7 from striped bass; piscidins 1, 5, and 6 from white bass; and piscidin 3 corresponding to both striped bass and white bass) were prepared by diluting the 4 mg/mL stocks to 2 mg/mL in 0.2% bovine serum albumin (BSA) + 0.01% acetic acid. These were then serially diluted to a concentration of 3.9 μg/mL in the same diluent. The piscidin dilutions were added to the first 10 columns of the 96-well plate containing bacteria, column 10 was loaded with diluent only to act as a positive growth control. The final concentrations tested were 200 μg/mL to 0.39 μg/mL. The plates were incubated with shaking at 200 rpm for 18 h, except for S. iniae, which was incubated for 48 h. Fish pathogens and human pathogens were incubated at 25°C and 37°C, respectively. Once visible growth was observed in the controls, the plates were read at OD₆₃₀ on a BioTek EL800 plate reader (BioTek, Winooski, VT). The MIC was taken as the lowest concentration of piscidin that reduced growth when compared to the growth control by more than 50%. To determine the MBC, 20 μL of the media from the wells of the three lowest piscidin concentrations that had no visible growth of bacteria were plated onto the appropriate media plates and incubated as described above. Colonies were counted to determine whether growth was equal to or less than 0.1% of the colony forming units (CFUs) in the original inoculum. The MBC was defined as the lowest concentration of piscidin that killed at least 99.9% of the CFUs in the original inoculum.