Antimicrobial Peptide Potency Assay

Bac8c (RIWVIWRR-OH), melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-OH) and its sub-fragment mel12-26 (bold type) were in L-isomer form and 95% pure by mass spectrometry (Mimotopes, Melbourne, VIC, Australia). Peptides were dissolved in molecular biology grade water and tested for their minimal lethal concentration (MLC) against S. aureus strains, using a variation of a previously described method (Dean et al., 2011 (link)). Briefly, 200-μl aliquots of bacterial culture (10⁶ CFU ml^-1 in 8 g l^-1 bactopeptone) were transferred to a polystyrene 96-well plate (Nunc, New York, USA) containing the test peptides at final concentrations from 1 to 170 μg ml^-1. The plates were incubated at 37°C with 200 rpm orbital shaking for 1 h, then bacteria from each well were plated on separate 1.5% agar plates, containing 25 g l^-1 bactopeptone medium, and incubated at 37°C for 24 h. Survival was measured by colony numbers, as a percentage of untreated controls. Negative controls contained mock-inoculated medium. The lowest dose that resulted in >99.9% cell death was recorded as the MLC. Experiments were repeated two or three times, each with triplicate samples.