The minimal bactericidal concentration (MBC) was determined by streaking 5-μL aliquots of the microtiter plate reaction mixtures used to determine the MIC onto a Mueller-Hinton (MHB) agar plate. The wells containing the three serial dilutions above and below the MIC were analyzed. The lowest concentration of peptide that ablated the bacterial colony growth on the agar plate was deemed the

CHIR99021 MBC. The in vitro antifungal activities of peptide solutions were determined by a quantitative micro-spectrophotometric assay [2]. Inhibition of growth was measured in 96-well microtiter plates at 595 nm. Routine tests were performed with 20 μL of a peptide test solution, 10 μL of a spore suspension (2 × 106 spores mL−1) and 70 μL of potato dextrose broth (PDB) (HiMedia, Mumbai, India). Microcultures containing 20 μL of sterile distilled water PCI-32765 purchase in place of test solutions were used as negative control. The commercial fungicide Captan (0.2 mg mL−1) [35] was used as the positive control. The plates were allowed to stand for 30 min at 27 °C to allow the spores to sediment, after which, the absorbance was measured at 595 nm in a Multiscan Spectrum microplate reader (Thermo Electron Corp., Varta, Finland). After a 48 h incubation at 27 °C, growth was recorded by

measuring absorbance. All assays for antifungal activity were performed, at a minimum, in triplicate. The growth inhibition percentage was determined based on the equation [(ΔC − ΔT)/ΔC] × 100, where ΔC was the corrected absorbance of the control microculture at 595 nm and ΔT was the corrected absorbance

of the test microculture. The corrected absorbance values equaled the absorbance at 595 nm of the culture measured after 48 h minus the absorbance at 595 nm measured after 30 min. A microplate method, as previously described [13], was used with slight modifications to determine the MIC of peptide test solutions. Briefly, 20 μL from a 500 μg mL−1 stock solution was G protein-coupled receptor kinase added to the first column of a microplate. Then, double serial dilutions were performed using distilled sterile water for the remaining columns. In each well, 20 μL of peptide dilutions were mixed with 180 μL of the fungal spore suspension (2 × 106 spores mL−1 in fresh PDB). The microplates were incubated for 48 h at 27 °C. All experiments were performed in triplicate. The MIC readings were measured as absorbance at 595 nm. MIC was defined as the lowest peptide concentration that inhibited 90% fungal growth. The in vitro minimal fungicidal concentration (MFC) was determined as described by Espinel-Ingroff et al. [12]. After a 48 h incubation, 20 μL from each well was subcultured onto PDA and incubated at 27 °C until growth was observed in the growth control subculture.