Proteolysis was assayed against gelatin on agar plates, using a
modified method (Vermelho et al., 1996). Briefly, a loopful of the
culture to be evaluated was inoculated on agar plates with the
detection medium (2% (w/v) sucrose (Merck), 0.5% (w/v) yeast
extract (Britania), 2% (w/v) peptone (Britania), 1.5% (w/v) agar (Britania)
autoclaved and supplemented with 1% (w/v) gelatin) and
incubated at 37 C for 48 h. Extracellular protease detectionwas done
after staining agar plateswith Coomassie blue (0.25%, w/v) for 1 h in
methanolacetic acid-water 5:1:4 (v/v/v) and destaining with methanolacetic
acid-water. Regions of enzyme activity were detected as
clear areas, indicating that hydrolysis of the substrates had occurred.