Extracellular Protease and Hemolytic Activity Assays

The extracellular protease production was determined by the plate assay in Luria Bertani (Merck, Darmstadt, Germany) agar medium supplemented with 1% of skimmed milk (w/v). A bacterial suspension of 0.5 McFarland was prepared and subsequently inoculated (5 μL), then all plates were incubated at 37 °C for 24 h in a normal atmosphere. The presence of extracellular proteases was revealed by the formation of clear halos around the colonies which were measured. The halos were classified as negative (−) in the absence of a halo, as weak positive (+/−) in the presence of a halo less than 11 mm, as positive (+) in the presence of a halo less than 13 mm, and as strongly positive (++) in the presence of a halo less than 15 mm [38 (link)].
The hemolytic activity was determined by the plate assay using a Columbia Agar with 5% sheep blood (Merck, Darmstadt, Germany). A bacterial suspension of 0.5 McFarland was prepared, and subsequently, 5 μL was inoculated and plates incubated at 37 °C for 24 h in a normal atmosphere. The production of hemolysins was identified by the presence of clear (β-hemolysis) or diffuse (α-hemolysis) halos around the colonies. The absence of a halo shows that there was no production of hemolysins [39 (link)].