Skim milk powder suspension

Production of the virulence factors caseinase, gelatinase, lipase, β-haemolysin and phospholipase were determined as described by Natrah et al. (2011) (link). For the lipase and phospholipase assays, marine agar 2216 (Difco, NJ, United States) (MA) plates were supplemented with 1% Tween 80 (Sigma-Aldrich, St. Louis, MO, United States) or 1% egg yolk emulsion (Oxoid, Hants, United Kingdom), respectively. The development of opalescent zones around the colonies after 2 days of incubation at 20°C was considered a positive result. The caseinase assay plate was prepared by mixing double strength MA with a 4% skim milk powder suspension (Oxoid, Hants, United Kingdom), and sterilized separately at 121°C for 5 min. Clearing zones around the bacterial colonies grown after 2 days of incubation at 25°C were considered a positive result. Gelatinase assay plates were prepared by mixing 0.5% gelatine (Sigma-Aldrich, St. Louis, MO, United States) into MA. After incubation for 4 days, saturated ammonium sulfate (80%) in distilled water was poured over the plates and after 2 min, clearing zones around the colonies were considered a positive result. β-haemolytic activity was determined using Columbia Blood agar (Oxoid, Hants, United Kingdom), and clearing of the agar around the colony after 2 days of incubation at 25°C was considered a positive result. All assays were performed in triplicate.

The caseinase and hemolysin assays were performed as described previously with some modifications (Zhang and Austin, 2000 (link); Austin et al., 2005 (link)). The caseinase assay plates were prepared by mixing double strength Marine Agar with a 4% skim milk powder suspension (Oxoid, Hampshire, United Kingdom), each sterilized separately at 121°C for 5 min. Clearing zones surrounding the bacterial colonies were measured after 2 days of incubation. Hemolytic assay plates were prepared by supplementing Marine Agar with 5% defibrinated sheep blood (Oxoid, Hampshire, United Kingdom) and clearing zones were measured after 2 days of incubation.

Protease and hemolytic activity assays were performed according to Natrah et al. (50 (link)) with some modifications. The protease assay plates were prepared by mixing double strength LB₃₅ agar (30g/L) with a 4% skim milk powder suspension (Oxoid, Basingstoke, Hampshire, UK) sterilized separately at 121°C for 5 min. Hemolytic assay plates were prepared by supplementing LB₃₅ agar with 5% defibrinated sheep blood (Oxoid). The selected indole analogues were added into agar before it was poured into petri plates at concentrations of 10 and 100 μM, respectively. All agar plates were opened at room temperature for 15 min to dry. Then the bacterial suspension was applied to the surface of an agar plate (at least 3 replicates per treatment). The plates were incubated upright at 28°C, and the clearing zone diameter and colony diameter were measured after 4 days of incubation.