Amido black

C. albicans isolates’ aspartyl protease activities were evaluated by a previously described method [27 (link), 29 (link)]. Aliquots (5 μL) of standardized fungal inoculum of the previously mentioned antifungal-treated and control suspensions were spot inoculated aseptically onto 1% w/v bovine serum albumin (Levochem, N.Y., U.S.A.) agar plates, which were left to dry out and then incubated at 37 °C for 5 days. Further proteolytic activity was stopped using 20% w/v trichloroacetic acid (S D Fine-Chem Limited, Mumbai, India) and plates were stained with 1.25% w/v amidoblack (Hi- Media, Mumbai, India) and checked for the presence of proteolysis zone around the colonies, which was not stained with amidoblack. The protease index (Pr_z) was measured in terms of the ratio between the diameters of unstained zone and the colony [23 (link)]. Each assay was conducted in quadruplicate on two individual experiments. C. albicans ATCC 10231 was the positive control strain for the experiment. Results for this test were expressed as the percentage reduction in aspartyl protease production applying the following formula: $$\text{Aspartyl protease reduction}\%=1‐\left[\left(\mathrm{Prz}\text{assay}/\mathrm{Prz}\text{control}\right)\right]\times 100$$

Candida isolates’ aspartyl protease activities were evaluated as previously reported [31 (link)]. The degradation of bovine serum albumin (BSA) was measured. Ten microliters of test isolate containing 10⁶ CFU/mL were inoculated onto 1% w/v BSA (Levochem, New York, NY, USA) agar plate. The incubation conditions were maintained at 37 °C for five days. Further protease activity was suppressed by adding 20% w/v trichloroacetic acid after incubation (S D Fine-Chem Limited, Mumbai, India), and the plate was stained with 1.25% w/v amido black (Hi-Media, Mumbai, India). Proteolytic activity was demonstrated by a zone of proteolysis encircling the colony that could not be stained with amido black. The protease index (Prz) was calculated by dividing the colony diameter by the diameter of the unstained zone. A Prz value of one indicates no protease activity, while a Prz value lower than one indicates that the tested isolate expresses aspartic protease. The higher the aspartic protease activity, the lower the Prz value [32 ]. The assay was repeated three times for each isolate. As a positive control, C. albicans ATCC 10231 was used.