Enzymatic activity plate assay—Determination of hydrolases

After 5 days of culture, pellets with 6.5 mm in diameter of each fungus was inoculated in an enzymatic media, which contained as an inductor of enzyme activity “Tween 20” (T₂₀); “Tween 80” (T₈₀) [9,10]; or “W”, as called by the author [11]; or “K” [12]. This modified medium (K) is composed by: 1.0 g.L^-1 Yeast extract, 1.0 g.L^-1 KH₂PO₄, 0.5 g.L^-1MgSO₄.7H₂O, 1.0 g.L^-1 (NH₄)₂SO₄ and 20.0 g.L^-1 bacteriological agar.

All the media was sterilized at 121°C for 15 minutes and the medium “K” was cooled to 60 °C and then the following compounds were added: 10.0 g.L^-1 of egg lecithin, CaCI₂ 100.0 mM, and 2.0 mL of aqueous solution 0.1% Rhodamine B (previously sterilized by filtration through 0.22 μm syringe filter). Each media was emulsified with intense shaking by 10 minutes and then 20.0 mL distributed in each 90 mm diameter Petri plates.

The fungi were inoculated and maintained in a bacteriological incubator for 10 days at 27 °C. The enzymatic activity (S1 Fig) of fungi was observed through the formation of precipitate crystals of calcium salts or the appearance of clear different color halos around the colonies, using stereomicroscope and microscope. Daily growths were observed and measured the size of the colony and the activity halo. The bacterial strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were used as a positive control in the detection of lipase’s presence test.