Modulation of Candida albicans Biofilm

Biofilm of C. albicans was developed by growing diluted overnight culture in YPD broth (OD_600nm=0.5) in a 24-well polystyrene plate for 24 hr in a static condition at 30 °C as described before (Bose et al., 2023 (link)). After, 24 hr of biofilm formation, the biofilms were either kept untreated or treated with 250 µM EDTA or mentioned concentrations of other metal chelators (DTPA, Aprotinin, TPEN, and CE), 8 µM MgSO₄, 8 µM ZnCl₂, 8 µM FeCl₂, 8 µM MnCl₂, 250 µM EDTA +8 µM MgSO₄, 250 µM EDTA +8 µM ZnCl₂, 250 µM EDTA +8 µM FeCl₂, and 250 µM EDTA +8 µM MnCl₂; and again, incubated for another 24 hr. After a total 48 hr of incubation, the supernatant was gently removed without disturbing the biofilm and the wells were washed with 1 X PBS twice. The biofilm so obtained was treated with 0.1% crystal violet and left for 20 min to stain at 25 °C. The plate was allowed to air dry and the bound dye was then re-suspended in 33% glacial acetic acid, and absorbance was recorded at 570 nm using a Perkin-Elmer plate reader. The experiments were repeated twice with biological triplicates. Similarly, biofilms were formed and treated in 8 well-chambered slides, images were obtained after staining with 1% acridine orange using Leica TCS SP8 confocal scanning system with where excitation at 483 nm and emission with 500–510 nm band-pass filter were used.