Visualizing BCA's Effect on Candida albicans Cell Wall

The aniline blue and calcofluor white (disodium salt of 4,4′-bis-[4anilino-bis-diethyl-amino-S-tri-azin-2-ylamino]-2,2′stilbene-disulfonic acid) staining methods were used to visualize the effect of BCA on the cell wall of C. albicans. Aniline blue has the ability to bind to (1,3)-β-D-glucans, whereas calcofluor white binds to chitin in the C. albicans cell walls. The yeast cells at the exponential phase were harvested by centrifugation at 4500× g at 4 °C for 5 min. Next, the cells were washed twice and resuspended in PBS. BCA at concentrations of 125 µg/mL and 250 µg/mL and 1% DMSO as a control were added to the cell suspensions and incubated at 37 °C for 4 h. The cells were harvested and washed in PBS. Next, the cell density in each experimental group was adjusted to 1 × 10⁸ cells/mL and the cells were resuspended in an aniline blue solution (0.1%) or calcofluor white (0.1%). The samples were stained for 30 min, washed with PBS, and fluorescence was measured in a black 96-well microplate using a spectrofluorometer at 370 nm excitation and 509 nm emission wavelengths for aniline blue and at 370 nm excitation and 440 nm emission wavelengths for calcofluor white [62 (link),81 (link),82 (link),83 (link)].