Inhibition of Candida Biofilm Formation by P. nigrum Extract

The effect of P. nigrum ethanolic extract on the biofilm formation of C. albicans strains was tested in 96-well microplates [57 (link),58 (link)]. One hundred µL of Candida cell suspensions (1 × 10⁶ cells/mL) in RPMI-1640 with MOPS were dispensed in 96 microdilution wells with or without P. nigrum extract (8 to 2048 μg/mL). The plates were then incubated at 37 °C and allowed to adhere for 1 h. The non-adherent cells were removed, and 200 μL of fresh RPMI was added. The plates were incubated further at 37 °C for 24 h. After incubation, biofilms were washed twice with PBS, and finally, 200 µL of RPMI-1640 plus 10 µL of 700 µM resazurin (Sigma–Aldrich) was added to each well and incubated at 37 °C for 2 h. The biofilm was quantified indirectly by measuring the fluorescent water-soluble resorufin product that results when resazurin is reduced by reactions associated with respiration. Fluorescence was measured at 560 nm with emission at 590 nm in an automated plate reader (Model 550 Microplate Reader Bio-Rad, Milan, Italy). Caspofungin (0.03 to 1 μg/mL) was used as a standard antifungal drug.