0.4 mm menadione

As described earlier, the biofilm formation in untreated (control) and Ag-Cu bimetallic NPs treated C. albicans cells was estimated [37 (link)]. In brief: freshly grown yeast cells were inoculated (1 × 10⁶ cells/mL) in 96-well plates and incubated for 2 h at 37 °C without shaking. Post-incubation period, the media was removed, and wells were two times washed with fresh PBS followed by addition of sterile growth medium containing desired concentrations of NPs (¼ × MIC and ½ × MIC) and incubation at 37 °C for 24 h and 48 h. The outcome of NPs on C. albicans biofilm formation was estimated by semi-quantitative 2,3-Bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) reduction assay. For this assay, 1 mg/mL XTT and 0.4 mM menadione (SigmaAldrich, St. Louis, MO, USA) were prepared in fresh PBS and acetone, respectively. Afterward, a freshly prepared solution of XTT:menadione (20:1) was prepared and added to PBS washed pre-formed biofilms and kept in the dark for 3 h at 37 °C. Next, the wells were aspirated, and the colorimetric changes were recorded at 490 nm using SpectraMax iD3 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The XTT reduction was directly proportional to the metabolic activity of cells embedded in C. albicans biofilms.