Under aseptic conditions in the laminar flow safety cabinet, each disc was sterilized with 95% ethanol on each side then subjected to 1 h ultraviolet illumination. The discs were placed in 12-well plates according the 5 groups with 1 mL of artificial saliva and incubated for 2 h. The overnight culture of C. albicans was adjusted to 0.5 McFarland, and 1 mL of yeast suspension was added to each well with different concentration of caffeine, except for the control samples. The plates were incubated at 37 ± 1 °C for two days to allow the biofilm formation phase of C. albicans. Nonadherent cells were removed by washing the discs twice with PBS and placed in new, sterile, 12-well plastic plates. Finally, the biofilms were scraped and vortexed for 2 min at 3000 rpm speed to dislodge the adherent cells from the denture [25 (link),26 (link)]. The adherent fungal cells were evaluated by microplate reader (Bio-Rad xMark TM Microplate Spectrophotometer, Hercules, CA, USA) at 620 and 410 nm.
Free full text: Click here
