Candida suspensions were spectrophotometrically standardised to a concentration of 1 × 106 cells/mL. The resulting suspensions were used for all the further procedures. Aliquots of 100 μL of Candida standardised suspension were individually transferred to separate wells of a 96-well microtitre plate. After inoculation, an equal volume of diluted Cur solutions (100 μL) was added to the appropriate wells to give final concentrations of 5, 10 and 20 μM.
After dark incubation of 1, 5, 10 and 20 min, the samples were irradiated on the LED device for 4 min, which corresponded to 5.28 J/cm2 (P+L+). 41 To determine whether LED light alone had any effect on cell viability, additional samples were made with no PS (P−L+). The effect of Cur alone was also determined by exposing the yeast suspensions to the PS in an identical manner to those described above, but with no Protease Inhibitor Library concentration light exposure (P+L−). The suspensions that were not exposed to LED light or Cur acted as overall control (P−L−). All experiments were performed five times on two independent occasions. The microtitre plate containing the no-light samples was kept in the dark for 24 min, corresponding to the pre-irradiation time plus light exposure time.
Ten-fold serial dilutions (10−1, 10−2 and 10−3) were generated from the fungal click here suspensions and plated on SDA in duplicate. The plates were then aerobically incubated at 37 °C for 48 h. After incubation, yeast colony counts of each plate were quantified and the colony forming unit per millilitre (CFU mL−1)
was determined. A loopful of recently cultivated yeast was subcultured in RPMI 1640 overnight in an orbital shaker (AP 56, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil) at 120 rpm and 37 °C. The cells grown were harvested by centrifugation at 4000 rpm for 7 min, and the supernatants were discarded. The pellet was washed twice in PBS, and finally resuspended in PBS. Candida suspensions were spectrophotometrically standardised to a concentration of 1 × 106 cells/mL. Aliquots of 100 μL of the resulting aminophylline standardised Candida cell suspensions were transferred to appropriate wells of a 96-well microtitre plate and incubated at 37 °C in an orbital shaker (75 rpm). After 90 min of the adhesion phase, the supernatants were removed from the plate wells and gently washed twice with 150 μL of PBS to remove the non-adherent cells. Next, 150 μL of freshly prepared RPMI 1640 were added to each well and the plates were incubated in an orbital shaker for 48 h at 37 °C in order to generate single-species biofilms. After incubation, the wells were carefully washed twice with PBS to remove non-adherent cells. Aliquots of 150 μL of Cur at 20, 30 and 40 μM were added to each appropriate well directly onto the biofilm. The experimental conditions were identical to those of the planktonic cultures: P+L+, P−L+, P+L− and P−L−. All experiments were performed five times on three independent occasions.