2.13. Antifungal Susceptibilty Testing of Biofilms

Biofilm forming ability in fungi was evaluated with the CV assay as previously described by O’Toole [37]; biofilm development was considered if the OD value at 540 nm was higher than 0.15. Candida isolates were suspended in RPMI-1640 broth in concentrations of 1 × 10⁶ cells/mL, and aliquots of 100 µL were inoculated onto flat-bottom 96-well sterile microtitre plates (TPP, Trasadingen, Switzerland) and then incubated statically at 37 °C for 24 h to produce one-day-old biofilms [38,39]. The examined EO concentrations for sessile MIC determination ranged from 0.045 to 12.5 v/v%. The biofilms were washed three times with sterile physiological saline. Afterward, MIC determination was performed in RPMI-1640 using XTT-assay. The percentage change in metabolic activity was calculated based on absorbance (A) at 492 nm as 100% × (A_well − A_background)/(A_{drug-free well} − A_background). MICs of biofilms were defined as the lowest drug concentration resulting in at least 50% metabolic activity reduction compared to control wells [38,39].