Novapath

Candida albicans strains (1 × 10⁶ cells ml⁻¹) were added to each well of 96-well tissue culture microtitre plate with MIC, 10⁻¹× MIC and 10⁻²× MIC of antifungals and tigecycline. The positive controls without antimicrobial agent and negative controls without cells were also added. The plates were incubated for 24 and 48 h at 37 °C. After incubation, the wells were washed twice with PBS and measured in PBS at 450 nm on microplate reader (Bio-Rad Novapath). Inhibition of biofilm formation was determined with comparing results with positive controls (Dosler & Karaaslan, 2014 (link)). Each experiment was performed in four wells and was repeated two times.

Biofilm formation of fifteen clinical C. albicans strains were quantified by crystal violet assay described by others (Djordjevic, Wiedmann & McLandsborough, 2002 (link)). Briefly, after biofilm formation, each well was washed twice with 200 µl of PBS and air dried for 45 min. Then, each washed well was stained with 110 µl of 0.4% aqueous crystal violet solution for 45 min. Afterwards, each well was washed four times with 350 µl of sterile distilled water and immediately distained with 200 µl of 95% ethanol. After 45 min of distaining, 100 µl of distaining solution was transferred to a new well and the amount of the crystal violet stain in the distaining solution was measured with a microtiter plate reader (BioRad Novapath) at 595 nm. The absorbance values of the negative controls (containing no cells) were subtracted from the values of the test wells to minimize background interference. Each strain was tested six times and biofilm production quantities were reported as the arithmetic mean of absorbance values of the six replicate tests.

Activities of antimicrobial agents on mature C. albicans biofilms, were studied using the standardized static microtitre plate model and measured by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[8phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT; Sigma-Aldrich, St. Louis, MO, USA) reduction assay which is the most commonly test used to estimate viable biofilm growth (Pierce et al., 2010 (link)). Doubling concentrations of antimicrobials were added to the pre-formed 48 h mature biofilms, as described above. Drug-free biofilm wells containing only RPMI 1,640 were used as controls. Biofilms were incubated at 37 °C for 48 h. After incubation, the medium was aspirated and washed with PBS, three times. XTT was prepared as previously published and added to each well. Microtitre plates were incubated in the dark for 3 h at 37 °C. Biofilm growth was measured spectrophotometrically at optical density 450 nm, on microplate reader (BioRad Novapath). SMICs were determined as the minimum antifungal drug concentration that caused 50% reduction of biofilm compared to drug-free untreated biofilm controls. Each experiment was performed in four wells and was repeated two times.