Activities of citral and thymol against planktonic cells were tested by broth microdilution method using Clinical and Laboratory Standards Institute (CLSI) document M27, guidelines [16
]. Planktonic cells were grown in SDB for 24 h at 30 °C with 200 rpm. The harvested cells were washed using sterile phosphate buffered saline (1 × PBS, 0.1 M, pH 7.4) and resuspended at 2 × 103
cells/mL concentration in the RPMI-1640 medium. Serially double diluted concentrations from 0 µg/mL to 1024 µg/mL of citral and thymol were prepared in RPMI-1640, respectively and the cell suspension of 100 µL was added to each well of 96-well microtiter plates so that the final working volume contains 1 × 103
cells/mL with the final DMSO concentration not exceeding more than 5% in any assay. Pre-sterile 96-well polystyrene microtiter plates (Tarsons, West Bengal, India) were set with 100 µL of each dilution as treatment and control well contained 5% DMSO dispensed in RPMI-1640. The plates were then incubated at 37 °C for 48 h and growth was measured using 96-well plate reader (SpectraMax, Molecular Devices, CA, USA), in terms of optical density (OD) at 600 nm [17
]. The biofilm formation assay was performed in 96-well polystyrene microtiter plates as described earlier [18
]. Briefly, the cells at a concentration of 2 × 106
cells/mL were suspended in a 100 µL volume in each well in RPMI-1640. Serial double dilutions of citral and thymol were made in RPMI-1640 and added to each well as treatment while acquiring a final concentration of cells as 1 × 106
cells/mL and concentrations of citral and thymol ranging from 0 µg/mL to 1024 µg/mL for each. Control wells were prepared with 5% DMSO in RPMI-1640. Plates were incubated at 37 °C for 48 h. For preformed biofilms, the cells were suspended at a concentration of 1 × 106
cells/mL, taking 100 µL of suspension in each well of 96-well polystyrene plates. The plates were then, incubated at 37 °C for 24 h. After incubation, non-adherent cells were removed through aspiration of media and the wells were washed thrice using 1 × PBS. Residual PBS was removed by inverting the plates over blotting sheets. A volume of 100 µL of each serially double diluted concentration (0 µg/mL to 1024 µg/mL) of citral and thymol, not exceeding DMSO by 5%, was added to each well with treatment, whereas, control wells were made using 5% DMSO in RPMI-1640. The plates were incubated at 37 °C for another 24 h. The metabolic activity of biofilm was quantitatively determined by colourimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide sodium salt] reduction assay.
After appropriate incubations of microtiter plates, media was aspirated and biofilm was washed using 1 × PBS. Colourimetric XTT reduction assay was performed as reported earlier [19
]. A stock solution of 5 g/L XTT tetrazolium salt (Sigma-Aldrich, MO, USA) was mixed in 10 mL of PBS to get 0.5 g/L working solution. The 1 µM final concentration of menadione (Sigma-Aldrich, MO, USA) was added to XTT. The working XTT-menadione solution of 100 µL was dispensed into the wells containing prewashed biofilm and to the control wells (for background subtraction). All the plates were incubated at 37 °C in the dark for 1 h and absorbance were read at 492 nm. Colour change due to formazan formation was directly correlated to the metabolic activity. Percentage killing of each component was calculated using the formula [1 − (OD492
sample / OD492
control)] × 100%.
(minimum inhibitory concentration of planktonic cells, biofilm inhibitory concentration and biofilm eradicating concentration, respectively at which the cell growth was reduced to 50% to that of the healthy cells) for C. tropicalis
were determined using microdilution method (as described above) in the treated/untreated wells from 0 µg/mL to 1024 µg/mL supplemented with 400 µg/mL of ergosterol and 0.8 M sorbitol as an osmotic supporter, obtained from Sigma-Aldrich, MO USA to study the ergosterol binding and sorbitol effect, respectively [20
]. Amphotericin B (1 µg/mL, 4 µg/mL and 8 µg/mL) was taken as the control drug for the ergosterol assay.