FIG 1.

Schematic of the caspofungin sensitivity screening assay. Steps and conditions: (a) frozen cells were transferred to YPD agar using a pronged replicator and incubated for 48 h at 25°C; (b) cells were transferred to 600 μL YPD in a deep-well plate using a multichannel pipettor and incubated for 48 h at 25°C; (c) 30 μL was transferred to 600 μL YPD in a deep-well plate and incubated for 48 h at 25°C; (d) cells were centrifuged, washed 2 times, and resuspended in 200 μL PBS; (e) cells were transferred to a clear, flat-bottom plate and optical densities measured at 650 nm; (f) cells were normalized to an OD₆₅₀ of 2 in a final volume of 120 μL PBS; (g) cells were diluted 1:20 in 200 μL YNB; (h) cells were diluted 1:10 in 100 μL YNB; (i) cells were diluted 1:10 in 100 μL YNB in 4 replicate plates; (j) caspofungin was added at 10 μg/mL to 3 replicates, PBS was added to the control plate, and the plates were incubated for 48 h at 25°C; (k) XTT reagent was added to all plates, the plates incubated for 3 h at 37°C and centrifuged, and 100 μL transferred to fresh plates; and (l) absorbance was read at OD₄₉₀. Plates incubated in the 2nd, 3rd, and 10th steps were covered with a Breathe-Easy membrane. Image created with BioRender.com.