FIG. 5.

Isolation of persister cells from a biofilm. C. albicans MC191 was grown as a biofilm for 48 h in RPMI 1640 medium in microtiter plate wells. A homogenous population of cells from disrupted biofilms was obtained by applying a forward scatter and a side scatter gate, as shown in panel A, for all subsequent analyses. (B) A biofilm was stained with 100 μg/ml fluorescein diacetate for 24 h, disrupted, washed three times with PBS, and analyzed with a MoFlo cell sorter. Single events representing individual cells were physically sorted directly on YPD medium and incubated for 48 h (D). (C) A biofilm was treated with 100 μg/ml amphotericin B, stained with fluorescein diacetate, and similarly analyzed with the cell sorter. Two distinct populations were separated based on green fluorescence intensity, as shown. (E) Particles representing 96 events from the dim population, R4, were sorted onto YPD agar and incubated for 48 h. (F) Particles representing over 6,000 events from R2 were sorted onto YPD agar and incubated for 48 h.