Persister cells are phenotypic variants of wild-type cells.
An overnight culture of C. albicans SC5314 (wild-type) was diluted to OD600 0.1 and seeded to a flat bottomed 96-well plate (CELLSTAR Greiner) containing RPMI-MOPS medium to allow biofilm formation. Biofilms were grown at 37°C for 24 hours, washed with 1× PBS and challenged with 100 μg/mL AmB dissolved in fresh RPMI-MOPS medium or left to mature in fresh RPMI-MOPS medium. After 24 hours, the medium was removed, and the remaining biofilm was again washed with 1× PBS and stained with 100 μg/mL fluorescein diacetate (Sigma Aldrich) and 500 μg/mL Texas Red conjugated to concavalin A (Molecular Probes) for 60 minutes. Fluorescein diacetate stains living cells green, while Texas Red conjugated to concavalin A was used to stain the fungal cell wall red. Pictures were taken using the scanning confocal microscope at a magnification of 600× (white scale bar represents 20 μm). (A) A C. albicans biofilm treated with AmB lacks green fluorescent cells (indicating that most of the cells are dead). However, a bright fluorescent cell is also present (indicated by the black arrow). This cell meets the definition of a persister cell. We were able to identify several persister cells in the treated biofilm but none of them were of the hyphal form. (B) A nontreated biofilm is thicker in appearance and therefore has a higher background of green fluorescence. Most of the cells in focus are green and thus appear to be viable (indicated by the black arrow). AmB, amphothericin B.
