Biofilm Growth and Metabolic Activity

Biofilms were grown as previously described^{36 (link),37 (link)}, with some modifications. The adhesin diploid deletion library was grown overnight in 96-well plates, in YPD media at 30°C. Subsequently, each strain was subcultured into RPMI media (supplemented to 2% glucose), in 96-well plate format. Plates used for biofilm growth were made of polystyrene (Corning, Inc), polyvinyl chloride (PVC; Corning, Inc), or silicone (E&K Scientific). Biofilms were allowed to form for 24 hours, at 37°C. The biofilms were then washed twice with PBS to remove non-adherent cells, and resuspended in fresh RPMI media and incubated at 37°C for another 24 hours. Biofilms were again washed twice with PBS to remove non-adherent cells, and metabolic activity was measured using XTT. 90 μl of 1 mg/ml XTT and 10 μl of 320 μg/ml phenazine methosulfate was added to each well and allowed to incubate for 2 hours at 37°C. The supernatant was transferred to a clean 96-well plate, and absorbance was measured at 490 nm. For each assay, two versions of the diploid efflux pump library, including wild-type control strains, were screened in each of the different 96-well plates (polystyrene, PVC, or silicone). Each assay was performed in duplicate, yielding a total of four replicates per strain.