Biofilm imaging and viability assay.

Biofilm was imaged using both an inverted light microscope (Evos Cell Imaging System; Thermo Fisher) and confocal laser scanning microscope. Light microscopy was done to visualize structure and formation differences of 3- and 5-day-old biofilms pre- and postexposure to disinfectants. Cells in the biofilms were also visualized using a confocal laser scanning microscope according to the protocol of Jurcisek et al. (34). Briefly, bacterial biofilms from all stages were formed on a four-well chamber slide. Following incubation, treated or untreated biofilms were washed, stained with BacLight live/dead stain, and fixed with neutral buffered formalin (8, 10, 34). A live/dead viability kit contained Syto9, and propidium iodide stain was added to visualize in vitro killing effects. Slides were fixed and washed with PBS, and plastic wells were removed from the slide. Saline was added to plastic wells and covered with coverslips. The edges of the coverslip were sealed with mounting medium and air dried for 1 h before microscopy. Samples were imaged under oil immersion using a laser confocal microscope (34). The excitation and emission wavelengths for Syto9 were 488 nm and 505 to 550 nm, respectively, and 543 and >650 nm, respectively, for propidium iodide (34). All assays were performed in duplicate.