Visualization of biofilm formation by confocal laser scanning microscopy

Overnight cultures of S. aureus strains were diluted with TSB supplemented with 1% glucose at a ratio of 1:200, then inoculated in cell culture dishes (23 mm diameter) with glass bottoms (FluoroDish, WPI, Florida, USA) and incubated at 37°C for 24 h. After removal of non-adhered cells, the biofilms were washed with phosphate-buffered saline, stained with a Live/Dead BacLight Viability Kit (Molecular Probes, Eugene, Oregon, USA), and subsequently analyzed with a Leica confocal laser scanning microscope (TCS SP8; Leica, Heidelberg, Germany) detecting the fluorescence intensities of SYTO9 and propidium iodide (PI). A series of images were acquired at 1 μm intervals in the Z section to measure the biofilm thickness. IMARIS 9.0 software (Bitplane, Zurich, Switzerland) was used to generate three-dimensional view of the biofilms.

To evaluate the effects of the antibacterial agents on 24-h biofilms, three biofilm-forming MRSA strains (strain USA300, strain 234 and strain 15098) were selected and cultured as described above for 24 h. After removal of the suspension cultures, four-fold MIC concentrations of the agents in fresh media were added and incubated at 37°C for an additional 24 h. The biofilms were then stained with the Live/Dead BacLight Viability Kit (Viable cells in the biofilms exhibited green fluorescence, and dead cells exhibited red fluorescence). The effects of the agents on 24-h biofilms were analyzed by visualizing three-dimensional biofilm structures with CLSM and calculating the ratio of dead/live bacterial cells with ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).