Materials and methods

Cement disks (diameter = 6 mm) impregnated with gentamicin and colistin were submerged in bacterial suspensions of Methicillin-resistant Staphylococcus Aureus(MRSA), Staphylococcus epidermidis (SE), and PA. Negative controls (specimen disks without antibiotic) were similarly prepared. Biofilm formation was visualized by confocal scanning laser microscopy (CSLM), after staining the discs with the live/dead BacLight viability stain containing SYTO 9 dye and propidium iodide. Images from five randomly selected areas were acquired for each disc. Sequential optical sections of 2 µm were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analyzed, quantified and rendered into three-dimensional (3D). The biofilm thickness on antibiotic bone cement compared with the controls was automatically evaluated.