Fv1000 confocal inverted microscope

For microscopy analysis, narrow glass coverslips (10.5 × 35 mm, ProSciTech, Townsville, Australia) were inserted in the biofilm culture tubes and P. aeruginosa biofilms were grown otherwise as described above. After 24 h incubation and 20 min treatment, the coverslips harbouring biofilms were carefully removed from the tubes by using sterile tweezers and rinsed twice with PBS before wiping clean one side and staining the other side with LIVE/DEAD BacLight bacterial viability kit reagents (Molecular Probes) according to the manufacturer’s procedure. Three hundred microliters of the staining solution (1:1,000 dilution of each SYTO9 and propidium iodide components in PBS) was trapped between the biofilm sample on the coverslip and a standard microscope slide. After 20 min incubation at room temperature in the dark, the samples were observed with an Olympus FV1000 Confocal Inverted Microscope, and imaged with Leica DFC 480 camera. Cells that were stained green were considered to be viable, those that stained red were considered to be dead.

For microscopy analysis, P. aeruginosa biofilms were grown in glass-bottom, 24-well plates (MatTek Corporation, Ashland MA, USA) as described above. After 7 h incubation including 1 h treatment, biofilms were rinsed twice with PBS before being stained with LIVE/DEAD® BacLight™ bacterial viability kit reagents (L-7007, Molecular Probes) according to the manufacturer's procedure. One microliter of each of the two components were mixed thoroughly in 1 mL of PBS, then 0.3 mL of this solution was trapped between the sample and the glass microscopy slide and allowed to incubate at room temperature in the dark for 20 min. The samples were observed with an Olympus FV1000 Confocal Inverted Microscope, and imaged with a Leica DFC 480 camera. Cells that were stained green were considered to be viable, those that stained red and stained both green and red were considered to be non-viable.