Bacterial Viability Assay using DAPI and PI

DAPI (4′, 6-Diamidino-2-phenylindole dihydrochloride) and PI (Propidium iodide) were used in the assay.^{29,29b (link)} DAPI is the dye that stains all dead and living bacteria, whereas the PI dye only stains the dead bacteria with damaged membranes as itself is not cell permeable and it has to interact with the nucleic acids of the bacteria and fluoresce in bright red color. Briefly, the bacteria were allowed to grow to the mid logarithmic phase and then incubated with the polymer P6 (10 μg/mL) at 37 °C for 3 h. The solution was centrifuged at 10,000 g for 10 min in an Eppendorf tube. The supernatant was removed and the bacterial pellets were washed with PBS three to four times. PI (5 μg/mL) was added and incubated for 15 min in dark at 0 °C. The excess of the dye was removed by PBS washes (×3). Next, the cells were incubated with DAPI (10 μg/mL in water) for 15 min in dark at 0 °C and excess of the dye was removed, followed by PBS washes (×3). The bacteria were then examined under oil-immersion objective (100×) by using the Zeiss Axio Imager Z1optical microscope.^{30 (link)}