Axio imager z1 optical microscope

Two dyes were used in the study: 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide (PI). DAPI has good ability to dye any bacterial cells; however, PI can only dye dead cells because it can only pass through damaged membranes. Bacteria were cultured to the mid-logarithmic phase at 37 °C and diluted to 1 × 10⁶ CFU mL⁻¹, then, incubated with compound 17 for 2 h at 37 °C (no compound was added to the untreated controls), the cell pellets were collected after centrifugation at 3000 rpm for 15 min at 4 °C, then washed with PBS and incubated with DAPI (10 μg mL⁻¹) for 15 min on ice in the dark. Next, the bacteria were washed twice with PBS to remove excess dye. The procedure was repeated with PI (5 μg mL⁻¹). A Zeiss Axio Imager Z1 optical microscope with an oil-immersion objective was then used to observe the cells.

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)}