Live dead baclight bacterial viability kits l7012

Cell viability was determined by LIVE/DEAD BacLight Bacterial Viability Kits L7012 (Molecular Probes, Invitrogen) according to the manufacturer’s instruction. Briefly, Xoo cells were grown to an OD₆₀₀ of 1.0 in NYG medium. Cells were collected by centrifugation at 10,000 rpm for 10 min and washed three times with fresh MMX medium. The bacteria were then inoculated into fresh MMX plus 100 μM FeCl₃ (control), Xinjunan (1/4 MIC), Xinjunan (1/4 MIC) plus 100 μM FeCl₃, Xinjunan plus 100 μM FeCl₃ and 150 μM DIPY for 30 min and 4 h. Then, the cells were collected by centrifugation at 5,000 rpm at 4°C for 10 min and the pellets were washed and resuspended in 10 mL of PBS buffer.
Next, 3 μL of the dye mixture (combined equal volumes of SYTO9 and PI) was added to each milliliter of the bacteria suspension and incubated at RT in dark for 15 min. A total of 5 μL of the stained bacterial suspension were put onto a glass slide and covered with a coverslip. Imaged cells with filters listed below: Ex/Em 480/550 for SYTO9, Ex/Em 490/635 for PI.

The percentage of live cells were determined using the LIVE/DEAD BacLight Bacterial Viability Kits L7012 (Molecular Probes, Inc., Eugene, OR, United States). The samples were harvested at the same turbidity at 600 nm after 3 to 5 h. The fluorescence emission spectrum (excitation 470 nm, emission 490 to 700 nm) of each cell suspension was measured using an Infinite M200 Pro plate reader (Tecan, Switzerland). The ratio was calculated of the integrated intensity of the portion of each spectrum from 510 to 540 nm (green, live cells) to that from 620 to 650 nm (red, dead cells) for each bacterial suspension. Isopropanol-treated cells (30 min) were used as the dead cell negative control.