Live-Dead Bacterial Cell Staining

The live–dead staining of bacterial cells was performed according to the protocol described by Manoharadas et al., 2021 [31 (link)]. Briefly, the staining for viable cells was performed by the addition of 5 μM of SYTO9 (ThermoFischer Scientific, Bedford, MA, USA), diluted in DMSO. The staining was performed for 10 m in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable cells, propidium iodide (ThermoFischer Scientific, Bedford, MA, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the cells. The staining was performed for 10 m, followed by washing with 1× PBS and final rinsing with double distilled water. The image was captured by confocal microscope (Zeiss, Jena, Germany) at an excitation/emission of 483/503 nm for SYTO9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Jena, Germany). The acquired image was processed by the Zen lite software (Zeiss, Jena, Germany).