Cell Membrane Integrity

To evaluate the integrity of cell membranes, we used the BacLight Live/Dead bacterial viability kit (L-7012; Molecular Probes). Briefly, we added approximately 2 × 10⁷ CFU/ml to tubes containing 10 ml of nutrient broth. Untreated and treated samples having BC (IC₅₀: 1.5 mg/L and MIC: 3 mg/L) were grown for 24 h at 37°C and then were centrifuged at 10,000 × g for 10 min. The supernatant was removed, and the pellet was resuspended in 2 ml NaCl (0.85%). We added 1 ml of the sample to 20 ml NaCl (0.85%), incubated the suspension for 1 h at room temperature, and then centrifuged the mixture at 10,000 × g for 10 min (repeated twice). We removed the supernatant and resuspended the pellet in 10 ml NaCl. We added 3 μL of Syto9 and 3 μL of propidium iodide (PI) to each sample. Samples were then incubated at room temperature for 15 min. We analyzed the integrity of the bacterial membranes using a fluorescence microscope (Reichert) equipped with a halogen lamp, Neoplan 100 × /1.25 oil objective and a 1,713 filter cube (fluorescein; 490/510/520 nm) at 1,000× magnification. In the assays, the fluorescent green nucleic acid stain Syto9 passes in living and dead bacterial cells, whereas PI cannot penetrate intact membranes. When the cellular membrane is damaged, PI can penetrate bacteria and cause the cells to appear red (31).