Bacterial Membrane Integrity Evaluation

Membrane integrity was assessed using the fluorescent probes SYTO 9 and propidium iodide (PI) (LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, USA). The test strains were grown to the mid-log phase. The obtained cultures were centrifuged at 10,000 rpm for 10 min, the supernatant was discarded and the pellet was dispersed in 10 mM HEPES buffer supplemented with 1.25 mM Ca ²⁺ to an optical density of 0.2 (OD ₆₀₀) that corresponded to 10⁸ CFU/ml. Bacterial cells in 50 μl were transferred to a 96-well microtiter plate (Eppendorf, US). SYTO 9 fluorescence kinetics was measured using Fluoroscan Ascent FL plate reader (Thermo Scientific, US) at 485 nm excitation and 535 nm of emission wavelengths as described earlier (Vasilchenko et al., 2020 (link)). The assay was performed twice in three technical replicates.
Microscopy was performed using a Zeiss Axio Imager A2 fluorescent microscope (Carl Zeiss, Germany) equipped with filter sets useful for simultaneous viewing of SYTO 9 and PI stains.