4.9. Bacteria Cell Membrane Permeability Assay

Each microorganism was inoculated in TSB (Sigma-Aldrich, St. Louis, MO, USA) for sixteen to eighteen hours at 37 °C in a shaking incubator at different revolutions per minute (rpm) according to different bacteria. Then 200 µL of initial growth cultures were transferred into 25 mL TSB and cultured in a shaking incubator at 37 °C. 2.5 h later, the cultures were centrifuged at 1000× g at 4 °C for 10 min to collect the bacteria and then washed twice by 30 mL 5% (v/v) TSB in 0.85% (m/v) sodium chloride (NaCl) solution. Then, the washed bacteria were resuspended by 5% (v/v) TSB in 0.85% (m/v) NaCl solution until a 1 × 10⁸ cfu/mL density obtained by testing the OD at 590 nm wavelength. The microorganisms were incubated with Der-PS4 in a range of concentrations based on MICs in a black 96-well flat bottom plate at 37 °C for two hours. For the positive control, the microorganisms were incubated with 70% (v/v) isopropanol at room temperature for 1 h and then centrifuged at 13,000 × g at 4 °C for 5 min. The supernatant was discarded and the precipitate was washed twice by 5% (v/v) TSB in 0.85% (m/v) NaCl solution and finally resuspended by 5% (v/v) TSB in 0.85% (m/v) NaCl solution. All groups were stained by SYTOX^TM green nucleic acid stain (Life technologies, Carlsbad, CA) to a final concentration of 5 µM and incubated in dark at 37 °C for 5 min. The fluorescent intensity was detected by a Synergy HT plate reader (Biotech, Minneapolis, MN, USA) with the excitation of 485 nm and emission of 528 nm.

For the time-curve bacteria cell membrane permeability assay, the microorganisms were processed as described above until a 1 × 10⁸ cfu/mL density obtained. After that, the positive control was treated with 70% (v/v) isopropanol at room temperature for 1 h first, then washed twice and resuspended by 5% (v/v) TSB in 0.85% (m/v) NaCl solution. All groups were incubated with SYTOX^TM green stain (5 µM) for 15 min and subsequently were treated by Der-PS4 with final concentration of 4, 8 and 16 µM respectively based on the results of last step. The fluorescent intensity was detected every 15 min for 120 min.