Analysis of membrane damage

Membrane damage of E. coli O157:H7 during the treatment was analyzed by using the fluorescence probe propidium iodide (PI) and through scanning electron microscope (SEM). PI is a red-fluorescent nuclear and chromosome counterstain that penetrates only bacteria with damaged membranes and is frequently used to detect cell membrane damage^{59, 60}. Test solutions consisting of bacteria (~1 × 10⁹ CFU/mL) suspended in GA solution (15 mM), PG solution (10 mM), EDTA solution (1 mM), or HCl solution (pH = 3.1) were treated under UV-A light exposure or incubated under the dark for 30 min. Bacteria in DI water alone in the dark was used as live control. After treatment, samples were washed with DI water and centrifuged for 2 min at 10,000 × g. Then, a volume of 50 µL of PI was added to each sample to reach a concentration of 5 µM, following dark incubation at room temperature for 15 min. Subsequently, samples were washed and suspended in 500 µL 1 × PBS. A volume of 100 µL of this sample were transferred to a 96-well plate, and the fluorescence intensity was measured using a plate reader with excitation and emission wavelength of 490/635 nm. The fluorescence intensity was corrected using equation (¹).

Membrane damage was also visualized by SEM imaging based on method described by Kihm et al.⁶¹. E. coli O157:H7 (approximately 1 × 10⁷ CFU/mL) suspended in 15 mM GA solution, 10 mM PG solution or DI water were exposed to UV-A light or incubated in the dark for 30 min. Then, cells were recovered by filtering through a 0.2 µm sterile filter, fixed by incubating in 0.25% glutaraldehyde for 1 h, rinsed three times in DI water, dehydrated six times in ethanol of increasing concentration, and stored in a desiccator overnight for dehydration prior to imaging. To observe cell morphology under SEM, bacteria were coated with gold (20 nm) with a sputter coater. After coating with gold, the morphology of bacteria was studied using a SEM at an accelerating voltage of 10 kV.