Action on Membrane Integrity and Esterase Activity in E. coli

To evaluate the damage caused by Mygalin to the cell membrane, E. coli (10⁸ CFU/mL) were cultured with Mygalin (0.5 mM) or ampicillin (0.5 mM) for 5 h and then washed and suspended in 50 mM phosphate buffer, pH 7.0. Then, the bacteria were incubated with PI at a final concentration of 60 μM and kept in the dark for 15 min (Nocker et al., 2011). Then, 20 μL of sample was added to a slide with 1% agarose, the slide was covered with a coverslip, and the membrane integrity was analyzed by confocal microscopy. For the esterase activity assay, untreated bacteria and those treated with the drugs for 4 h were washed once in PBS and suspended in 50 mM phosphate buffer. A total of 180 μL of bacterial suspension was placed on black COSTAR^® 96-well microplates with the addition of 20 μL of CFDA (250 μM). The samples were incubated for 30 min in the dark, and fluorescence was measured at 485/535 nm excitation/emission wavelengths (Nocker et al., 2011; Hyldgaard et al., 2015).