Quantifying Bacterial Viability Under Mepyramine Stress

To determine the amount of viable and dead bacteria under mepyramine stress conditions (30, 100 and 1000 μg/ml mepyramine, incubation time 24 h at 37 °C) the LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to manufactures manual. The assay contains two fluorescent nucleic acid stains (SYTO 9 and propidium iodide). Bacteria with intact membranes (living bacteria) are stained fluorescent green, whereas cells with damaged membranes (dead bacteria) are stained fluorescent red. Bacteria cultures without adding any agent served as positive control. Bacteria with addition of isopropylalcohol (70%, Honeywell, Morristown, NJ, USA) were used as negative control. The stained bacterial suspension was analysed using fluorescence microscopy (Leica, Wetzlar, Germany) by counting the stained bacterial colonies in two visual fields (100 x magnification) at 490 and 546 nm [16 (link), 17 (link)].