Observer z1 inverted widefield microscope

To investigate if cell membranes of non-culturable bacteria remained intact after exposure to the respective antimicrobial treatment, we used the LIVE/DEAD^TMBacLight^TM bacterial viability kit (Molecular Probes, Eugene, OR, United States). Therefore, 100 μl of the bacterial sample was removed from the respective wells, centrifuged at 8,000 ×g for 5 min, washed with PBS twice, and diluted 1:100. Then 1.5 μl of SYTO 9 dye and 1.5 μl of propidium iodide were added to the sample, followed by vortexing and 15 min incubation in the dark. After incubation, cells were trapped with a 0.2-μm polycarbonate membrane filter (Sterlitech, Kent, WA, United States), which were subsequently placed between a slide and a coverslip and used for fluorescence microscopy. Viable cells with intact membranes will appear green by fluorescent SYTO 9, which permeates intact or damaged plasma membrane, while propidium iodide can only permeate damaged and disrupted plasma membranes and competes with SYTO 9 for DNA-binding sites. Therefore, cells with intact membranes show green fluorescence while cells with damaged membranes appear with red fluorescence. Those were considered to be dead. Pictures were taken with the Zeiss Observer Z1 inverted widefield microscope and ZEN 2012 (blue edition) software (Oberkochen, Germany).

In order to determine if cell membranes of non-culturable bacteria remained intact after exposure to the respective treatment conditions, the LIVE/DEAD^TMBacLight^TM bacterial viability kit (Molecular Probes, Eugene, USA) was used. Correspondingly, 1 ml of the bacterial sample was centrifuged at 8,000 × g for 5 min, washed with PBS twice and diluted 1:1,000. Subsequently, 1.5 µl of SYTO 9 dye and 1.5 µl of propidium iodide were added to the sample, followed by mixing and 15 min incubation in the dark. After incubation, cells were captured with a 0.2 µm polycarbonate membrane filter (Sterlitec, Kent, USA). The filters were trapped between a slide and a coverslip and used for fluorescence microscopy. This test is based on analysis of cell membrane integrity by fluorescence microscopy. Viable cells with intact membranes are stained green by fluorescent SYTO 9, which can pass the intact or damaged plasma membrane, while propidium iodide can only pass through damaged and disrupted plasma membranes and competes with SYTO 9 for nucleic acid binding sites. Therefore, cells with intact membranes exhibit green fluorescence and cells with damaged membranes exhibit red fluorescence. The latter were considered to be dead. Photographs were taken with the Zeiss Observer Z1 inverted widefield microscope and ZEN 2012 (blue edition) software (Oberkochen, Germany).