Bacterial Viability Assessment using Fluorescent Stains

Fluorescent propidium iodide (PI) stain was used with the SYTO^® 9 (Live/Dead^® BacLight™ Bacterial Viability Kit, Life Technologies GmbH, Darmstadt, Germany) to determine the number of viable and dead bacterial cells [53 (link)]. Intact cells and those with disrupted membranes can be penetrated by the green fluorescence stain SYTO^® 9, whereas the red-fluorescent PI can only penetrate disrupted cell membranes. Hence, viable and active bacterial cells fluoresce green and non-intact cells fluoresce red. The PI and SYTO^® 9 were diluted in 0.9% NaCl to achieve a final concentration of 0.1 nmol/mL. The different materials covered with the initial bacterial adhesion were then transferred to multiwell plates and stained with 1 mL SYTO^® 9/PI solution in 0.9% NaCl per well, for 15 min at room temperature, in a dark chamber. The stained materials were subsequently placed with the contaminated side on a drop of 0.9% NaCl solution in an 8-chambered cover glass (µ Slide 8 well, ibidi GmbH, Munich, Germany), and analyzed using an inverse epifluorescence microscope (ApoTome.2, Axio Observer.Z1, ZEISS, Oberkochen, Germany) with a 63 × oil immersion objective (Plan-Apochromat 63x/1.4 Oil DIC, ZEISS, Oberkochen, Germany).