Bacterial viability assays in comparison. (a) Conventional endpoint dual staining bacterial viability assay and (b) eGFP/PI assay optimized for in situ bacterial viability monitoring. The dual staining assay commonly employs two DNA stains, SYTO® 9 (green triangle) and propidium iodide (PI, red rectangle) that both intercalate into the bacterial DNA. SYTO® 9 diffuses passively into living bacteria whereas PI cannot pass intact bacterial membranes and only enters permeabilized dead bacteria. The use of constitutive eGFP expression rather than SYTO® 9 provides direct detection of viable bacteria without the addition of a fluorescent dye and circumvents the competitive displacement of SYTO® 9 by PI that can result in a dual staining of dead bacteria.