Mechanism and use of the commercially available viability stain, BacLight

Abstract

Background BacLight (Molecular Probes, Eugene, OR, USA) is a popular fluorescence-based two-component stain for determining bacterial cell viability. The main purpose of this work was to fully elucidate the mechanism and to determine why it is sometimes reported that cells stain simultaneously live and dead. Methods Solutions of DNA were stained with the two components, propidium iodide (PI) and SYTO9, in different combinations, and fluorescence spectra were collected. Results K_PI and K_SYTO9 were approximately 3.7 × 10⁵/M and 1.8 × 10⁵/M. SYTO9 emissions were stronger and overlapped those of PI. Fluorescence resonance energy transfer from SYTO9 to PI was observed. It was, even under normal conditions, possible for DNA bound SYTO9 to have a component in the red region equal to that of DNA bound PI. Potentially confusing emissions were also found to occur when PI was not in sufficient excess to saturate nucleic acid (>0.4 M PI to 1 M DNA base pairs). Conclusions The mechanism is a combination of displacement of SYTO9 by PI and quenching of SYTO9 emissions by fluorescence resonance energy transfer. Confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for.