New methodology for viability testing in environmental samples.

Environmental samples can be complex and are comprised of microorganisms and a matrix of decaying organic matter as well as an inorganic phase such as sand or precipitated material (waste water, sludge, soils, etc.). Nucleic acid dyes have recently been developed to address the growing need for environmental analyses (cell staining, counting, viability testing and specific organism identification). However, certain dyes may not be ideally suited for testing of environmental samples, because they readily adhere to the substrate material as well as their target molecule, resulting in increased non-specific binding and background fluorescence. The aim of this study was to address the limitations of the widely used and commercially available Live/Dead BacLight Bacterial Viability kit (Molecular Probes, Eugene, OR). A new combination of nucleic acid dyes, i.e. SYTO13 and SYTOX Orange (Molecular Probes, Eugene, OR), was proposed as an alternative. The dyes were carefully chosen for their spectral separation and increase of fluorescence quantum yield. A protocol for this combination was first designed and optimized and the two staining assays were compared against suspensions of live and dead E. coli, mixed in different proportions and it was shown that both protocols performed equally on pure cultures. However, when testing activated sludge samples, the commercial kit showed greater background fluorescence and non-specific binding than the alternate combination. Therefore, the proposed dye combination and its corresponding protocol are deemed more suitable for use on complex environmental samples than the Live/Dead BacLight Bacterial Viability kit.