Faculty of Biotechnology and Food Sciences in Nitra Journal of Microbiology, Biotechnology and Food Sciences 1338-5178 vol. 6 2016/17 2016-12-01 2016-12-01 EVALUATING VIABLE CELL INDICATORS FOR FILAMENTOUS CYANOBACTERIA AND THEIR APPLICATION 886 893 EN Tylor J. Johnson Tylor J. Johnson Michael B. Hildreth Michael B. Hildreth Liping Gu Liping Gu Emily L. Baldwin Emily L. Baldwin Ruanbao Zhou Ruanbao Zhou William R. Gibbons William R. Gibbons Filamentous cyanobacteria, such as Anabaena 7120 have great industrial potential due to their capability to be genetically engineered to produce next-generation biofuels while using minimal nutrients. One challenge of working with these microbes is that classical methods of quantifying cell viability are not effective due to their filamentous morphology. Therefore, fluorescent assays were evaluated to determine if they could be used as a reliable indicator of cell viability. Initially, a dual-stain assay using SYTO® 9 and SYTOX® Blue was investigated. Fluorescence from SYTO® 9 was accurately correlated with viable cells; however, SYTOX® Blue did not work as a non-viable cell indicator due to non-specific binding in both viable and non-viable cells. Autofluorescence from light harvesting pigments was also evaluated as a viable cell indicator, but unfortunately these pigments resulted in several emission peaks that couldn’t be captured by a single emission filter. Moreover, certain light harvesting pigments continued to fluoresce after the cell became non-viable. SYTO® 9 was then compared to absorbance and chlorophyll content to quantify viable Anabaena 7120 in a chemical inhibition testing protocol. This protocol requires a low initial biomass concentration to prevent binding of the chemicals to cell biomass, and at low cell densities SYTO® 9 was superior to absorbance and chlorophyll content in quantifying viability. It was also determined that SYTO® 9 allows for the evaluation of different cultivation media on the growth of cyanobacteria in photobioreactors. SYTO® 9 is a reliable, accurate indicator of viability of filamentous cyanobacteria and can be used in a high-throughput manner via a microplate reader.
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