Utilizing a Dual-Fluorescence Assay to Quantify Viability of Filamentous Cyanobacteria

Abstract. Filamentous cyanobacteria are currently being engineered to produce high-value chemicals, including 3^rd generation biofuels. Because these strains grow in a filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. With an appropriate mixture of the SYTO 9 and propidium iodide stains, viable cells with intact cell membranes stain fluorescent green, whereas non-viable cells with damaged membranes stain fluorescent red. Known percentages of viable and non viable cells were mixed and then evaluated for green vs. red fluorescence. While initial trials did develop a linear correlation between viable and non-viable cells, this calibration was off-set due to inaccuracies in generating solutions of viable and non-viable cells that contained equal numbers of cells based on optical density determined after isopropyl alcohol treatment. In subsequent trials we omitted this optical density-based equilibration step and instead relied on an initial, equivalent split of cells used to prepare viable and non-viable calibration solutions. This adjustment to the protocol was successful in generating linear and accurate correlation equations for 3 species of filamentous cyanobacteria. These equations will be useful in assessing the effects of specific chemicals on cell viability and to optimize photobioreactor system performance.