The last couple of years have shown an increased research interest in microalgae and cyanobacteria, especially in the field of biofuels. Therefore simple and fast culture monitoring methods to determine the viability of the cells are needed.
As a simple method for the monitoring of fitness, absorption spectra give an overview of the amount of pigments and their distribution in the culture. Also the chlorophyll concentration (and the chlorophyll-concentration/OD750) can be used to analyze the culture condition . Nevertheless, both methods only allow the quantification of growth and viability on the culture level, giving an average over all cells, but not a quantification of the viable cells. A differentiation of viable and non-viable cells on a cell by cell level allows for a causal evaluation of cell viability in regard to changing conditions, such as stress, cell aging or respiration rate. The standard method for the determining of cell viability is plating of the culture and subsequent counting of the colonies. This method however, requires plate preparation and at least one week until colonies can be identified. An alternative method for the determination of cell viability is fluorescent staining. In a dual-fluorescence assay the autofluorescence of chlorophyll is used to identify viable cells, and the fluorescent dye SYTOX Green is used to identify non-viable cells . Although staining is faster than plating, it is vastly more expensive, and there is still need for culture preparation. For an automation of fluorescent staining, a flow cytometric approach for Microcystis has been previously reported . The automation of the viability analysis via flow cytometry has the advantage of being less time consuming and more reliable. Nevertheless staining of the sample is still required. In contrast, the method presented in this paper could be adapted for use with flow cytometry and would allow the differentiation of viable cells without any sample preparation.
The new method is a fast and simple alternative using red chlorophyll fluorescence and an unspecific green autofluorescence for the determination of cell viability. Red autofluorescence of chlorophyll is only present in viable cells. For higher plants and filamentous cyanobacteria it is known, that there is a steady state between the building and degradation of chlorophyll. Senescence or dying results in a switch from equilibrium (with high turnover) to a massive degradation of chlorophyll [4, 5]. Therefore the red autofluorescence fades and a green unspecific fluorescence, which can be observed at the same excitation wavelength but is superimposed by the red chlorophyll fluorescence in viable cells, becomes visible. This green fluorescence has mostly been described in higher plants and is caused by a variety of different molecules like flavonoids, flavins (e.g. FAD), cinnamic acids (e.g. ferulic acid), betaxanthine and pyridine nucleotides [e.g. NAD(P)H]. For algae this phenomenon has barely been analyzed and the molecules responsible for the green autofluorescence are mostly unidentified [6, 7].
Nevertheless this effect allows one to distinguish between viable and non-viable cells (For this method non-viable cells are defined as cells that still show an intact cell shape but have lost the ability for growth and/or division.) at the same excitation frequency. By the use of a long pass filter for the emission wavelength both fluorescent signals can be observed simultaneously. A direct determination of the number of viable and non-viable cells is now possible without any sample preparation.
By the use of an automated fluorescence microscope and subsequent analysis of the generated images, an automated procedure for the determination of cell viability was created. For the image analysis, a plugin was written for ImageJ, an Open Source project which allows easy expansion [8, 9]. The plugin enables the determination of the total cell concentration and the number of viable and non-viable cells by counting fluorescent cells and classing them by color.
We tested the red-green fluorescence assay for Synechocystis sp. PCC 6803, as it is a model for cyanobacteria and photoautotroph organisms in general . The results for the automated viability analysis via ImageJ were validated with values obtained by plating, chlorophyll-measurement, and the analysis of the absorption spectra. Furthermore a temperature stressed Synechocystis culture was analyzed with the fluorescence assay and absorption spectroscopy. Results were compared with those of the viable culture, to test the applicability for a vitality analysis. Additionally the automated determination of the cell concentration by the plugin was investigated and compared to the optical density at 750 nm, which is often used to determine cell concentration for unicellular cyanobacteria.