Single Cell Analysis of Microalgae and Associated Bacteria Flora by Using Flow Cytometry

Microalgae are usually cultivated in mixed cultures with associated bacteria flora. Despite bacteria contaminants can strongly influence microalgal growth, they are rarely monitored, mainly because standardized and quick methods are missing. The aim of this study was to develop a quick flow cytometric method to quantify coenobia-forming microalgae (Tetradesmus obliquus) and associated bacteria. Staining conditions and sonication pretreatment were optimized to obtain the most accurate microalgal and bacterial cell counting. To pre-treat microalgae before counting, pulsed sonication (ton/toff = 30/60) was better than continuous sonication since it allows obtaining 100% single cells by minimizing cell lysis. On the contrary, sonication was not required for bacteria, because they were mainly found as single cells and only a relevant cell lysis was obtained when sonication was applied. To analyze samples as those tested in this study, bacteria should be analyzed directly after fixation and DNA staining, without sonication (duration of analysis: 90 min). Instead, sonication was mandatory for microalgae, while fixation and DNA staining could be avoided (duration of analysis: 30 min). Future studies should investigate the effect of the biological variability, that seems to be the most relevant factor affecting the accuracy and reproducibility of the flow cytometric analysis