A Simple Protocol for Periodic Live Cell Observation of Flagellate Stages in the Lichen Alga <em>Trebouxia</em>

Flagellate stages of green microalgae such as <i>Trebouxia</i> are only partially characterised, with recent evidence suggesting that they are involved in both sexual and asexual reproduction. Conventional methods based on fixed samples in light, confocal, or electron microscopy provide only static observations and prevent real-time monitoring of living cells. To overcome this limitation, we have developed a simple and cost-effective protocol for observing <i>Trebouxia</i> flagellate cells over several days by coating microscopy slides with Bold's basal medium. The method preserves cell viability and allows repeated imaging of motile cells in the same areas so that their behaviour and development can be continuously observed. In this way, qualitative observations, such as flagellate cell release, motility, and gamete fusion, can be combined with quantitative analyses of cell morphology. The protocol has proven to be robust and reproducible and was applied to several <i>Trebouxia</i> species. Compared to existing techniques, it allows the monitoring of dynamic processes and provides a powerful tool to study specific life stages not only in <i>Trebouxia</i> but also in other unicellular and colonial green algae. Key features • This protocol allows real-time monitoring over several days of <i>Trebouxia</i> flagellate cells with standard light microscopy. • This protocol preserves cell viability and motility for repeated daily observations of the same cell groups. • This protocol is simple, low-cost, and adaptable to other motile algal cells. • This protocol is based on the methodology described in [1], where it was originally applied and validated.