Microalgae are essential food for the aquaculture of molluscan shellfish and larval finfish, and the species from genus Tetraselmis are excellent candidates due to their ease of culture, biochemical composition, and high levels of EPA, ARA, 24- meth sterols, starches, and existing lipids. This genus of microalgae is also a candidate for biofuels and pharmaceuticals due to its anti-inflammatory, antimicrobial, and antioxidant properties. Over time, microalgae cultures are susceptible to risks, such as contamination, degradation, mutation, or complete loss of cultures; Therefore, cryopreservation could be applied to preserve the germplasm in perpetuity. In our previous work, effective protocols for the cryopreservation of Tetraselmis suecica had been developed (Aquaculture 2023, 566, 739172) where neutral red was used to stain cells for viability analysis. However, due to the large number of samples involved in protocol development, staining and microscope counting for viability analysis became the biggest obstacle. Flow cytometry is an important tool for single-cell sample analysis using fluorescent dyes which could be applied to viability analysis.
The goal of this study is to cryopreserve Tetraselmis species with a focus on the development of viability analysis and application of the existing protocol for Tetraselmis strata across six Tetraselmis species. The objectives are to: 1) Develop an effective method for viability analysis of Tetraselmis species using flow cytometry and fluorescent dye of Propidium Iodide (PI); 2) Apply the existing cryopreservation protocol for T. striata to other species in this genus; 3) Evaluate the effect of microalgae age and lipid content on cryopreservation over 14 days, and 4) Verify the cryopreservation protocols across the six species.
Staining of PI at concentrations ranging from 0 to 80 µg/ml for 10 min indicated the effective concentration was 30 µg/ml or beyond for viability analysis, and this concentration is applicable for six Tetrasemis species. This study is still in progress. The quick and accurate method for viability analysis using flow cytometry and PI can speed up the post-thaw sample viability process for this project. Cryopreservation protocols to be established in this study would be useful for long-term germplasm preservation for the aquaculture industry and stock centers.