Cryopreservation of Pyramimonas mucifera: effect of DMSO and the slow cooling method on viability

Mokoena, Karabo Thato
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Microalgae are diverse and important for scientific research and applicability to industry. Microalgal cultures are expensive to maintain for such purposes and require frequent batch culturing which has adverse effects that could lead to the loss of algal diversity. To preserve this diversity, cryopreservation has been identified as an appropriate conservation measure. The aim of this study was to determine the effect of 10 % dimethyl sulfoxide (DMSO) and the two-step cooling method on cell viability and ultrastructure of Pyramimonas mucifera (Chlorophyta, Pyramimonadophyceae, Pyramimonadales, Pyramimonadaceae). Viability was inferred using membrane integrity and mitochondrial function. Fluorescent stains propidium iodide (PI) and Rhodamine123 (Rh123) were used, respectively, to determine these aspects. The ultrastructural response to this protocol was characterised by focusing on the membrane integrity of the plasma membrane, chloroplast, and mitochondrion of P. muciferacells. DMSO was proven to be an appropriate cryoprotectant as it caused minimal damage to cell viability (<30%) and ultrastructure, and cells were uncompromised upon its removal (>90% recovery). The two-step cooling method caused a decrease in cell viability, mechanical damage, and signs of apoptosis in some cells compared to untreated and DMSO treated cells. However, the method was found to be acceptable for this species as viable cells were always encountered and could re-establish into vigorous cultures after cryopreservation.
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master’s in Science (Animal, Plant and Environmental Sciences), 2022