Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 °C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at −0.3 °C min−1 to −60 °C and osmotic shock invariably resulted in damage to the organism’s pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 °C, cooling at 0.5 °C min−1 to −60 °C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to −130 °C followed by relatively rapid warming (∼90 °C min−1) to ambient temperature (ca. 25 °C).