Fleck, Roland A. and Pickup, Roger W. and Day, John G. and Benson, Erica E. (2006) Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy. Cryobiology, 52 (2). pp. 261-268. ISSN 0011-2240
Full text not available from this repository.Abstract
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).