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

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

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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).

Item Type:
Journal Article
Journal or Publication Title:
Cryobiology
Uncontrolled Keywords:
/dk/atira/pure/subjectarea/asjc/1300/1300
Subjects:
?? cryomicroscopycryopreservationeuglena gracilisflow-cytometryviability assessmentgeneral biochemistry,genetics and molecular biologygeneral agricultural and biological sciencesgeneral medicineagricultural and biological sciences(all)biochemistry, genetics ??
ID Code:
208855
Deposited By:
Deposited On:
01 Nov 2023 15:20
Refereed?:
Yes
Published?:
Published
Last Modified:
16 Jul 2024 12:10