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

Fleck, Roland A.; Pickup, Roger W.; Day, John G.; Benson, Erica E.. 2006 Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy. Cryobiology, 52 (2). 261-268. 10.1016/j.cryobiol.2005.12.003

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Abstract/Summary

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: Publication - Article
Digital Object Identifier (DOI): 10.1016/j.cryobiol.2005.12.003
Programmes: CEH Programmes pre-2009 publications > Biodiversity
CEH Sections: _ Ecological Processes in Freshwater & Soils
ISSN: 0011-2240
Format Availability: Electronic, Print
Additional Keywords: Cryomicroscopy, Cryopreservation, Euglena gracilis, Flow-cytometry, Viability assessment
NORA Subject Terms: Biology and Microbiology
Date made live: 28 Jun 2007 10:02
URI: http://nora.nerc.ac.uk/id/eprint/324

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