TY - JOUR

T1 - Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

AU - Fleck, Roland A.

AU - Pickup, Roger W.

AU - Day, John G.

AU - Benson, Erica E.

PY - 2006/4/30

Y1 - 2006/4/30

N2 - 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).

AB - 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).

KW - Cryomicroscopy

KW - Cryopreservation

KW - Euglena gracilis

KW - Flow-cytometry

KW - Viability assessment

U2 - 10.1016/j.cryobiol.2005.12.003

DO - 10.1016/j.cryobiol.2005.12.003

M3 - Journal article

C2 - 16455069

AN - SCOPUS:33644793728

VL - 52

SP - 261

EP - 268

JO - Cryobiology

JF - Cryobiology

SN - 0011-2240

IS - 2

ER -