title: Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy.
abstract: Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree 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 degrees C min(-1) to -60 degrees 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 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees 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 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK. rfleck@nibsc.ac.uk
authors.forename: Roland A
authors.initials: RA
authors.lastname: Fleck
authors.forename: Roger W
authors.initials: RW
authors.lastname: Pickup
authors.forename: John G
authors.initials: JG
authors.lastname: Day
authors.forename: Erica E
authors.initials: EE
authors.lastname: Benson
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D020285:Cryoelectron Microscopy; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D005056:Euglena gracilis / Q000166:cytology / Q000254:growth & development*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D005615:Freezing; D050296:Microbial Viability; D009997:Osmotic Pressure; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D005452:Fluoresceins; D012965:Sodium Chloride; C018506:diacetylfluorescein