| Marine phytoplankton cultures are needed for reproducible results in basic research and mariculture. Cryopreservation, or storage at extremely low temperatures, will ease culture maintenance effort and expense, will reduce accidental loss of culture strains, and will eliminate the genetic drift that occurs during more traditional culture maintenance methods. The objectives of this research have been to develop a cryopreservation protocol for marine diatoms and to understand the nature of freezing injury such that a logical approach can be applied to the cryopreservation of novel species.;In order to assess viability accurately, a reproductive viability assay was developed for the planktonic diatom Thalassiosira weissflogii in which a dilute culture is pipetted onto micro-test plates and the number of reproductive cells calculated from a Poisson distribution. T. weissflogii can now be recovered from liquid nitrogen with >70% reproductive viability. This success is accomplished by using the cryoprotectant combination of 1 M proline (in which the cells are incubated for 24 h) and 2 M methanol, by decreasing the sample volume to 15 (mu)l, and by employing a novel two-step warming protocol that allows rapid warming through low sub-zero temperatures while also permitting slow warming at temperatures of greatest change in liquid volume.;Cold shock was found to be an increase in plasma membrane permeability, to be caused by rapid cooling, and to be a factor in freezing injury. Proline ameliorates cell injury incurred during slow freezing; methanol, which reduces cold shock injury, is needed to confer cell survival from liquid nitrogen.;Flow cytometry was used to compare freezing and thawing protocols. Autofluorescence and rhodamine 123 fluorescence were measured and were considered indicative of carotenoid association with light harvesting centers and membrane potential, respectively. Higher fluorescences followed higher viabilities. However cells killed by rapid cooling but not initially cold shocked showed high fluorescences but low viability. These cells are presumed to have suffered from spontaneous intracellular ice formation.;Significant progress has been made in the study of cryopreservation in marine diatoms, not only in comparison to the dearth of published work on the cryopreservation of marine algae, but also in consideration of the contribution this study represents toward the cryopreservation of other algae and in the field of cryobiology. |
