Introduction: Vitrification is the method of choice for preserving biological specimens prior to cryo- electron microscopy. Infectious agents prepared in this way carry the risk of disease transmission. Some cells can resume growth after the high-pressure freezing-thaw cycle [1,2]. Using fluorescent indicators of metabolic activity, we determined the percentage of active bacteria and yeasts after high-pressure freezing (HPF).
Methodology: A Leica HPM 100 high-pressure freezer was used to vitrify Saccharomyces cerevisiae (Baker’s yeast) and vegetative cells of Geobacillus stearothermophilus. Numbers of metabolically active yeast cells were determined by FUN-1 assay based on fluorescence microscopy of individual cells [3]. This was compared to recovery via classical, culture-based techniques. The effects of different freezing rates on metabolic activity were determined by immersing cells in liquid nitrogen, freezing to -20°C, or controlled-rate (isopropanol bath) freezing to -80°C. We also compared the protective effects of extracellular/intracellular cryoprotectants on recovery of active cells.
Results & Conclusion: Activity in vitrified and thawed S. cerevisiae was affected by cell density, presumably as a result of volume exclusion. In cells cryoprotected with 10% dimethylsulfoxide (DMSO), original activity levels were restored only after 6 hours; this was not the result of regrowth. At the same timepoint, cells cryoprotected with 20% dextran had only recovered to just over half of original levels. DMSO, however, reduced activity in Control samples from 95% to 65%. In the absence of additional cryoprotectant, activity was critically dependent on freezing rate in the order [liquid nitrogen < -20°C < -80°C (controlled-rate) < high-pressure freezing]. Comparisons of culturable, active and total cell counts imply a sub-lethal injury phenomenon. Interspecies differences in tolerance to freezing damage suggest that infection risk must be assessed on a case-by-case basis.
Acknowledgement: We acknowledge the facilities of the Australian Microscopy & Microanalysis Research Facility at the Australian Animal Health Laboratory, CSIRO.