Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.

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Cryoinjury in individual ram spermatozoa was investigated in cells cooled at 10 degrees C/min on a programmable cryomicroscope. In physiological buffer and cryoprotective media, there was a smooth decline in sperm swimming speed with decreasing temperature; cooling in buffer caused a marked decline in the proportion of cells displaying forward progression, especially once the temperature fell below 16 degrees C. Spermatozoa cooled in the presence of rhodamine 123, a mitochondrial-specific dye, showed that abolition of sperm motility by cold shock in buffer was not due to mitochondrial inactivation. Temperature decline through the region of 10 degrees C caused a number of spermatozoa in buffer to undergo a sudden asymmetric bending of the flagellum in the region of the midpiece. Ultrastructural studies suggest that this was caused by an unstable, asymmetric membrane lesion. Spermatozoa cooled in the presence of cryoprotective media showed better recovery of motility after rewarming and failed to exhibit the bending effect described above.

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The changes in morphology and viability of 20 species of fungi during freezing were examined in relation to cooling rate and the presence of glycerol. All of the hyphomycetes examined, the ascomycete Sordaria, the zygomycete Mucor, and the basidiomycete Schizophyllum, survived freezing and thawing in the absence of glycerol. Cryomicroscopy demonstrated that for these fungi the formation of intracellular ice at rapid rates of cooling was not lethal. Isolates from the oomycetes and some basidiomycetes required glycerol for survival. The morphological response of Phytophthora, Aschersonia and Volvariella differed from other genera, with shrinkage occurring at all rates of cooling. The conventionally employed cooling rate for the cryopreservation of fungi, 1 "C min-l, was found not to be optimal for all of the strains studied. Serpula recovered best after cooling at 0.5 "C min-l followed by rapid thawing, and all the oomycetes examined gave highest recovery after cooling at rates between 5 and 10 "C min-l followed by rapid thawing.