Summary: The changes in morphology of Link and Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0·5–1·0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 °C in the presence or absence of glycerol. In shrinkage occurred in all hyphae at rates of cooling of less than 15 °C min; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18°C minto 55 °C min. This species was particularly resistant to freezing injury and recovery was>60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of in the growth medium, shrinkage occurred and no samples survived on thawing from -196 °C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 °C minthe loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.


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