The relative efficiency of various fluids in the rapid freezing of protozoa

The cooling efficiencies of various fluids at low temperature were compared by measuring the temperature decay in 3 microliter water samples plunged into them. A simple model of cooling was used in order to discuss the results. Liquid ethane was found to produce a cooling rate of 660 KS-1, about twice that of liquid propane, while ethanol was almost as effective as ethane between 273 to 223 K. The heat-transfer coefficient of liquid ethane was estimated to be between 1500 and 5000 W m-2 K-1, depending on the physical state assumed for the water sample. Samples of flagellated organisms, after being frozen rapidly in the above way, were freeze-substituted by the method of Barlow & Sleigh (1979). Although this fixation did not give good definition of the microtubules of the flagellar axoneme, it exhibited reasonable tissue preservation in thin sections of the cell body. The fixation method resulted in preserved flagellar wave shapes, which were observed under the light microscope and in critical-point dried cells examined by scanning and conventional electron microscopy. It was concluded (a) that methods for preserving the wave shape of the flagellum and for preserving its internal structure may not be compatible, and (b) that although the present cooling method (with ethane) approaches the speed required to arrest a flagellar wave, further improvements in the speed of the method are desirable.