Revival of Frog's Spermatozoa Vitrified in Liquid Air

Abstract

It is now known that water in aqueous colloids takes the crystalline state at temperatures extending from 0°C to some tens of degrees below zero and that, when exposed, while still liquid, to lower temperatures, it takes the vitreous state. 1 Using the method of immersion in liquid air for vitrification it was found that a colloid which contains 90% water when spread on a microscope coverslip 1/10 mm thick, can be vitrified if the thickness of the layer treated does not exceed a few micra, while when the water content decreases to 50%, the vitrifiable thickness increases to 0.2 mm. 2 The assumption that vitrification does not disintegrate living matter as crystallization does was previously verified in some plant cells. 3 In this paper it will be shown that in animal cells, in which the motility can be taken as a sign of vitality, life is not destroyed by the vitrification treatment.

In a first series of experiments, excised frog testes were cut open and the spermatozoa expressed through the cut end were smeared on an ordinary coverslip. This was then immersed in liquid air for about 10 seconds and immediately after in pond water at +20°C in order to insure a rapid warming and in that manner avoid crystallization during the warming through the dangerous zone of temperatures. The spermatozoa left on the slide or dispersed in the pond water were all dead.

One might have suspected that the 20 degree interval (from 0° to +20°) allowed for rapid warming did not constitute a large enough temperature difference, but since our experiments with plant epidermis 3 and with moss leaves 4 were successful with a warming bath at + 20° we suspected the high heat capacity of the too thick coverslips to be responsible for a too slow cooling or warming.