SUMMARY We have developed three instruments to freeze different biological specimens for various purposes in the easiest and most economical way: a punching device, a rapid immersion device and a device to freeze the specimens against a copper block cooled by liquid helium. Cooling rate measurements are made by a thin flat thermocouple. Freezing against a copper block cooled down to 4 K gives cooling rates 3–5 times higher than freezing by immersion into propane at 85 K.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OSO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nudeosomes. With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space. The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space. The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria. The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas. The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature. The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OSO4 but more confined than its very dispersed form found after aldehyde fixation. 960 on July 16, 2020 by guest

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