In the preceding paper of this series we have described an efficient and versatile apparatus for freeze-drying by vacuum sublimation. This apparatus makes it possible to dehydrate biological materials at low temperatures and, by the use of ionization gauges, to determine the end-point of dehydration of material maintained at temperatures ranging down to --80°C. It is so designed that different samples of the same material can be subjected simultaneously to various experimental conditions. Time, rate, and degree of drying have been shown to have a marked influence on the physical properties of lyophilized biological materials other than viruses by Elser, Thomas, and Steffen (1); Greaves and Adair (2); Flosdorf, Hull, and Mudd (3); Greaves (4) and others. Proom and Hemmons (5) have published working details for the freeze-drying preservation of a collection of more than 1500 strains of bacteria. Various methods of freezing, the degree of drying, and the effect of storage were tested by viability counts. Recently, Hutton, Hilmoe, and Roberts (6) have reported on the effects of these factors on the quantitative survival of Brucella abortus. Similar studies dealing with the survival of viruses have not been reported.The present investigations deal with the quantitative survival of influenz a virus suspensions, after repeated freeze-thaw cycles, freezing at various speeds, storage in the frozen state at various temperatures, and vacuum sublimation at various temperatures following different types of preliminary treatment. Subsequent investigations are planned which will be concerned with the effects of these factors on the survival of complex cells.

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After freezing, suspensions of influenza virus were dried by sublimation of water in vacuo to contents of residual moisture of 3.2, 2.1, 1.7, 1, or 0.4%. The stability of the several suspensions was determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at different temperatures to lose 1 log of infectivity titer, the order of stability in relation to residual moistures was as follows: 1.7% > 2.1% > 1% > 3.2% > 0.4%.

A method for the quantitation of neuraminidase in the presence of N-acetylneuraminic acid aldolase is described. The neuraminidase content of Diplococcus pneumoniae was found to be dependent on the media employed for growth; the highest enzyme activity per milligram of bacterial protein was obtained with Todd-Hewitt broth. Neuraminidase production was stimulated in D. pneumoniae by the addition of N-acetylneuraminlactose, N-acetylneuraminic acid, or N-acetylmannosamine to the growth medium. Three rough strains of D. pneumoniae, which were nonpathogenic for mice, lacked neuraminidase activity. Seven of 12 smooth strains contained neuraminidase; enzyme activity was not detected in the remaining 5 smooth strains. There was no correlation between the presence of neuraminidase activity and the capsular type or between neuraminidase production and animal virulence.