On the Behavior of the Rous Tumor Virus to Freezing

In the twenty-three years that have elapsed since the announcement by Rous of his discovery of a fowl sarcoma transmissible by a presumably cell-free filtrate, the exact nature of the causative agent is still undetermined. Of the three principal possibilities, that of filter-passing tumor cells would seem to be excluded rather definitely. But whether we are dealing with a living agent, or with a product of metabolism apparently of enzymatic character capable of inciting the changes which in turn produce it, is still open to question. The production by Fischer of a tumor of this type, by the prolonged treatment of cultures of fowl fibroblasts with weak solutions of arsenic oxide, constitutes strong evidence in favor of the latter view. The principal evidence of contrary character lies in the work of Nakahara, who found that repeated freezing and thawing of the tumor cells greatly impaired the transmissive effect of the agent. His work has been questioned, however, on the ground of neglect to guard against oxidation, to which the agent is peculiarly susceptible. Partly for the purpose of checking this work of Nakahara, and partly to determine the limits of the possibly lethal effect on the agent of repeated extreme temperature changes, the work reported here was instituted. In it the agent, as well as suspensions of tumor cells, were subjected to repeated freezing by means of carbon dioxide ice, and to rapid thawing, both in practically complete absence of oxygen. The procedure was relatively simple. The virus, after filtration in physiological salt solution through a sterile “W” Berkefeld filter, was placed in glass ampoules with slender, drawn-out necks. The air was then exhausted by means of a strong water pump, and replaced by pure hydrogen. This procedure was repeated ten times, and the ampoules were finally sealed in the exhausted condition. They were then frozen a variable number of times by means of “dry ice” kept in a large Dewar jar, and after freezing were rapidly thawed by immersion in boiling water exactly to the point of liquefaction of the virus. For comparison, various bacterial suspensions were similarly treated, except as to filtration, at the same time. The tumor material was obtained from Plymouth Rock chickens originally inoculated with tumor desiccate, and this was transplanted by cell and filtrate inoculation into successive fowls until it was ascertained that the tumor was in the state of cell-free transmissibility. The tumors so obtained were ground in a meat-grinder, and then by hand with the addition of powdered carborundum. The suspension, diluted ten times with physiological salt solution, was centrifuged for