08/11/2021
A HIGH SPEED VACUUM CENTRIFUGE SUITABLE FOR THE STUDY OF FILTERABLE VIRUSES.
By JOHANNES H. BAUER, M.D., and EDWARD G. PICKELS, Ph.D
From the Laboratories of the Rockefeller Foundation, New York
June 6, 1936.
The study of filterable disease-producing agents has been much handicapped by the lack of suitable apparatus. The microscope, the ordinary laboratory centrifuge, and culture medium, all of which are invaluable in the study of disease producing bacteria, are of no assistance in the study of viruses.
This centrifuge was primarily intended for the separation and concentration of the yellow fever virus.
A series of experiments was carried out with a view to studying the operating characteristics of the centrifuge as well as the behavior of yellow fever virus in an intense centrifugal field. In most of these a neurotropic strain ( attraction to nerve tissue) of the virus derived from mouse brain was used. This was chosen because of its greater virulence for mice and its presence in the material under study is therefore more easily determined than that of the viscerotropic ( internal organs) form derived from the blood of infected monkeys.
A 20 per cent mouse-brain suspension was prepared by grinding finely fifteen mouse brains in a sterile mortar and suspending them in 25cc of undiluted normal monkey serum. After centrifugation the tubes containing the virus showed a definite whitish sediment at the bottom, and while most of the supernatant fluid was clear, the topmost layer of the column, about 5 mm. thick, showed a distinct grayish turbidity and apparently contained much finely dispersed fat.
Although the material was centrifuged at a higher speed for a longer time than in the preceding experiment, the separation of the virus was less satisfactory.
Experiment 4. Dr. T.P Hughes of this laboratory in his chemical studies on the virus found that the fat from INFECTED mouse brains can be removed by extraction with ether without a material loss of the virus activity. He very kindly furnished us with 25 brains which had been dried in vacuo in the frozen state and from which the fat had been extracted in a Soxhlet apparatus with ether and petroleum ether. These brains were finely ground with quarts and in a sterils mortor and suspended in 25 cc. of a diluent made up of equal parts of ascitic fluid and distilled water. As in the previous experiments, the material was rendered clear by centrifuging first for 30 minutes at 3,000 R.P.M., and afterward for 1 hour at 13,000 R.P.M. The clear material was then centrifuged in the vacuum centrifuge for 3 hours at a speed of 27,300 R.P.M. The virus content in the supernatant fluid and sediments was tested in exactly the same manner as in the preceding experiments. As seen from this table there was still a considerable amount of virus present in the supernatant fluid. It is obvious from these results that factors other than the presence of fat are responsible for the failure to obtain a complete separation of the virus in the centrifuge.
DISCUSSION
The centrifuge described above was successfully applied for the separation and concentration of yellow fever virus. The major portion of the virus was sedimented out of the suspensions when centrifuged for 3 hours at a speed of 25,000 R.P.M.; in fact, it became so firmly packed to the bottom that the supernatant fluid could be poured off and the sediment resuspended in fresh diluent without appreciable loss of virus activity. A certain small proportion of the virus remained persistently in the supernatant fluid even when hemoglobin which was present in the virus suspension had been effectively separated. We are unable as yet to find a satisfactory explanation for the failure to secure a complete separation of the virus under the conditions described.
Although we have not had an opportunity to study the behavior of other viruses in this centrifuge, we feel that it should prove a useful instrument in the study of filterable disease-producing agents, including the smallest known.
Jim's comment. These doctors took the infection from a person with yellow fever and tried to separate the poison from the infection. They were experimenting with the idea that they could isolate the poisonous particle from the poison. That would be like throwing acid on your arm, and after it eats away the flesh trying to isolate the poisonous particle of the acid. It was all poison. This craziness started with snake venom. Trying to isolate the part of the snake poison that was poisonous. It was all poisonous. And Thomas M. Rivers was hard at work in convincing the medical profession that this poison (virus) was alive and the cause of disease. He succeeded beyond his wildest dreams.
Jim Dandy O'Kelly Founder of the Anti-Vaccination League of America and discoverer of the virus lie.