Ultra-Filtration Experiments with the Encephalitis Virus from the St. Louis Epidemic

Webster and Fite 1 showed recently that the virus isolated from cases of encephalitis in the St. Louis and Kansas City epidemics of 1933, passes through Berkefeld N candles in a relatively high concentration. While these findings indicate that the virus particles are exceedingly small, it was considered of interest to determine their size more accurately by filtration through graded collodion membranes of known porosity.

The membranes used in these experiments were prepared according to the method of El ford 2 with certain minor modifications adopted by Bauer and Hughes. 3 They were sterilized by steaming for one hour at 100°C. The average pore values were calculated according to Poiseulle's law from data obtained by measurement of the thickness of the membrane, its water content, the rate of flow of water through a known area, and the pressure producing the flow. These measurements were made after the sterilization.

The virus-containing material used consisted of fresh brain tissue from mice which had succumbed to the experimental disease. In most of the experiments 4 brains were ground in a sterile mortar, suspended in 100 cc. of a diluent of equal parts of hormone broth (pH 8.0), ascitic fluid, and distilled water. The suspension was centrifuged for 30 minutes at about 3000 r.p.m. The supernatant fluid was passed through a Seitz filter, and then through a collodion membrane with an average pore diameter of about 0.25μ. This stock filtrate was then passed through a series of membranes of different pore sizes. From 6 to 8 different membranes were used in each experiment. The effective filtration area for each membrane was about 5 sq. cm., and the amount of the virus suspension passed through such area was usually 10 cc. To reduce the amount of adsorption of proteins from the virus suspension by the membranes, 3 cc. of sterile broth was passed through each membrane before filtration of the virus suspension.