Procedures in the Isolation and Cultivation of an Organism from Berkefeld N Filtrates of Poliomyelitis Virus

I. Culture Mediums. A. “E B” Medium (Sheep brain after extraction with ethyl alcohol and benzol). Sheep brain, removed aseptically at the slaughter house, was washed thoroughly in sterile physiological salt solution, freed from blood and membranes, and ground finely in a meat chopper. The material was extracted once with 2 volumes of 95% ethyl alcohol overnight (12-16 hours) at room temperature (20-22°C.) and dried in air. The tissue residue was extracted in the same manner twice with 2 volumes of U.S.P. benzol. The resulting finely powdered dry material, in the proportion of 2 gm. to 10 cc. of 0.85% sodium chloride solution, was mixed thoroughly and sterilized in the autoclave at 15 pounds pressure for 20 minutes. The final pH was adjusted to 7.6.

B. “V B” Medium (Minced sheep brain in veal infusion broth). This substrate consisted of sterilized minced sheep brain contained in a veal infusion broth to which no peptone had been added.

Fresh sheep brain, removed aseptically at the slaughter house, was washed thoroughly several times in sterile physiological salt solution, freed from blood and membranes, and one kilo of tissue added to one liter of sterile veal infusion broth, pH 8.0, to which no peptone had been added. The mixture was coagulated thoroughly by boiling for 2-3 minutes and 0.2% glucose by volume was added. The cooled material was next passed 2 to 3 times through a meat chopper and minced. Test tubes measuring 1.5×15 cm. were filled to a depth of approximately 6 cm. (roughly 10 cc. by volume) and sterilized in the autoclave at 15 pounds pressure for 20 minutes. A second sterilization at 15 pounds pressure for 10 minutes was given on the following day, whenever the technical procedures in the preparation of the material warranted such added precaution.