The growth of a virulent Type I pneumococcus in an automatic transfer device on a medium which aerobically maintains virulence results in a decrease in the presence of pure oxygen or pure carbon dioxide, but in no change in the presence of nitrogen. Whereas partial pressures of oxygen, that is 5 parts with 95 parts nitrogen by volume, and 10 parts with 90 parts nitrogen, resulted in a decrease in virulence, with 20 parts oxygen and 80 parts nitrogen, the decrease was almost negligible. Media which under aerobic conditions failed to furnish suitable constituents for maintenance of virulence, that is spleen, aminoid peptone, powdered milk, beef infusion, whole rabbit, and rabbit muscle, also failed under nitrogen. Media from whole guinea pig kept the organism virulent for 246 transfers under nitrogen, but with some decrease. Media made from discard mice, survivors of pneumococcus infection, furnished conditions under nitrogen, which caused first a decrease of virulence, but later after 246 transfers, a restoration to a more virulent state. Gradual increase in temperature over a period of 10 days from 36.5°C. to 42°C. in a sample of medium, which was otherwise suitable for maintenance of virulence under aerobic conditions, resulted in a decrease in virulence.

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