1. Penicillin was found to be actively spirocheticidal in vitro against the Reiter, Kazan, Nichols, and Noguchi strains of so called S. pallida, and a strain of mouth spirochetes. The threshold concentration was 0.01 unit per Cc. (1–160,000,000 penicillin). The rate and degree of action increased with the concentration of penicillin up to a level of approximately 0.1 to 0.25 unit per cc., which rendered more than 99 per cent of the organisms non-viable within 12 hours. Higher concentrations did not appreciably accelerate the effect.

2. Within the range 4 x 104–107 organisms per cc., the initial rate at which the spirochetes were killed was not affected by their number. Consistent with that observation, no demonstrable penicillin was bound or inactivated by thick suspensions. The amount of penicillin required to sterilize suspensions of varying density nevertheless varied to a large extent with the initial number of organisms. This was only in part due to the progressive deterioration of the penicillin with prolonged incubation; and the persistence of organisms resistant to the drug, and perhaps an adaptative change after prolonged exposure to penicillin, may be contributing factors.

3. The organisms remained actively motile for a period of 8 to 24 hours after they had been rendered non-viable by the action of penicillin. Even 500 units of penicillin per cc., or approximately 10,000 times an effectively spirocheticidal concentration, did not accelerate that delayed immobilization. It follows that, although penicillin rapidly renders the organisms non-viable, the metabolic system affected is not immediately essential to the life of the cell, and the motility and presumably other vital functions remain unaffected for a significant number of hours.

4. The rate at which the organisms were killed by penicillin increased with temperature in the range 8–40°C. With an original inoculum of 106 spirochetes per cc., the percentage of organisms surviving after 24 hours at 39–40°, 36–37°, 32–33°, 22–23°, and 8°C. was 0.02, 0.2, 1, 10, and 100 respectively; and those results were independent of the concentration of penicillin in the range 0.25 to 250 units per cc. If these observations with a non-pathogenic organism in vitro are applicable to the pathogenic organism in vivo, they suggest that the combined use of fever and penicillin in the treatment of syphilis may be more effective than either alone.

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