STUDIES ON PNEUMOCOCCUS GROWTH INHIBITION : II. A METHOD FOR DEMONSTRATING THE GROWTH-INHIBITORY AND BACTERICIDAL ACTION OF NORMAL SERUM-LEUCOCYTE MIXTURES.

Somewhat discordant results which have been reported by others who have investigated the property of the whole blood of resistant animals to cause inhibition of growth or death of pneumococci have led us to investigate this matter and to develop a new technique in which the conditions as they are present in the animal body are more nearly imitated. The observations already made have rendered it probable that phagocytosis plays some rôle in any destructive power for pneumococcus which whole blood possesses. We have, therefore, employed mixtures of serum and leucocytes in our tests, since when blood is coagulated the conditions become highly artificial. Furthermore, in order to imitate more nearly the conditions in the circulating blood the mixtures have been constantly, though gently, agitated. For this purpose a specially devised apparatus has been employed. The mixtures of serum and leucocytes have been inoculated with varying numbers of pneumococci in the active growth phase and after varying intervals of time the tubes containing the mixtures of serum, leucocytes, and bacteria have been opened, examined microscopically, and cultures made. Employing this technique it has been found that the growth of pneumococci having low virulence for cats is markedly inhibited in mixtures of cat serum and cat leucocytes. It was impossible to recover pneumococci from the tubes showing no apparent growth, either when the contents were transplanted into various kinds of culture media, or when the contents were injected into mice of a variety highly susceptible to pneumococcus infection. 10,000 times the number of pneumococci sufficient ordinarily to kill a mouse failed to do so after a 24 hour sojourn in the cat serum-leucocyte mixture. Mixtures of dog serum and leucocytes exert a similar action. The serum and leucocytes of animals susceptible to pneumococcus infection (rabbits and guinea pigs,) on the other hand, failed to injure pneumococci even in extremely small quantities. These results indicate that the blood of resistant animals, at least of the dog and cat, possesses destructive properties for pneumococci, and that this destructive power is not possessed by the blood of certain susceptible animals. The experiments suggest that natural immunity depends chiefly, if not entirely, upon this property. The leucocytes play an active part in this process, but whether the destruction of the pneumococci occurs entirely within the leucocytes or not is not determined. That the serum also plays a part is shown by the fact that when the serum of resistant animals was inactivated before being used in the serum-leucocyte mixture, the growth of even very small numbers of pneumococci was not prevented. Further experiments with cat serum and leucocytes were carried out to determine the optimum rate and time of agitation, the amount of serum and leucocytes required, and also the period of incubation necessary for the inhibition of growth and death of the pneumococci to occur.