SUMMARY: Four methods were employed to study the immediate fate of staphylococci within leucocytes after phagocytosis. These were: The method of Cohn & Morse where leucocytes and staphylococci were mixed in tubes and kept agitated. Periodically samples were removed and lightly centrifuged to sediment the leucocytes. Plate counts were made on the lysed leucocytes to measure the intracellular organisms, the supernatant to measure the extracellular population, and a non-centrifuged sample to determine the total number of viable cocci present. Concentrated suspensions of leucocytes and staphylococci were packed together by brief centrifugation to permit rapid phagocytosis without antibodies then diluted in cold Hanks solution to stop further phagocytosis. The suspension was then rapidly passed through a Servall centrifuge with the continuous flow attachment to remove excess extracellular organisms. The infected leucocytes were placed in suspension and samples removed and treated as in the previous procedure. Leucocytes and staphylococci were placed in plastic chambers containing a number of small coverslips. The cells were allowed to sediment and adhere to the coverslips, then washed to remove most of the extracellular organisms. Coverslips were removed at intervals and attached leucocytes lysed to liberate intracellular cocci which were enumerated by plate counts. Companion coverslips were washed, fixed, and stained in order to count the number of leucocytes present. The extracellular staphylococcal population was estimated by making plate counts on the tissue culture medium in the chambers. Suspensions of infected leucocytes were diluted and placed in a series of Petri dishes. The cells were permitted to settle and attach to the glass. Periodically the plates were washed and melted trypticase soy agar was added. After incubation the plate counts afforded an estimation of the viable cocci remaining within the leucocytes at each sampling.

The results obtained with these procedures were in fairly good agreement with each other. It was found that (18-Z and Smith strains) survived in significant numbers within monocytes and polymor-phonuclear leucocytes of normal rabbits for several hours. Little destruction of this organism during the first hour after phagocytosis could be demonstrated.


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