FIG. 1.

Experimental protocol for evaluating Tetrahymena-phage interactions. (A) Experiments began by adding T4 bacteriophage to T. thermophila in medium, usually Osterhout's solution, and incubating this culture in 75-cm² tissue culture flasks that were maintained on an orbital shaker (50 rpm) at room temperature. (B) At various times afterwards, a 1-ml sample of the culture was transferred to a 15-ml sterile plastic centrifuge tube. (C) A long glass Pasteur pipette was used to layer 1 ml of Histopaque on this sample, after which the tube was centrifuged at 400 × g for 10 min at room temperature. (D) After centrifugation, a pellet was observed and a distinct top layer (the medium) and a distinct bottom layer (the Histopaque) were evident. The distribution of Tetrahymena cells, which are indicated as ovals in the diagram, was monitored by phase-contrast microscopy, and the distribution of phage, which are indicated by X, was monitored by double-agar overlay. After the step gradient centrifugation, all of the Tetrahymena cells were found as a pellet, regardless of the experiment. In contrast, the titer of the phage in panel D, which was usually expressed relative to the titer added in panel A, varied with the experiment.