FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

Abstract

The reaction between an antistaphylococcal phage and the homologous bacterium has been studied, applying the following exptl. technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining (phage). (b) Sampling was done at short intervals, i.e., every 0.1 hour, (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of >95% of free phage. Using these technics, the reaction was followed: (a) with pH maintained at 6.1 and temp, at 28[degree]C to slow the process; (b) with pH maintained at 7.2 and temp, at 36 [degree]C. In addition, separate expts. were performed on the sorption of phage by bacteria at 30[degree], 23[degree], and 0[degree]C. At pH 6.1 and 28 [degree]C the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant, (b) When 90% of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques, i.e., phage is formed intracellularly, but is retained within cellular confines, (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque), (d) With the concs, of phage and bacteria used in the expt. carried out at pH 6.1 and 28[degree]C, there are 2 further increments in (phage)act. before massive lysis begins, (e) During terminal lysis there are sharp rises in the curves for (total phage)plaq., (extracellular phage)act., and (extracellular phage)plaq. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being insufficient size to be removed by the super-cel filters. At pH 7.2 and 36[degree]C the steps in the phage production curve, as detd. by activity assay and plaque count, are much less prominent than those observed at pH 6.1 and 28[degree]C. However, the plateaus described by Ellis and Del-bruck for Escherichia coli and coli phage can be detected also in the present case if frequent samples are taken. The sorption expts. show a significant rise in the rate of phage uptake with increase in temp., again supporting the view that the reaction involves more than a purely physical adsorption. Delbruck''s objections to: (a) the use of the activity assay for determining (total phage) in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in soln. or phage bound to living susceptible cells, under the conditions of the expts. reported here and in earlier work. The titration values obtained in the expts. designed to exhibit intracellular phage precursor are not the result of artifacts as Delbruck has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.