Effect of Phage on Electrokinetic Potential of Susceptible Cells.,∗

Abstract

Earlier work 1 , 2 has served to establish the following essential facts concerning the phage-bacterium reaction:

A. Phage-production is conditioned by bacterial growth.

B. There is at all times a normal distribution of phage between susceptible cells and the surrounding medium providing the cells are alive, i. e.,

C. Lysis of bacteria depends upon the attainment of a critical ratio of phage to bacteria. In our experiments this threshold is approximately 100 activity units 3 per bacterium.

While the above relationships are significant for the development of a rational mechanism of phage action on bacteria 4 and have been proved to apply to more than one organism and the corresponding phage, 5 they give no evidence as to how phage induces cellular dissolution. Apparently phage does not measurably alter the normal bacterial growth-rate or the rate of cellular metabolism; 4 , 5 , 6 it may or may not bring about swelling of susceptible bacteria just before lysis begins. Bronfenbrenner 8 has found that phage produces hydrolytic cleavage of bacterial proteins although this does not seem to be a constant concomitant of phagic action. 9 Since the site of action has never ken clearly demonstrated and it is not even known whether phage taken up by a bacterium is inside the cell or merely held on the cell's surface, we felt that measurements of the electrokinetic potential of susceptible staphylococci which had been exposed to anti-staphylococcal phage would be of interest.

I. Measurement of the rate of electrophoresis. To estimate the electrokinetic potential the rate of electrophoresis of untreated and phage-treated staphylococci was determined. Two types of suspensions were employed:

A. Live Cells. A suspension of living staphylococci was made from a 16-hour culture of organisms which had been grown on agar, thoroughly washed, and resuspended in sterile water. From this dense suspension the following mixtures were prepared: 12±107 staphylococci per ml. in undiluted phage (1±10 10 activity units/ml.) and 12±10 7 staphylococci per ml. in broth. Both mixtures were kept in ice-water for 2 hours to allow equilibrium between intracellular and extracellular phage to become established. They were then centrifuged, the supernatants decanted, and the discarded solution replaced with an equal volume of secondary sodium phosphate and primary potassium phosphate buffer-mixture of pH 7.0.