Production of Phage in the Absence of Bacterial Cells. ∗

Abstract

In recent publications 1 2 it was shown: (a) that during one phase of the reaction between phage and bacteria in the presence of 0.25M NaCl phage is formed without concomitant bacterial growth; (b) that when pH and temperature are properly adjusted the bacterial substrate can be maintained in the resting state while [phage] rises logarithmically with time at the rate of a ten-fold increase per hour.

The obvious inference of these experiments is that bacterial growth, long held to be the prime conditioning factor for phage-formation, is actually not essential at all. Instead it would appear quite possible that resting cells may produce some sort of phage-precursor which in the presence of phage is promptly converted into more phage. This hypothesis is supported to a considerable extent by our observations during the past 3 years on the increase in phage-titer that occurs when cell-free ultrafiltrates of bacterial suspensions are added to known quantities of phage. Young cultures of susceptible organisms are washed in saline solution to free them from phage-inhibitor. 3 4 Dense suspensions of the washed cells are placed in infusion-broth of pH 7.4 at 36°C. for 2 hours. The fluid portion of the mixture is then separated from the cells by ultrafiltration through a 2% or 2.5% acetic-collodion membrane. 5 When equal quantities of the ultrafiltrate and known phage-preparations are mixed an increase in phage-titer occurs. For example when 1 ml. of ultrafiltrate is added to 1 ml. of phage containing 1×10 activity-units/ml.₆ the final titer remains 1 × 10 activity-units/ml. Such yields, 100% increases in the amount of phage originally added, are quantitatively significant inasmuch as the method of titration is capable of detecting differences in [phage] of ± M5%. Furthermore, when a solution of phage is subjected to serial dilution, using the ultrafiltrate as diluent, the actual titers are consistently higher than the concentrations calculated from the original amount of phage added and the difference between these values becomes progressively greater as the range of dilution is extended.