The Intracellular Growth of Bacteriophages

Abstract

Direct studies of bacteriophage reproduction have been handicapped by the fact that the cell wall of the infected bacterium presents a closed door to the investigator in the period between infection and lysis. As a result it was impossible to demonstrate the presence of intracellular phage particles during this so called latent period, and, much less, to estimate their number or to describe them genetically. This barrier has now been penetrated. It is the purpose of the first two papers of this series to describe two methods for disrupting infected bacteria in such a way that the intracellular phage particles can be counted and their genetic constitution analyzed.The first method used to liberate intracellular bacteriophage depends on the induction of premature lysis in infected bacteria by "lysis from without" which occurs when a large excess of phage particles is adsorbed on bacteria (1). It was found by nephelometric tests that T6 lysates are efficient in disrupting cells when moderately high multiplicities are used (2). The further observation was made that the addition of a large number of T6 particles to bacteria previously infected with T4, would, under some conditions, cause liberation of T4 particles before the expiration of the normal latent period of these cells. It therefore seemed hopeful that a method of reproducibly disrupting infected bacteria could be developed on the basis of this preliminary knowledge.

The Intracellular Growth of Bacteriophages

“…1 are quite analogous to those obtained with T4 (1). When the lysing medium is added to the infected bacteria before 12 minutes of their latent period have elapsed, practically no active virus particles can be recovered, not even the infecting ones.…”

“…The media used and the host strain of Escherichia coli, B/r/l, have been described previously (1). As before, all virus assays were done by the agar layer technique, making use of the mutant B/6 on which the T6 in the lysing medium does not form plaques.…”

“…Daughter T4 particles can be recovered from infected bacteria by poisoning their metabolism during development and adding an excess of the virus T6 to induce them to lyse prematurely (1). However, since the yields of virus depend on which poison is used, it seemed desirable to develop and test some radically different method for arresting metabolism and disrupting infected cells.…”

“…For arresting metabolism it was noted that the simple procedure of chilling infected cells would have the advantage of slowing down almost all reactions in the cells rather than blocking only certain enzyme systems. Furthermore, chilling does not induce infected cells to lyse as certain poisons do (1).…”

Effect of Infection with Phage Lambda on the Synthesis of Protein, RNA and DNA in Escherichia coli

“…a sample of the diluted culture was incubated for a further 30 min. in TB +0.02 M-KCN (Doerman, 1952;Weigle & Delbruck, 1951), and then assayed for phage particles. KCN+a drop of chloroform for 30 min.…”