INTRACELLULAR MULTIPLICATION OF BACTERIAL VIRUSES 247 



parental bacteriophages is lost at the outset of the reproductive process, since no 

 infective particles whatsoever can be found in any of the lysates in which the 

 infected bacteria have been opened within 10 minutes following infection. 

 After more than ten minutes have elapsed, however, an ever-increasing 

 number of infective particles make their intracellular appearance until the 

 final crop of progeny has been attained which would have been released by 

 the spontaneous lysis of all the infected bacteria at the end of the normal 

 latent and rise periods. The time intervening between infection and first 

 intracellular appearance of infective progeny particles, i.e., the stage of 

 intracellular bacterial virus growth during which the infected host cell 

 contains no material capable of infecting another bacterium, is called the 

 eclipse (Luria, 1950). The discovery of the eclipse period confirmed an earlier 

 observation of Wollman and Wollman (1937), who opened up phage-infected 

 megatherium bacilli by lysozyme digestion of the cell wall and noticed that the 

 infectivity associated with the parental phage particles could not be recovered 

 from the artificial lysate. The WoUmans already inferred from their finding 

 that the infecting bacteriophage enters a noninfective, or "cryptophagic," 

 phase in the course of its intracellular growth. 



Other methods for premature lysis of phage-infected bacteria have been 

 devised since Doermami's experiment. Among these may be mentioned 

 sonic oscillation (Anderson and Doermann, 1952), explosive decompression 

 through sudden release from a high-pressure nitrous oxide bomb (Fraser, 

 1951), and treatment of the phage-infected bacteria with eithev glycine (Kay, 

 1952; DeMars, 1955), chloroform (Sechaud and Kellenberger, 1956), or 

 streptomycin (Symonds, 1957). In the case of phage-infected bacterial 

 protoplasts, whose cell wall has already been digested away by enzyme 

 treatment, immediate lysis can be induced at any moment by changing the 

 suspending medium of the protoplasts from one of high to one of low osmotic 

 pressure (Brenner and Stent, 1955; Salton and McQuillen, 1955). All these 

 methods reveal exactly the same sequence of events of mtracellular phage 

 growth, i.e., disappearance of the infectivity of the parental phage particle, 

 appearance within the cells of infective progeny particles after an eclipse 

 lasting for about half of the latent period, and increase in the number of 

 intracellular progeny at a constant rate. Such Hnear kinetics of appearance of 

 infective progeny naturally represent only an average over the entire infected 

 culture, and it was possible that in individual infected cells the increase in 

 intracellular phages follows rather different, e.g., exponential, kinetics. In 

 order to study the time course of phage growth in individual infected bacteria, 

 therefore, the techniques of premature lysis and single burst have been 

 combined. That is, a very large number of tubes, each containing less than 

 one infected bacterium, were incubated and premature lysis induced at 

 various times after infection. This experiment showed that the moment at 



