240 G. S. STENT 



particles, release of the progeny by lysis of the infected cells, reinfection of a 

 much greater number of bacteria, and repetition of the intrabacterial 

 multipUcation-lysis-reinfection cycle until a sufficient number of phage 

 particles have been produced so that all of the cells of the culture are infected 

 and finally lysed. Although this idea now appears simple enough, it did not 

 seem to be clearly understood by d'Herelle's critics. Bordet, for instance, 

 remarked at the occasion of his Croonian Lecture (Bordet, 1931): "It would be 

 difficult to suppose that an intrabacterial virus, originally present in small 

 amount and at first allowing the microbes to develop, attacks them all at a 

 given moment, almost simultaneously." D'HereUe, however, had some 

 experimental support for his view, since he could show that the multiplication 

 of the bacteriophages from a small inoculum of a few particles to the final 

 yield of several milhon progeny proceeds in a stepwise manner in the culture, 

 each step requiring about 75 minutes and leading to an approximately 20-fold 

 increase in the total concentration of phage particles. Burnet (1929) was able to 

 adduce more convincing proof concerning the stepwise nature of phage 

 multiplication by an improved experimental arrangement. He placed a 

 sufficiently small aliquot of a phage suspension into each of a large number of 

 tubes containing a small volume of a growing bacterial culture, so that each 

 tube received, on the average, one or two phage particles. This set of tubes 

 was then incubated and the total content of a number of single tubes plated 

 for assay at one-minute intervals on agar plates seeded with sensitive indicator 

 bacteria. The result of this experiment, the progenitor of the present day 

 "single-burst" technique, was that for the first 20 minutes after the bacteria 

 are first infected, each tube contains either none or only one or two plaque- 

 forming infective centers and that after more than 20 minutes have elapsed, 

 many of the tubes suddenly contain anywhere from 20 to 100 infective centers. 

 Burnet concluded from this result that: "the phage particle has clearly for 

 the first 20 minutes been multiplying under some spatial constraint which is 

 then suddenly released. When we combine this information with the fact that 

 when a large excess of phage is added to multiplying bacteria in broth or in 

 agar, visible lysis as judged microscopically or macroscopically occurs about 

 20 minutes after the addition, one must admit that the liberation from 

 spatial constraint can only be synonymous with the disruption of the invaded 

 bacterium and the associated liberation of (progeny) phage particles into the 

 medium." 



In 1939, EUis and Delbriick devised the one-step growth experiment, which 

 clearly demonstrated that the progeny of the infecting bacteriophage particle 

 appear only after a period of constant virus titer and provided also the proof 

 that no further production of bacteriophages takes place if reinfection by 

 phage progeny of any uninfected bacteria in the culture is prevented. The 

 one-step growth experiment, which today represents the basic procedure for 



