:ui 



CHAPTER 26 



of this DNA pool is assembled into mature 

 phage, each genome surrounded by a newly 

 synthesized coat (head and tail) (Figure 



26 2D). About 20 to 40 minutes after in- 

 fection, the infected bacteria produce en- 

 zymes called endolysins which rupture the 

 bacterial cell wall and liberate infective 

 phage into the medium. 



This last step completes the lytic cycle 

 of a bacteriophage — the only one possible 

 for intemperate or virulent phages, such as 

 T. For temperate phages, this is one of 

 the two possible cycles — upon entering a 

 bacterium, the alternative is integrating with 

 the bacterial chromosome as a prophage, 

 thereby making the bacterium lysogenic and 

 immune. Even in this event, remember, 

 prophage occasionally dissociates from the 

 chromosome and replicates to produce in- 

 fective phage liberated by lysis of the host 

 cell. 



Virulent Phages 



Methods for assaying the amount of phage 

 present in a solution are based upon the 

 virus's capacity to lyse sensitive bacteria. 

 In one commonly-used method, the surface 

 of an agar-containing plate is heavily seeded 

 with sensitive bacteria which, upon incuba- 

 tion, will grow to form a continuous and 

 somewhat opaque lawn. When a few in- 

 temperate phage particles are mixed with 

 the sensitive bacteria before incubation, 

 each particle enters a different bacterium, 

 grows there, subsequently lyses the host, 

 and releases up to several hundred daugh- 

 ter phage. These particles proceed to at- 

 tack bacteria near the original host, caus- 

 ing them to lyse later. The repetition of 

 this cycle produces a progressively increas- 

 ing zone of lysis that is detected as a clear- 

 ing or plaque in the bacterial lawn. Under 

 these conditions each plaque is derived from 

 one ancestral phage and a count of plaques 

 therefore corresponds to a count of phage 

 in the infecting sample. 



The detailed appearance of a plaque de- 

 pends upon the medium, host, and phage. 

 When other factors arc controlled, dilTcrent 

 mutants of a phage may produce plaques 

 with characteristically dilTcrent morphology. 

 Plaque differences can involve size, turbid- 

 ity, presence or absence of a halo, nature 

 of the edges, and — when a dye is added to 

 the agar in the plate — color. The investi- 

 gator can. therefore, detect and maintain 

 phage mutants affecting plaque type. Ge- 



figure 26-3. Plaques produced by parental 

 and recombinant phage types. Progeny phage 

 of a cross between hr+ and h ' r were tested 

 on a mixture of suitable indicator bacteria. 

 The small clear and the large turbid plaques 

 are made by the parental types of phage prog- 

 eny (h r f and h • r, respectively). The large 

 clear and the small turbid plaques are produced 

 by the recombinant types of progeny (h r and 

 h + r+, respectively) . (Courtesy of A. D. 

 Hershey.) 



