646 



INTRACELLULAR GROWTH OF BACTERI0PH.4GES. I 



The first experiments in devising a method of this kind were made with 

 phage T5 (3). It was found that T5 is Hberated before the end of the latent 

 period if the infected cells are exposed to a high excess of T6. However, the 

 extremely low rate of adsorption of T5 coupled with difficulties in inactivation 

 of unadsorbed phage by specific antisera indicated that this phage was a poor 

 choice. Hence T4 was chosen because of its fast rate of adsorption and because 

 of the availability of high titer antisera against it. The first experiments with 

 T4, along with the T5 results, showed conclusively that, by itself, lysis from 

 without is not sufficiently rapid for the purpose of this investigation. It is likely 

 that phage growth continues after the addition of the lysing agent T6. There- 

 fore the attempt was made to stop phage growth while T6 was allowed to ac- 

 complish lysis from without. Low temperature could not be used for this purpose 



TABLE I 



Composition of the Growth Medium 



Material 



KH0PO4 



Na2HP04 (anhydrous) 



NH4CI 



MgS04-7HOH 



Glycerol 



Acid-hydrolyzed casein 



rf/- Tryptophan 



Gelatin* 



Tween-80 



* To reduce surface inactivation of free phage particles (4) 



Amount 



gm. per liter 

 1.5 

 3.0 

 1.0 

 0.2 

 10.0 

 5.0 

 0.01 

 0.02 

 0.2 



since it also inhibits lysis from without. A search for a suitable metabolic in- 

 hibitor was therefore undertaken, and cyanide was eventually chosen as the 

 most suitable one. 



Materials and Methods 



The experiments described here were carried out with the system T4r48^ growing at 

 37°C. in Escherichia coli, strain B/r/l. The latter is a Tl resistant, tr>ptophan-de- 

 pendent mutant of B/r obtained from Dr. E. M. Witkin. 



Two media were used in these experiments, namely the growth medium and the 

 lysing medium. The composition of the growth medium used for both bacterial and 

 phage cultures, is given in Table I. The lysing medium consists of growth medium with 



^ The subscript refers to a particular r mutant of T4 which arose by mutation and 

 was numbered after the system of Hershey and Rotman (12) using the high subscript 

 number to avoid confusion with the mutants already described by Hershey and 

 Rotman. 



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