1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 61 



This result may also be stated in the following way : complexes formed by infec- 

 tion of a cell with two phage particles do not have a survival curve corresponding 

 to the two-target curve of figure 1 ; at any given dose of ultraviolet the probability 

 of the complex being infective is greater than given by that curve. This effect 

 occurs to a marked degree with phage T2 and would be expected to cause anom- 

 alous results in an experiment of the Luria-Latarjet type, where intracellular 

 multiplication is going on. However, there are other phages, T7 for instance, 

 with which multiplicity reactivation does not occur, and it seemed of interest 

 to extend the experiments to such a phage. 



Another possible cause of anomalous results is "photoreactivation" of phage 

 (Dulbecco, 1950). Ultraviolet inactivated phage particles may be reactivated, 

 after adsorption to a sensitive bacterium, by exposure to white light. Thus, we 

 may expect the infectivity of a phage-bacterium complex to have a higher 

 resistance to ultraviolet if exposed to light (after ultraviolet irradiation) than 

 if kept in the dark. Precautions are therefore necessary in order to avoid this 

 effect. 



MATERIALS 



Phages: T7, T2, and T2r, prepared from lysates in broth, purified by centrif- 

 ugation, and resuspended in buffer. 



Bacterium: Escherichia coli, strain B, grown in broth. 



Growth medium (broth): bacto-tryptone, 1 per cent plus 0.1 m NaCl. 



Buffer: 1/15 m phosphate buffer, pH 7, plus 0.1 m NaCl, plus lO"' m MgS04. 



METHODS 



A Luria-Latarjet experiment involves the following steps: 



1. Phage particles are added to a suspension of bacteria and time is allowed 

 for infection of the cells to occur, then unadsorbed phage is eliminated. 



2. The complexes are allowed to develop and samples are removed at various 

 stages of the latent period. 



3. Each sample is exposed to several doses of ultraviolet. 



4. Aliquots are plated to determine the fraction of infective centers surviving 

 each dose. These operations must be completed before the end of the latent 

 period. Furthermore, since the radiation resistance of the infective centers 

 changes rapidly with time, it is essential for accurate results that growth start 

 almost simultaneously in all cells and that it be halted during irradiation. 



In an attempt to best satisfy these requirements, the following procedure is 

 used : 



1. Adsorption of phage without growth. The bacteria are prepared from an 

 aerated broth culture in the logarithmic phase (1 X 10^ cells per ml). The cells 

 are centrifuged, resuspended in buffer, centrifuged again, resuspended in buffer 

 at a concentration of 1 X 10^ per ml, and aerated by bubbling at 37 C for one 

 hour in order to exhaust intracellular nutrients and bring the bacteria to a starved 

 condition. A purified suspension of phage particles in buffer is then added. 

 Under these conditions adsorption takes place, but no lysis or phage liberation 



300 



