-loS It ADI AIION moLOGY 



ill ilaikiicss and the other iiiidcr a .source of white hglit, tlie j^hilc; kept in 

 the hf^ht shows after incubation a much greater numl)er of bacteriophage 

 coh)nies; this is the basic experiment of photoreactivation of bacterio- 

 phages. A simihir experiment can be done by mixing bacteria and bac- 

 teriophages in a nonnutrient medium, usually a buffer solution, so that 

 adsorption of tiie bacteriophages onto the sensitive bacteria can take 

 place, but not growth. The mixture is exposed to a white light and then 

 phited with sensitive bacteria on a plate of nutrient agar, which is incu- 

 bated in darkness. 



lioth these methods are useful. The first has been called the method 

 of photoreactivation on the plate; the second, the method in liquid. The 

 choice of the method to be used depends on the type of information 

 sought. The method on the plate is used for orientation experiments, for 

 testing for c|ualitative effects, and for some quantitative studies, par- 

 ticularly with phages which adsorb slow^ly (e.g., Tl and T5). The 

 method in li(iuid is generally used in quantitative work; it has been suc- 

 cessively applied to bacteriophages T2, T3, T4, and TO. 



3-3. ADSORl'TIOX OF I'lIAGJ-: OX BACTERIA A NECESSARY 

 PREREQUISITE FOR PHOTOREACTIVATION 



Free phages, inactivated with ultraviolet and then exposed to the 

 photoreactivating light in the absence of bacteria, are not reactivated ; for 

 photoreactivation to occur the particles must be adsorbed on the sensitive 

 bacteria. Demonstration that adsorption is required for photoreactiva- 

 tion has been obtained by mixing sensitive bacteria and irradiated bac- 

 teriophages under conditions which do not permit adsorption, exposing 

 the mixture to light, and then plating a sample from the mixture; the 

 fraction of active particles remains unchanged. The condition for non- 

 adsorption can be realized either by suspending phage and sensitive bac- 

 teria in a solution which has an ionic compo.sition unfavorable for adsorp- 

 tion or by mixing a phage with cells of a mutant of E. coli B resistant to 

 the phage (e.g., phage Tl and bacteria B/1). 



Two types of adsorption of bacteriophage particles on the sensitive 

 bacteria occur, depending on the ionic strength of the medium: when the 

 ionic concentration of the medium is too low, adsorption is reversible — 

 the adsorbed phage can be eluted with distilled water (Puck et al., 1951); 

 at high ionic concentration adsorption becomes irreversible. Photo- 

 reactivation takes place only if the phage has been adsorbed irreversibly 

 (Dulbecco, unpul)lished). 



Bacteriophages adsorbed on bacteria in a medium which does not per- 

 mit growth remain perfectly photoreactival)le over a period of several 

 hours. The medium may l)e either a hulTcr, in which case the bacteria 

 are taken from a logarithmic pha.se culture, washed and resuspended in 

 the buffer, or a .synthetic medium containing a limited amount of glucose 



