1950] PHOTOKE ACTIVATION OF INACTIVATED BACTERIOPHAGES 331 



filled with distilled water and passed through suitable filters (see later section); 

 for white light experiments infrared rays were absorbed by a filter of CuS04- 

 5H2O (5 per cent in water, 1 inch thick) and ultraviolet rays shorter than 330 

 m/i by a Corning glass filter no. 738. A mixture of phage and bacteria was ex- 

 posed to light in a small beaker (5 ml of mixture in a beaker 4 cm in diameter) 

 kept in a thermostatically regulated water bath and shaken by a reciprocating 

 motion in a horizontal plane to ensure uniform distribution of the material and 

 uniform illumination. Some experiments were done with a 100-watt General 

 Electric Ii-4 lamp without a condenser. 



In the experiments with illumination in liquid the ratio "phage particles : bac- 

 teria" was kept very low (about 10"^) to decrease the probability of multiple 

 infection of bacteria and the occurrence of reactivation by multiplicity (Luria, 

 1947). 



EXPERIMENTAL RESULTS 



Role of the Bacteria in the PHTR of Inactive Phage 



Phage particles inactivated by UV (UVP) can be reactivated by light only if 

 the particles are mixed with sensitive bacteria during illumination. Illumination 

 of UVP alone is without effect, as is shown by the following experiment : Phage 

 T2 was irradiated with the germicidal lamp for 30 seconds (dark survival = 

 2 X 10"^) and divided into two equal samples. The first sample was immediately 

 plated and incubated in darkness; the second one was exposed to the light of a 

 fluorescent lamp (80 watts at a 12-inch distance) for 1 hour at room temper- 

 ature and then divided into two parts, one of which was plated and incubated 

 in darkness, the other under the same light. The sum of plaque counts of two 

 plates for each sample are given in table 1 (I). 



In another similar experiment the UVP was first spread on the surface of a 

 nutrient agar plate and then exposed to the light; after illumination sensitive 

 bacteria were spread on the same plate in darkness. In this condition also PHTR 

 was not produced. 



These experiments clearly indicate that illumination of UVP in the absence 

 of bacteria has no reactivating effect; they do not show, however, whether PHTR 

 occurs only for adsorbed phage or also for nonadsorbed phage in the presence of 

 bacteria. This point was investigated by mixing UVP with bacteria in nutrient 

 broth without added NaCl (under these conditions the adsorption is slight), 

 illuminating the mixture, and testing for reactivation of the nonadsorbed phage 

 particles. A sample of phage irradiated with the germicidal lamp for 30 seconds 

 was mixed with a culture of bacteria in broth without added NaCl, containing 

 10^ cells per ml. The mixture was exposed to the light of an H-4 lamp at a 6- 

 inch distance for 10 minutes at 28 C, then centrifuged; samples from the super- 

 natant were plated and incubated both in darkness and in the light. The plaque 

 counts (two plates for each sample) are given in table 1 (II), together with an 

 assay of the irradiated phage diluted in broth by a factor equal to the one used 

 in the experiment. The result of this experiment clearly indicates that the un- 

 adsorbed phage particles are not reactivated by light. 



230 



