1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 333 



From these experiments with phage T2 one may conclude that PHTR occurs 

 only for UVP adsorbed on sensitive bacteria and that illumination either of 

 UVP or of bacteria before infection has no detectable effect. 



To test how soon after phage adsorption PHTR can occur, UVP and bacteria 

 were mixed on several plates, and the plates were immediately exposed to the 

 light of an H-4 lamp at an 8-inch distance at 28 C. The exposure was continued 

 for 10, 20, 30, or 50 seconds. The plaque count was found to increase even after 

 10 seconds, showing that no measurable delay exists between adsorption and the 

 beginning of PHTR and that PHTR has no measurable latent period. 



Action of Bacterial Extracts on PHTR 



Some attempts were made to obtain PHTR by illuminating mixtures of UVP 

 with cell-free bacterial extracts. Bacteria were grown in nutrient broth to a 

 concentration of about 5 X 10^ cells per ml and harvested in a Sharpies centri- 

 fuge. Two extraction procedures were used: (a) the thick bacterial suspension 

 was frozen at —30 C, the frozen paste was then ground with carborundum pow- 

 der and extracted with phosphate buffer (pH 7.5) for about 10 minutes, and the 

 extract was clarified by centrifugation ; (b) the bacteria were broken in a sonic 

 vibrator after the bacterial paste was diluted with an equal volume of phosphate 

 buffer, and the extract was clarified in the centrifuge. In both cases the super- 

 natant was a thick, yellowish liquid, which showed a high degree of enzymatic 

 activity (methylene blue reduction, tryptophanase). Both extracts still con- 

 tained a few living cells, which could be eliminated either by filtration or by 

 repeated freezing at —30 C. 



UVP was mixed into various dilutions of the extracts, and the mixtures were 

 kept either in light or darkness and assayed for active phages at different times. 

 Only extracts still containing living cells gave some PHTR. Removal of almost 

 all living cells eliminated PHTR. 



PHTR as a Function of the Dose of the Inactivating UV Light 



For several phages of the T group (T2, T4, T5, T6) the curve obtained by 

 plotting the logarithm of the active fraction against the UV dose approaches 

 a straight line (Latarjet and Wahl, 1945), at least for high values of the dose, 

 whereas an inflection with downward concavity, of dubious origin, may appear 

 for low doses. Three other phages (Tl, T3, T7) show, on the contrary, an inflec- 

 tion with upward concavity of unknown origin. 



If the inactivated phages are adsorbed on bacteria and exposed to light of 

 high intensity for a sufficient length of time, the active fraction increases and 

 reaches a maximum (see later section). After this maximum is reached, the 

 curve showing the logarithm of the active fraction against the UV dose has for 

 each phage the same shape as the curve obtained in darkness, but for a given 

 UV dose the slope of the curve obtained after PHTR is lower than the slope of 

 the curve in darkness. 



The fact that both curves in the light and in darkness tend to be straight 

 lines with different slopes for high UV doses is an indication that absorption of 



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