64 s. BENZER [vol. 63 



halt; the resistance remains constant for hours provided the sample is kept 

 chilled. The pipettes used for addition of broth and removal of samples are 

 previously equilibrated at 37 C in an incubator. 



In this manner, growth may be started and stopped in all cells simultaneously, 

 and the timing controlled to within a few seconds. Furthermore, many samples 

 may be taken at close intervals within the same growth experiment and the 

 irradiation conducted afterwards at leisure. 



For samples chilled very close to the end of the latent period (after 16 minutes 

 for T2r or 9 minutes for T7) lysis is not prevented by chilling. After a delay, 

 there appears a gradual increase in plaque count, presumably due to slow lysis 

 of cells containing completed phage particles. 



3. Irradiation. One or two ml of the suspension to be irradiated are placed in 

 a shallow watch glass. The suspension is transparent to ultraviolet by virtue of 

 the large dilution (100 X) of the broth. Ice in a small dish is used to chill the 

 suspension from below (figure 4). This prevents growth during the irradiation. 

 Chilling also serves to reduce greatly the rate of photoreactivation (Dulbecco, 

 1950) which might otherwise be caused by the visible light emitted by the 

 ultraviolet lamp. 



Figure 4- Arrangement for chilling sample during irradiation. 



Ultraviolet is supplied by a 15 watt "germicidal lamp" (General Electric 

 Company). The energy emitted in the 2537 A line accounts for almost all the 

 antiphage activity. The intensity of ultraviolet is such that T2r survives to the 

 extent of 10~- after an exposure of 40 seconds. This same intensity is used 

 throughout the experiments, and the doses are, therefore, given in units of sec- 

 onds. All manipulations after irradiation are conducted in dim yellow light to 

 minimize the possibility of photoreactivation. 



4. Plating. Aliquots of samples subjected to various doses of ultraviolet are 

 plated in a top layer of soft agar, seeded with unirradiated B, on broth agar 

 plates. The resultant plaques are counted to determine the fraction of complexes 

 whose infectivity has survived the irradiation. 



Note on multiflicity of infection. It is essential that the proportion of phage 

 particles to bacteria be kept quite small in order that very few multicomplexes 

 (i.e., bacteria infected with more than one phage particle) be formed. This is 

 particularly important in the case of T2, where the phage exhibits multiplicity 

 reactivation and the multicomplexes are much more resistant to ultraviolet 

 than monocomplexes. Thus, if one assumes a Poisson distribution of phages 

 per bacterium, and a survival value of 10"^ is to be accurate within 10 per cent, 

 the average multiplicity of infection must be 2 X 10~^ or less. In order to have 



303 



