EMORY L. ELLIS AND MAX DELBRUCK 367 



2 to 3 minutes, thus giving no opportunity for the multiplication of phage in the 

 liquid phase. Each step of each dilution was done with fresh sterile glassware. 

 Tests of the amount of phage adhering to the glass spreaders showed that this 

 quantity is negligible. 



The time of contact between phage and bacteria in the final dilution before 

 plating has no measurable influence on the plaque count, up to 5 minutes at 25°C. 

 Even if phage alone is spread on the plate and allowed to soak in for 10 minutes, 

 before seeding the plate with bacteria, only a small decrease in plaque count is 

 apparent (about 20 per cent). This decrease we attribute to failure of some 

 phage particles to come into contact with bacteria. 



Under parallel conditions, the reproducibility of an assay is limited by the 

 sampling error, which in this case is equal to the square root of the number of 

 plaques (10 per cent for counts of 100; 3.2 per cent for counts of 1000). To test 

 the effect of phage concentration on the number of plaques obtained, successive 

 dilutions of a phage preparation were all plated, and the number of plaques 

 enumerated. Over a 100-fold range of dilution, the plaque count was in linear 

 proportion to the phage concentration. (See Fig. 1.) 



Dreyer and Campbell-Renton (8) using a different anti-co/i phage and an anti- 

 staphylococcus phage, and a different technique found a complicated dependence 

 of plaque count on dilution. Such a finding is incompatible with the concept 

 that phage particles behave as single particles, i.e. without interaction, with 

 respect to plaque formation. Our experiments showed no evidence of such a 

 complicated behavior, and we ascribe it therefore to some secondary cause inherent 

 in their procedure. 



Bronfenbrenner and Korb (9) using a phage active against B. dysenteriae Shiga, 

 and a different plating technique found that when the agar concentration was 

 changed from 1 per cent to 2.5 per cent, the number of plaques was reduced to 

 1 per cent of its former value. They ascribed this to a change in the water 

 supplied to the bacteria. With the technique which we have employed, variation 

 of the agar concentration from 0.75 per cent to 3.0 per cent, had Httle influence 

 on the number of plaques produced, though the size decreased noticeably with 

 increasing agar concentration. (See Table L) 



Changes in the concentration of bacteria spread with the phage on the agar 

 plates had no important influence on the number of plaques obtained. (See 

 Table I.) The temperature at which plates were incubated had no significant 

 effect on the number of plaques produced. (See Table L) 



In appraising the accuracy of this method, several points must be borne in 

 mind. With our phage, our experiments confirm in the main the picture proposed 

 by d'Herelle, according to which a phage particle grows in the following way: 

 it becomes attached to a susceptible bacterium, multiplies upon or within it up 

 to a critical time, when the newly formed phage particles are dispersed into the 

 solution. 



In the plaque counting method a single phage particle and an infected bac- 

 terium containing any number of phage particles will each give only one plaque. 



39 



