VIRULENCE OF THE BACTERIOPHAGE 147 



recorded as + + + +, meaning sterility, + ++, meaning confluent 

 plaques, ++, many plaques, or +, indicating that plaques are few. 



This is an excellent method for approximating quickly the degree of 

 virulence of a bacteriophage, but it is not sufficiently precise for a study 

 of the detailed features of the process of bacteriophagy. The authors 

 who devised the method have used it for studies to which we will give 

 considerable attention, and for which the method yields fully adequate 

 results. 



Pfreimbter, Sell and Pistorius^°" follow a procedure which consists in 

 adding a drop of the bacteriophage suspension to be titrated to a sus- 

 pension of the susceptible bacterium and spreading a definite quantity 

 of this mixture upon agar plates, immediately after preparing the mix- 

 ture, and again after intervals of 3, 6, and 24 hours. After the plates 

 are incubated the presence of plaques and the number found provide 

 the significant data. 



This is a very good method, especially for comparing the virulences 

 of races that are weak. With highly active races it is necessary to work 

 with dilutions. 



According to the technic of Otto and Munter,^**" a culture of the sus- 

 ceptible bacterium is spread over the surface of an agar plate and upon 

 this surface drops of the suspension to be titrated, diluted to different 

 degrees, are deposited. After incubation the areas where the drops of 

 bacteriophage were deposited are observed and the results are recorded 

 as 0, meaning a normal bacterial culture, that is, no bacteriophage, 

 ±, indicating a few plaques,* +i and +2 implying that plaques are 

 numerous or confluent, +3 means that only isolated bacterial colonies 

 develop, and +4 denotes that the surface of the ac,ar is sterile. A 

 record of such a titration, employing this notation, is : 



Dilution, 1 : 1000, + 4 

 Dilution, 1:1,000,000, +2 

 Dilution, 1:100,000,000 ± 



In effect, this procedure is simply a modification of the method of 

 counting colonies of the bacteriophage. But it lacks accuracy, and it is 

 precisely this defect which has caused those who devised the method to 

 arrive at certain erroneous deductions.^^* For example, they observe 

 that when greater and greater dilutions of a fluid containing the bacterio- 

 phage are made, a degree of dilution is reached where spreadings no 

 longer show plaques, although in this dilution active principle is un- 



* A rather curious notation, for the sign ± usually indicates a doubtful result. 

 If there are plaques, there is no chance for doubt, the bacteriophage is present. 



