VIRULENCE OF THE BACTERIOPHAGE 157 



This method of purification by successive passages is the first one that 

 I employed. ^^^ But this method permits, however, the reasonable 

 objections that although it may be true that all of the bacteriophage 

 corpuscles avirulent for the bacterium at the expense of which the pas- 

 sages are made become ehminated it is none the less true that not all 

 of those which are virulent and which have multiplied necessarily belong 

 to the same race. As a matter of fact it is quite possible to assume that in 

 a filtrate of feces or of water not all of the corpuscles virulent for a given 

 bacterium are derived from a single corpuscle. According to this, 

 then, in order to be sure of working with a pure race it is necessary, as 

 is the case in bacteriology, to start with a single corpuscle. There are 

 two methods which will permit this; the method of dilution in liquid 

 media of Pasteur, and the method of colony isolation of Koch. 



We have seen that when we make serial dilutions, by tens, of a sus- 

 pension of the bacteriophage and add susceptible bacteria to each of 

 these dilutions bacteriophagy occurs up to a certain dilution. In the 

 higher dilutions it does not take place. If we prepare a second series of 

 comparable dilutions in sterile bouillon and distribute the 10 cubic centi- 

 meters of the last active dilution among 10 suspensions of the susceptible 

 bacterium, adding 1 cc. to each suspension, bacteriophagy will take 

 place in some of these 10 suspensions, wherever a single bacteriophage 

 corpuscle had been present. To be more certain of attaining the de- 

 sired end we may filter one of these suspensions and repeat the same 

 operation. This can be repeated even a third time. Such is the method 

 of "successive dilutions" which I have most frequently employed. I 

 prefer it to that of plaque isolation, for reasons which will be explained 

 later. Let us observe that with very virulent bacteriophages determina- 

 tion of the presence or absence of bacteriophagy is simple, inasmuch as 

 when it is present the suspension becomes completely cleared. With 

 less virulent bacteriophage races the formation of "secondary cultures" 

 frequently masks the clearing of the medium. Here, in order to as- 

 certain whether bacteriophagy has occurred it is necessary to spread the 

 suspected suspension on agar and the culture developing there will 

 provide the answer.* 



finite amount of the original filtrate involves the calculation of that degree of 

 dilution where the volume of the original material present is less than the volume 

 of one electron. The electron being the smallest possible mass of matter, to say 

 that as the result of the successive dilutions there remains in the liquid a quantity 

 of material less than one electron, is to say that there remains a hypothetical 

 but not an actual amount. 



* The characters of secondary cultures will be discussed in a following chapter. 



