158 THE BACTERIOPHAGE AND ITS BEHAVIOR 



The dilution method can be appKed even in the case of races of low 

 virulence, provided bacteriophagy in the hmiting suspensions is con- 

 trolled by spreading them on agar. However, with races of low viru- 

 lence a purification by means of isolation through plaques is certainly 

 less compHcated. 



We have seen that it is only necessary to touch the centre of a plaque 

 with a sterile platinum wire and then immerse this wire in a suspension 

 of the susceptible bacterium to cause bacteriophagy of this suspension. 

 Each plaque on the agar being a colony of bacteriophage corpuscles 

 resulting from the multipHcation of a single corpuscle, we have here a 

 method of isolation absolutely analogous to that discovered by Koch 

 for the isolation of bacteria. But we may mention the fact that as is 

 often the case with bacteria, the colony developing at a given point 

 on the agar may be derived, not from a single corpuscle, but from a group 

 of two or more. This being the case, it is unwise to rely upon a single 

 isolation. Certainty demands that the process of isolation be repeated 

 two or three times. 



The method of isolation by plaques has been employed especially by 

 Bail and his collaborators^^ who were the first to call attention to the 

 possibiUty that a filtrate possessing activity for a single bacterial species 

 could contain a mixture of bacteriophage races. Wolif and Janzen''^*' 

 have likewise made this observation in studying different races of 

 Typho-bacteriophage. 



It may be well to emphasize the fact that if we would effect isolation 

 by the plaque method it is necessary to proceed in the following manner. 

 Inoculate a heavy suspension, 250,000,000 bacteria per cubic centimeter, 

 with a very small quantity of the raw suspension of bacteriophage. 

 When this mixture is spread immediately upon agar the surface should 

 show, after incubation, but very few plaques. If too many corpuscles 

 are inoculated into the bacterial suspension it is possible that several 

 will become attached to a single bacterium and the plaque will be formed 

 of elements not the issue of a single corpuscle. Furthermore, for this 

 purpose Petri dishes should not be used. Agar slants are much better, 

 since they may be placed in a vertical position immediately after plant- 

 ing in such a way that it will insure that none of the liquid remains on 

 the surface. The presence of fluid facilitates the development of the 

 corpuscles, as in a Hquid medium, and if this occurs the isolation will 

 be fictitious. 



