VIRULENCE OF THE BACTERIOPHAGE 151 



0.05 cc. on another plate, inoculating the 0.5 cc. into a third suspension. 

 This will give a 10~^ dilution. If there is reason to beheve that the race 

 is very active continue in this same way up to the twelfth tube (dilu- 

 tion 10~^2)_ If i\^Q pace is of but moderate potency it is unnecessary to 

 go beyond the tenth dilution. It is also unnecessary to prepare plates 

 corresponding to the last two dilutions, 10~^^ and 10"^^^ qj.^ jf ^j^g pg^^jg 

 is of less virulence, those corresponding to the 10"^ and lO-^" dilutions. 

 In these extreme dilutions the number of corpuscles is too small for them 

 to appear, except by accident, in the 0,05 cc. spread over the agar. 



I would recommend placing the suspension dilutions in the incubator 

 at 32°C. To obtain plaques of the largest possible diameter, and for 

 this reason more readily counted, even in the case of weakly active 

 bacteriophage races* it is preferable to hold the plates at a temperature 

 of 20° to 22°C., in an incubator such as is used for gelatin media. 



After incubation the series of dilutions will permit an evaluation of 

 the intensity of bacteriophagy in relation to the dilution of the active 

 principle. But this is but a tentative measure, the less trustworthy 

 in that in many cases "secondary cultures" associated with the phenom- 

 enon of bacterial resistance, develop, rendering such a titration com- 

 pletely false and obscuring the true result. The exact measure of 

 virulence can be obtained only by counting the colonies of the bacterio- 

 phage. In the series of plates there are always two, sometunes three, 

 plates upon which the plaques are separated and easy to count. Since 

 the quantity of suspension spread upon the surface and also the degree 

 of dilution are known, it is easy to calculate the number of corpuscles 

 contained in the original suspension. 



This method, so well suited to virulence determinations, is also the 

 most practical procedure for studying the various phases of the phe- 

 nomenon of bacteriophagy. If one wishes to follow, for example, the 

 course of the phenomenon under definite determined conditions, it is 

 only necessary to remove at suitable intervals 0.5 cc. of the medium 

 in which bacteriophagy is taking place and to carry out the technic 

 which has been outHned in order to know the number of corpuscles 

 present at the moment when the specimen was removed. 



Can the method be used to measure the relative degrees of virulence 

 of different bacteriophages, when by serial passages we know that the 

 materials in question contain the active principle? Here is a typical 



* We have seen that the diameter of the plaque is proportionate to the viru- 

 lence, at least when the bacteriophage is acting upon strains of the same bacterial 

 species. 



