148 THE BACTERIOPHAGE AND ITS BEHAVIOR 



doubtedly still present, since it may be revealed by passages. The 

 cause for this difficulty is readily explained. Let us suppose that the 

 hmiting dilution is one where there is but a single corpuscle in the whole 

 volume of the dilution. This is, as we know, sufficient to cause bac- 

 teriophagy. Let us remove a drop of this dilution and spread it upon 

 agar. Either the corpuscle will be in the drop taken or it will remain 

 in the rest of the fluid in the tube, and naturally, the second possibility 

 is the greater. What will happen? In the first case we will have a 

 plaque when the spreading is made on the agar, and as no bacteriophage 

 corpuscles will be left in the dilution bacteriophagy will not take place 

 there. The deduction here would be that bacteriophagy in the fluid 

 medium does not take place when but few corpuscles are present, even 

 though they are very active. In the second case, it may be concluded, 

 as Otto and Munter decided, that bacteriophagy in a fluid medium takes 

 place, even when plaques do not appear after the material is spread upon 

 agar. Both conclusions are equally false, and a defective method of 

 titration is responsible for the errors. 



Maitland^^- has proposed a method of titration also based upon the 

 principle of counting the plaques found on agar. He prepares 6 dilu- 

 tions, from 10~^ to 10~*', in suspensions of the susceptible bacterium. 

 A fixed and uniform quantity of each of these dilutions is spread upon 

 agar, and after incubation he denotes the appearance of the agar cul- 

 tures by numbers, as follows : 



1 = 1 to 10 plaques 



2 = 10 to 20 plaques 



3 = numerous plaques 



4 = confluent plaques 



5 = culture broken up into irregular masses 



6 = traces of culture growth 



7 = culture debris 



8 = isolated colonies 



9 = but a single colony, or medium sterile 



Here is an example of such a titration, showing how, by repeated 

 platings he observes the multiplication of the bacteriophage in the course 

 of bacteriophagy. The titration is performed with a culture of B. 

 dysenteriae 24 hours old, to which is added an appropriate quantity of 

 the filtrate prepared from the feces of a patient convalescent from bacil- 

 lary dysentery. From time to time (as indicated) specimens of the 

 mixture are removed, dilutions are made, and agar plates are spread. 

 The results obtained are given in table 13. 



