144 THE BACTERIOPHAGE AND ITS BEHAVIOR 



2. With bacteriophage races of average activity ''secondary cultures" 

 (of which we will speak in the next chapter) develop, and where this is 

 the case the method of dilutions gives results which are entirely false. 



3. For very virulent bacteriophages the precision of the method is in 

 inverse proportion to the degree of dilution. Yet, with such races, it 

 is in the high dilutions that precision is most essential. As illustrating 

 this, here is a titration of a suspension of Staphylo-bacteriophage made 

 by the dilution method. Duphcate titrations are made. 



First titration. The last dilution in which bacteriophagy takes place 

 is l-lO^i". From this one might conclude that in 1-10"^° cc. there is 

 but one corpuscle, and that the original suspension contained, therefore, 

 10,000 million per cubic centimeter. 



Second titration. In this the last dilution to show bacteriophagy 

 is 1-10~". Applying the same reasoning, this time the suspension 

 should contain 100,000 million per cubic centimeter. Obviously these 

 two determinations present a very considerable discrepancy. 



By means of counts of the colonies of the bacteriophage developing 

 on agar we find an explanation of the difference. As a matter of fact 

 these counts show that the original suspension contained 81,000 million 

 corpuscles per cubic centimeter. There were in reality 8 corpuscles 

 in the 10 cc. of the 1 •10"^'' dilution. Consequently, the l-10~^i dilu- 

 tion shows, or does not show, bacteriophagy, depending upon whether 

 the particular fraction removed from the 10~^° tube contained, or did 

 not contain, a corpuscle. 



In the first dilutions, the error resulting from this fact is negligible. 

 It would be a maximum of 90 corpuscles between 10~^ and 10"^, of 900 

 corpuscles between 10~2 and 10"^. But it becomes enormous in the 

 higher dilutions. The error may amount to 900 million between 10~^ 

 and 10-9; to 9000 million between 10-^ and IQ-i"; and to 90,000 milhon 

 between lO-^" and lO-^i. 



This error of technic makes it readily apparent that errors of inter- 

 pretation of considerable significance may result from the use of such a 

 method, a fact all the more unfortunate since the procedure has an 

 atmosphere of precision. We have mentioned in the preceding chapter 

 one of the mistakes made by Doerr in relation to the multipHcation of 

 bacteriophage corpuscles. 



With reference to this method it is interesting to cite an observation 

 of Gratia and de Kruif^^ which, to them,, seemed "curious," although in 

 reahty it is not so strange. These authors prepared a series of in- 

 creasing dilutions of a suspension of Coli-bacteriophage, and found the 



