VIRULENCE OF THE BACTERIOPHAGE 143 



analogous to that devised by Miquel for counting bacteria, that is, the 

 procedure known as the ''method of successive dilutions." 



In accordance with this procedure 1 cc. of the bacteriophage suspen- 

 sion to be "titrated" is added to 9 cc. of bouillon. This gives an initial 

 dilution of 1:10, each cubic centimeter containing 1-10~^ cc. of the 

 original suspension.* One cubic centimeter of this first dilution is 

 removed and introduced into 9 cc. of bouillon. This second dilution is 

 1:100, containing per cubic centimeter 1-10"^ cc. of the original sus- 

 pension. One cubic centimeter of this dilution is then carried on into 

 9 cc. of bouillon, giving a third dilution, 1 : 1000, each cubic centimeter 

 containing 1-10^^ cc. of the original suspension. Continuing thus, by- 

 tens, the dilution up to the twelfth tube, a series of dilutions is obtained 

 in which in each cubic centimeter of the individual tubes there is 1-10~^, 

 1-10-2, 1.10-3, 1.10-4^ 1.10-5, M0-», MO-7, MO-S MO-9, MO-i", 

 1-10~", and 1-10-^2 ^c. of the original suspension. 



Having prepared these dilutions Appelmans next seeds each of them 

 with a culture of the susceptible bacterium and allows bacteriophagy 

 to proceed. The greater the dilution with which bacteriophagy takes 

 place the more active was the original bacteriophage suspension. 



This method has proved very attractive, doubtless because of its 

 appearance of "mathematical" precision. In reahty it has exerted an 

 unfortunate influence upon the study of bacteriophagy, for this pro- 

 cedure is, in great part, responsible for the errors which have been com- 

 mitted by those who have employed it. Several circumstances render 

 results obtained by this method invalid, among which we may mention 

 the following. 



1. The method can not be employed for measuring the virulence of a 

 race of bacteriophage of weak virulence. The difficulty here rests in 

 the fact that such a bacteriophage in no way inhibits the development 

 of bacteria. The presence of such races in a fluid medium can be dis- 

 closed only by the development of plaques when this fluid is spread 

 upon agar.^i^ I am aware that many authors (Otto,^^^ and later Doerr'^^^ 

 and their collaborators) have supported the reverse opinion, namely, 

 that a bacteriophage of weak virulence may be detected by "lysis" 

 in bouillon when plaques are lacldng. But these authors have fallen 

 into a double error, as wiU be pointed out shortly. 



* Obviously, one might take 4.5 cc. of bouUion and add to it 0.5 cc. of the bacteri- 

 ophage suspension. Then proceding by removing 0.5 cc. of this first dilution and 

 introducing it into 4.5 cc. of bouillon, a second dilution would be obtained. 

 Continuing thus a series could be prepared in all respects comparable to the series 

 described. 



