162 THE BACTERIOPHAGE AND ITS BEHAVIOR 



velops somewhat more rapidly at a temperature of 37°C., but even at 

 the lower temperature of 30°C. the bacterium reacts vigorously. For 

 this reason it is essential to restrict the period of contact to that just 

 sufficient to afford the corpuscles opportunity to multiply. And at the 

 same time a limited period of contact will prevent the bacteria from 

 acquiring a resistance through processes of adaptation. As a matter 

 of fact practical experience shows that this end is obtained by effecting 

 two passages every day; one in the morning upon arrival at the labora- 

 tory, the other in the afternoon just before leaving. 



An excellent procedure which provides through serial passages for the 

 increase in the virulence of the bacteriophage by adaptation and at the 

 same time for the selection of those corpuscles most apt at acquiring 

 such a virulence consists in carrying out a series of isolations by the 

 dilution method. The following scheme indicates how this can be 

 effected. 



Among 10 suspensions, each inoculated with 1 cc. of the last active 

 dilution of a bacteriophage, a certain number undergo bacteriophagy. 

 Select the tube in which the phenomenon has been the most marked.* 

 This suspension is filtered through a candle and the isolation procedure 

 is begun again. Once more that suspension in which bacteriophagy 

 is the most intense is filtered. This procedure is repeated up to the 

 point where the maximum virulence has been obtained. 



The only difficulty with this resides in the determination of the limit- 

 ing dilution, especially with very weak races of the bacteriophage, or 

 where bacteriophagy can only be disclosed by agar cultures. More- 

 over, in carrying out the passages one can not take time to make the 

 preliminary studies necessary to ascertaining the limiting dilution. In 

 order to obviate this difficulty the increase in virulence may be started 

 by a few simple passages and later, when a degree of virulence has been 

 obtained sufficient to cause bacteriophagy such as can be detected by 

 microscopic examination, the limiting dilution may be determined. 



When this is once determined the filtrate is utifized and an isolation 

 by dilution is made. The choice from among the bacteriophage sus- 

 pensions falls on the one where the phenomenon occurred most intensely. 

 This one is filtered, and the filtrate serves for a new isolation, arbitrarily 

 choosing as the Hmiting dilution the same as that disclosed by the pre- 



* Needless to say, if for any reason it is desired to make a selection from among 

 a very great many corpuscles, in the place of utilizing 10 cc. of the limiting dilu- 

 tion in 10 suspensions, one might use 20 cc. of the limiting dilution, or even more, 

 distributing it among 20 or more suspensions. 



