BACTERIOLYSIS 31 



phage. However, filtration has always appeared to give more 

 satisfactory results. In a later chapter, under a paragraph en- 

 titled "Multiple Cultures" a third procedure will be considered. 



In certain cases the virulence of the bacteriophage can be in- 

 creased in vivo. A guinea pig is injected intraperitoneally with 

 two cubic centimeters of the filtrate containing the bacteriophage 

 whose virulence it is desired to increase and with a few cubic 

 centimeters of the bacterial culture against which the bacterio- 

 phage is active. After twelve to eighteen hours, ten cubic centi- 

 meters of sterile bouillon is injected into the peritoneum and 

 a few minutes later the peritoneal exudate is removed by puncture 

 with a trocar. The fluid is collected in a few cubic centimeters 

 of citrate solution and after a few hours' incubation the material 

 is filtered (infusorial earth and bougie) . This filtrate frequently 

 shows that a bacteriophage is present which is significantly 

 more virulent than that which was introduced into the guinea pig. 



Usually it is relatively easy to increase the virulence of a weak 

 strain of the bacteriophage, but at times it may become very 

 difficult, particularly when working with strains active against 

 the Gram-positive cocci. In such cases it is necessary to effect 

 a great number of passages, and there is considerable risk of 

 losing the bacteriophage altogether, particularly during the first 

 few passages. I might cite as an example an anti-staphylococcic 

 strain with which Eliava was forced to make passages during 

 four months in order to obtain sufficient virulence to induce 

 complete lysis of a suspension containing 500 million staphylo- 

 cocci per cubic centimeter. 



ENUMERATION OF THE BACTERIOPHAGOUS ULTRAMICROBES 



A trace of a filtrate containing a bacteriophage very active 

 for a given bacterium introduced into a broth suspension of 

 this bacterium causes a lysis of the organisms there present within 

 a few hours. The medium becomes as clear as broth which 

 has never been inoculated. A trace of the lysed suspension intro- 

 duced into a new suspension similar to the first causes a similar 

 lysis, a trace of this second lysed suspension introduced into a 

 third tube reproduces the same phenomenon, and so on. Dur- 

 ing the past three years with a single strain daily passages have 



