VIRULENCE OF THE BACTERIOPHAGE 161 



a third suspension is inoculated with four or five drops of the filtrate. 

 Such transfers are continued until evident dissolution occurs. During 

 the process it is easy to verify the presence of the bacteriophage in each 

 passage, and to detect any increase in virulence, simply by spreading 

 the successive cultures on agar slants. Comparison of the cultures 

 secured with each passage reflects the degree of virulence. For ex- 

 ample, the agar growth obtained from the first passage shows a culture 

 growth with ten plaques, the second passage shows 100, with the third 

 the layer of bacillary growth is broken up with an abundance of the areas, 

 while with the fourth passage only a few isolated colonies of bacteria 

 are seen. It can be readily seen that the virulence of the bacteriophage, 

 that is, its ability to develop at the expense of the bacteria, increases 

 with each transfer until a point is reached where complete dissolution 

 of the suspension is obtained.* 



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

 of the bacteriophage, but at times it may become very difficult, partic- 

 ularly when working with races 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, partic- 

 ularly during the first few passages. I might cite as an example an 

 anti-staphylococcic race with which Eliava was forced to make passages 

 during four months in order to obtain sufficient virulence to induce 

 complete dissolution of a suspension containing 500 million staphylo- 

 cocci per cubic centimeter. 



It may be well to emphasize here a point of some importance. We 

 will see that the virulence of the bacteriophage becomes considerably 

 weakened when it is held in contact with bacteria which resist its action. 

 For this reason it is wise to avoid, in each passage, contact with those 

 organisms which have had time to acquire a resistance. To accomphsh 

 this it is best at first to conduct the passages at a relatively low tem- 

 perature; 30°C. is adequate to permit the active development of the 

 corpuscles and it retards somewhat the acquisition of a resistance to the 

 process of bacteriophagy by the bacterium. Bacterial resistance de- 



* The bacteriophage is not destroyed until a temperature of about 76°C. is 

 reached. Bordet and Ciuca^e have proposed to utilize this fact to avoid filtration. 

 They hold the bacteriophage suspension at 58°C. for an hour during which time 

 the bacteria are killed while the bacteriophage resists. This method of isolating 

 a virulent bacteriophage should never be employed during the course of a series 

 of passages designed to increase virulence. For although heating may not kill, 

 it attenuates the virulence of the bacteriophage with the result that the advantage 

 gained by each passage is lost through the heating. 



