34 THE BACTERIOPHAGE 



planted upon agar, reveals characteristic clear plaques. If, in 

 the bouillon, lysis is allowed to proceed for some hours, there are 

 more plaques. The plaque, then, to all appearances sterile, 

 is in reality a colony of the bacteriophage. 



MULTIPLICATION OF THE ULTRAMICBOSCOPIC BACTERIOPHAGE 



The multiplication of the bacteriophage in the course of its 

 activity can be followed by the method of counting. With a 

 definite suspension of Shiga bacilli, always 250 million per cubic 

 centimeter, two extreme cases are very interesting: (1) that 

 which occurs when a large number of ultramicrobes are 

 introduced, 4 and (2) what transpires when but few, or only a 

 single one, is inoculated. 



1. Mass inoculation. Ten cubic centimeters of a suspension 

 of Shiga organisms are inoculated with 0.04 cc. of the culture of 

 the bacteriophage. The culture of bacteriophage contains 3000 

 million germs per cubic centimeter. Observed macroscopically 

 from time to time, it will be seen that the turbidity gradually 

 increases up to about the third hour, 5 and from that time the 

 liquid clears little by little. Between the fourth and sixth hours 

 from the time of the inoculation the suspension has become limpid. 



4 As already stated, study of the bacteriophage is always the study of a 

 complex problem, because it is essential to consider the mutual actions 

 and reactions of three variable factors medium, bacterium, and bacterio- 

 phage. The complexity is rendered more difficult to express clearly because 

 of the lack of suitable words. Here, for example, what shall we term the 

 act of introducing a definite quantity of the bacteriophage into a bacterial 

 culture? Obviously the term "inoculation" can be employed, but the 

 medium has already been inoculated with the bacterium; thus an equivocal 

 or ambiguous meaning may result . The proper term would be ' 'contamina- 

 tion" but unfortunately this term has already been appropriated in bac- 

 teriology as a synonym for "pollution". The term "inoculation", then, 

 must be employed. A culture medium may be "seeded" with bacteria, 

 and then "inoculated" with the bacteriophage. 



Frequently it will be observed, and the conditions for this reaction 

 are not exactly determined, that the dissolution of the bacteria is preceded 

 by a very marked agglutination. This reaction is noted particularly 

 when the bacterial culture is inoculated with a relatively large amount 

 (twelve drops for example) of a bacteriophage of average virulence. 



