216 THE BACTERIOPHAGE AND ITS BEHAVIOR 



bacterium prevails, on agar, the virulence of the bacteriophage. But this 

 inhibition on agar certainly has a cause. What is it? The thought that 

 comes immediately to mind is that some products having an inhibitory 

 effect upon the bacteriophage are Hberated in the liquid, and that on 

 agar, although they must necessarily be present also, they may diffuse 

 into the substratum; they do not accumulate on the surface where the 

 conflict between the bacteria and the bacteriophage corpuscles takes 

 place. This hypothesis can be proved very readily, for if it is a correct 

 explanation, it is only necessary to conduct the experiment in such a way 

 that these products can not diffuse into the substratum. This should 

 inhibit bacteriophagy on the solid medium, just as in bouillon. 



Spread 0.1 cc. of a secondary culture of B. dysenteriae which has devel- 

 oped at 40°C. in spite of the presence of a bacteriophage of maximum 

 virulence upon each of the following media: 



(a) Upon an agar layer, about 10 mm. in depth, in a deep Petri dish. 



(6) Upon an agar layer (same medium as the above) that is only 2 

 to 3 mm. thick. 



(c) Upon another agar layer (again the same medium) that is only 

 about 1 mm. in depth.* 



After incubation, we find that no colonies have appeared on the deep 

 agar layer, that a few isolated colonies are on the agar of moderate 

 depth, and that on the thin layer the colonies are confluent. 



The same results are obtained if this experiment is performed with 

 any other bacterium, the staphylococcus in particular. If, instead of 

 taking secondary cultures which have developed despite the presence 

 of a potent bacteriophage, secondary cultures consequent to bacteri- 

 ophagy by races of strong or even moderate virulence are spread over 

 the agar layers of different depths, a similar result will be obtained : A 

 few colonies on the thick layer, many colonies on the agar of moderate 

 thickness, a continuous layer of bacterial growth on the thin layer. 



Since in such experiments only the thickness of the substratum is 

 varied, the only deduction is that the cause of the inhibition is some 

 product which is effective if it remains in contact with the bacteriophage 

 corpuscles. Furthermore, it is evident that the harmful action is most 

 pronounced when its concentration is high. 



In the section dealing with "Bacteriophagy on SoUd Media" I have 

 demonstrated experimentally that although bacteriophagy does not 

 take place on gelatin it is by no means because of the nature of the 



* To avoid rapid drying in the incubator, invert the plate, and place 1 or 2 

 cc. of sterile water in the cover. 



