46 THE BACTERIOPHAGE 



It sometimes happens, especially when using agar somewhat 

 dried out, that a few colonies of Shiga are obtained, always lo- 

 cated at the extreme edge of the layer of agar. We will return 

 to this extremely interesting particular in the discussion of sec- 

 ondary cultures. 



If, instead of a continuous covering of the bacteriophagous 

 growth the ultramicrobes are deposited in limited areas and 

 this is readily accomplished by placing drops of culture on the 

 sterile surface of a tube of agar, or again, by drawing lines over 

 the surface with a platinum loop dipped in the culture of bacterio- 

 phage, and after the tubes have remained inclined for a few hours 

 in the incubator to secure drying we find that the areas impreg- 

 nated with the bacteriophagous culture remain free of Shiga 

 bacilli, but that these organisms grow, on the contrary, perfectly 

 well on the parts not covered by the bacteriophage. 



When in the suspension planted upon agar the number of 

 bacilli is infinitely great and the number of the ultramicrobes is 

 sufficiently small, the bacteriophage culture as individual units 

 is distributed over the surface of the agar, and under such cir- 

 cumstances the bacterial layer will appear studded with apparently 

 sterile areas. These areas, or plaques, have a circular form with 

 a diameter of from 1 to 5 mm. The plaques are in general of the 

 greatest extent when the suspension is somewhat weak although 

 sufficiently concentrated to give a continuous layer of growth 

 rather than isolated colonies. On such a tube the areas are 

 larger as the subjacent medium becomes thicker, that is, toward 

 the bottom of the tube. Upon a Petri dish, where the agar layer 

 is of essentially the same thickness throughout, all of the plaques 

 of a given culture are of approximately the same diameter. As 

 will be seen, the area of the plaque bears a relationship to the 

 virulence of the bacteriophage which causes it. 



If a tube or plate presenting plaques is held in the incubator 

 at 37C., or at an entirely different temperature, no change 

 occurs in the plaques; their diameter remains indefinitely what 

 it was at first. They are never covered or encroached upon by 

 the bacterial culture. At no time does there exist within the 

 extent of the plaque, whatever its size may be, microscopically 

 visible bacterial cells. The plaque is always rigorously sterile. 



