THE BACTERIOPHAGE CORPUSCLE 93 



does not grow it is only because the bacteriophagous corpuscles 

 remain on the surface of the agar and exercise their dissolving action on 

 the bacteria deposited thereon. This is readily confirmed. If we take 

 a tube of agar which has remained apparently sterile after having 

 been inoculated with a suspension of the bacteria containing a bacterio- 

 phagous filtrate, and if the surface of the medium in such a tube is 

 washed with a few drops of sterile bouillon and to this is added a fresh 

 suspension of bacteria, this suspension will be dissolved within a few 

 hours. 



It sometimes happens, especially when using agar somewhat dried 

 out, that a few colonies of Shiga are obtained, always located at the 

 extreme edge of the layer of agar. We will return to this extremely 

 interesting particular in the discussion of secondary cultures. 



If, instead of spreading the bacteriophage filtrate over the entire 

 surface the corpuscles are deposited in Kmited areas — and this is readily 

 accomphshed by placing drops of filtrate on the sterile surface of a tube 

 of agar, or again, by drawing lines over the surface with a platinum loop 

 dipped in the suspension of bacteriophage, and after the tubes have 

 remained inclined for a few hours in the incubator to secure drying — ■ 

 we find that the areas impregnated with the bacteriophagous filtrate 

 remain free of Shiga bacilh, but that these organisms grow, on the con- 

 trary, 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 corpuscles is sufficiently small, 

 the bacteriophage principle as individual units is distributed over the 

 surface of the agar, and under such circumstances the bacterial layer 

 will appear studded with apparently sterile areas. These areas, or 

 plaques, have a circular form varying in size from those spoken of as 

 "pin-point" up to those with a diameter of 8 mm. The plaques are in 

 general of the greatest extent when the bacterial 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 essen- 

 tially the same thickness throughout, all of the plaques of a given cul- 

 ture are of approxunately 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 37°C., 

 or at an entnely different temperature, no change occurs in the plaques; 



