THE BACTERIOPHAGE CORPUSCLE 95 



comprised of organisms of increasing age. In other words, a thicker 

 and thicker layer of bacilh always becoming more and more resistant 

 to dissolution develops. This can be readily demonstrated by direct 

 experimental proof. 



If the agar surface in a Petri dish is heavily seeded with a Shiga 

 culture and at some point on this a drop of the bacteriophage filtrate 

 is placed, and after a three-hour incubation period another drop of the 

 bacteriophage is placed on the surface and this same process repeated 

 after six, twelve and twenty hours, with continuous incubation of the 

 plate during the intervals, it wiU be found fifteen hours later that the 

 areas upon which the first three drops were placed have remained ster- 

 ile — no bacillary growth has taken place. At the point where the fourth 

 drop was placed, that is, after the culture had been incubated for twelve 

 hours, there is a thin layer of growth composed of dead bacilli. The 

 area where the drop of bacteriophage was placed after twenty hours 

 presents an appearance practically normal. These five spots, then, 

 represent the diverse aspects of an isolated colony of the bacteriophage, 

 as from the centre to the periphery. 



The second reason is of a more general nature, representing a pheno- 

 menon common to the majority of cultivable organisms. The colonies 

 of the bacteriophage act absolutely Hke colonies of those bacteria which, 

 except for organisms such as B. proteus, never progressively invade the 

 surface of sohd media. Thus, if the Shiga bacillus is planted upon 

 agar in an amount suitable to yield isolated colonies, after 18 to 24 

 hours, each colony will be from two to four miUimeters in diameter, the 

 largest colonies to be found at the points where the medium has the 

 greatest depth, that is, toward the bottom of the tube. Such colonies 

 increase in size but very slowly, always more and more slowly as time 

 progresses, and even after two months, the zone of increase will not be 

 greater than a few millimeters. From the bacteriological point of view 

 it is not peculiar, as has been suggested, that the bacteriophage does not 

 invade the entire bacterial layer. Far from being dissimilar to other 

 cultivable organisms, an isolated colony of the bacteriophage behaves 

 exactly like a colony of bacteria. 



Why does the bacterial colony fail to increase in size and invade the 

 entire surface of the medium? Because the soluble substances result- 

 ing from the vital activity of the bacteria diffuse into the agar and these 

 substances constitute a true specific antiseptic which limits the growth. 

 The medium is "vaccinated" around the colony. The deeper the agar 

 layer, or the farther the colonies are separated, the greater the volume 



