48 THE BACTERIOPHAGE 



layer of bacilli always becoming more and more resistant to lysis 

 develops. This can be readily demonstrated by direct experi- 

 mental 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 culture of 

 the bacteriophage 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 

 will be found fifteen hours later that the areas upon which the 

 first three drops were placed have remained sterile — 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 

 phenomenon common to the majority of cultivable organisms. 

 The colonies of the bacteriophage act absolutely like colonies 

 of those bacteria which, except for organisms such as B. proteus, 

 never progressively invade the surface of solid media. Thus, 

 if the Shiga bacillus is inoculated upon agar in an amount suitable 

 to yield isolated colonies, after 18 to 24 hours, each colony will 

 be from two to four millimeters 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 bacteriologi- 

 cal point of view it is not peculiar, as has been suggested, that 

 the bacteriophage does not invade the entire bacterial layer. 

 It must be borne in mind that the bacteriophage, far from being 

 dissimilar to other cultivable ' organisms, behaves, when in iso- 

 lated colonies, exactly like a colony of bacteria. 



Why does not the bacterial colony continue to increase in size 

 and to invade the entire surface of the medium? Because the 



