4 THE BACTERIOPHAGE AND ITS BEHAVIOR 



This, of course, made it at once apparent that my first hypothesis 

 was of necessity false, the truth of the matter being that the fecal 

 material used in preparing the filtrate contained something which dis- 

 solved the dysentery bacilli. Nevertheless, my first hypothesis had 

 one virtue, since, as it had led me for such a long time to consider the 

 question of a virus pathogenic for the man or the animal it offered the 

 suggestion that the dissolving principle might be a virus pathogenic 

 for the bacterium. And because of this last hypothesis the following 

 experiment was devised and carried out. 



A drop of a dissolved culture was added to a fresh bouillon culture 

 of Shiga bacilli. About 15 hours later the bouillon was clear, all of the 

 bacilli originally present had been dissolved. Thus, several succes- 

 sive passages were effected in the same way, employing each time a 

 drop of the culture previously dissolved added to a fresh culture of 

 Shiga bacilli. In this repetition of the process, instead of becoming 

 weaker the activity became more and more pronounced, that is, the 

 disappearance of the bacilli was effected with greater and greater 

 rapidity. 



This time the guiding idea appeared to be correct. The principle 

 present in the intestinal contents became regenerated at the expense 

 of the Shiga bacilli. It behaved like a filtrable organism, parasitic of 

 bacteria. 



The next step was an attempt to reproduce the cultural irregular- 

 ities upon a solid medium. To do this, a very small amount, about 

 0.0001 cc. of one of the dissolved cultures was added to a young broth 

 culture of Shiga bacilli and the mixture was subcultured immediately 

 to an agar slant. Similarly, subcultures were made after incubation 

 for 1, 2, and 3 hours, a drop from the inoculated tube being used in the 

 transfers. After incubation of these agar subcultures the growth 

 revealed some of the "abnormal" appearances which had formerly per- 

 plexed me. But this time, the characteristics of these abnormalities 

 were so outspoken that their significance could not be overlooked. 



In the first tube the surface of the agar was well-covered with a 

 normal layer of B. dysenteriae, except that in the midst of the culture 

 there were two little islands, two "plaques," perfectly circular in form, 

 where the agar was bare, entirely free of all traces of culture. The 

 second agar tube, planted 1 hour after the original combination was 

 made, revealed 6 of these plaques. In the third, prepared after the 

 dissolving principle had acted for 2 hours, there were about 100 of 

 the plaques. The fourth tube remained without any evidence of 

 bacillary growth. 



