36 THE BACTERIOPHAGE 



37°C. the opacity is about two times as great as at the beginning; 

 after three hours, it is about two and one-half times as great; 

 after four hours, about three times; and then it begins to diminish, 

 so that after five hours the density is about twice as great as at 

 the beginning of the incubation. This clearing continues gradu- 

 ally, so that after fourteen hours the culture is almost entirely 

 clear. If immediately after the inoculation with the bacterio- 

 phage and then every thirty minutes, 0.02 cc. of each of these 

 two suspensions is transferred to agar slants, these tubes will 

 show, after incubation, the following: 



Plantings after one-half, one, one and one-half, and two hours 

 yield normal growths of B. dysenteriae Shiga. After two and one- 

 half hours the subcultures show three plaques in one tube, five 

 in the other (average four). Therefore, after two and one-half 

 hours the inoculated suspension contains 2000 bacteriophagous 

 ultramicrobes. 



After three hours the tubes show five and four respectively. 

 Hence, there has been no material increase between two and 

 one-half and three hours. 



The three and one-half hour plantings show nine and five 

 areas (average seven). The number of bacteriophagous ele- 

 ments has slightly increased. 



After four hours, the agar tubes show 101 and 111 plaques re- 

 spectively (average 105). After four hours, therefore, the num- 

 ber of ultramicrobes is between fifty and sixty thousand. 



After four and one-half hours, the counts are 145 and 160 

 (average 152), indicating that the suspension contains 75,000; 

 a number but slightly different from the count after four hours. 



After five hours, the agar tubes are sterile. When diluted to 

 1 : 1000 in a suspension of Shiga bacilli and transferred immediately 

 to agar in the same way, the tubes give four and six areas. Thus, 

 it appears that after five hours the suspension contains about 

 1,500,000 bacteriophagous germs. 



From this it is readily apparent that the multiplication of 

 the ultramicrobes is extremely rapid, and, what is most remark- 

 able, it is associated with successive jumps, each augmentation 

 being separated by an interval of about seventy-five minutes. 

 We will refer elsewhere to this experiment when we consider the 

 mode of reproduction of the bacteriophage. 



