BACTEKIOLYSIS 59 



The material of all three flasks is incubated at 37C. Immediately after 

 the inoculation, after thirty minutes, and again after 1 hour, 20 cc. are 

 taken from each of the three flasks and centrifuged at 4,000 revolutions per 

 minute for ten minutes. 



There are thus nine tubes which have been centrifuged. From the 

 supernatant fluid of each of these 0.02 cc. is taken and introduced into 

 other tubes containing suspensions of the Shiga bacillus, and the counts 

 of the ultramicrobe are made by plating 0. 02 cc. of each of these nine tubes 

 on six plates of medium. In this way an average of the counts can be ob- 

 tained. The results of these counts indicate the number of ultramicrobes 

 remaining in the medium, since those which have penetrated the bacterial 

 cells before the centrifugation have been thrown down with the cells during 

 this procedure and in consequence are to be found in the sediment. 



The results of the counts are as follows : 



Tube 1. Shiga suspension plus bacteriophage. 



a. Counts of the material made immediately after the preparation are 

 214, 193, 187, 221, 229, and 183 plaques. The average is 204, representing 

 5,000,000 germs per cubic centimeter in the original suspension immediately 

 after inoculation. 



6. Counts on the suspension after incubation for 30 minutes are 3, 7, 

 4, 6, 6, and 3 plaques. The average is 5. This indicates that there are 

 125,000 bacteriophagous germs in the suspension thirty minutes after the 

 inoculation. That is to say, of each 41 ultramicrobes inoculated 40 have 

 disappeared. 



A direct count of the suspension without centrifugation gives 

 5,000,000,000 elements per cubic centimeter. It is therefore certain that 

 the ultramicrobes which have disappeared from the fluid during the cen- 

 trifugation have gone down with the bacteria. And, as we will see in the 

 two control experiments, in the absence of Shiga bacilli this sedimentation 

 of the bacteriophage does not occur (at least, when centrifuged at a speed 

 of 4000 revolutions). 



c. After 1 hour, the count, made as before on the supernatant fluid 

 gives an average of 8 plaques, or 200,000 ultramicrobes per cubic centimeter, 

 a number essentially the same as that secured after thirty minutes. At 

 this time a count of a suspension which has not been centrifuged 

 gives 6,500,000, a number very close to that secured immediately after the 

 inoculation. 



It should be noted that in the hypothesis formulated with regard to 

 intrabacterial growth, each colony forming within the interior of a bac- 

 terium gives rise to but a single plaque ; just as in a bacterial count made by 

 the same method a whole clump of bacteria seeded upon agar will yield 

 only a single colony. 



d. Counts made upon the suspension with and without centrifugation 

 after one and one-quarter hours of incubation give the same number of 

 ultramicrobes, about 90,000,000. The inoculated organisms have therefore 

 increased from 5 to 90 millions; the increase being in a proportion of about 



