NATURE OF THE BACTERIOPHAGE 151 



cent glycerine, for example. This proves again that the survival 

 of a certain number of bacteria does not constitute a factor in 

 the serial activity, for, as this factor would fail the series could 

 not be continued. 



4. On agar the bacteriophage gives colonies at the expense of 

 the bacteria, and this permits counting the active elements. A 

 soluble ferment, diastase or toxin, can not concentrate its action 

 in definite points. It may be objected that the lytic diastase 

 is provided by the bacteria themselves and that each plaque on 

 agar represents the area where is to be found, after the inocula- 

 tion, a bacterium particularly able to furnish the diastase under 

 the influence of a force "X." 



The following experiments demonstrate that this objection is 

 not valid. 



Experiment LV. (A}. Take 10 tubes. Into the first place 10 cc. of a 

 suspension of B. dysenteriae Shiga containing 100,000,000 bacilli per cc., 

 into the second place 10 cc. of a suspension containing 200,000,000 bacilli 

 per cc., into the third place 10 cc. of a 300,000,000 suspension, and so on, 

 increasing by 100,000,000 the concentration of the suspensions introduced 

 into each tube of the series. The tenth tube, then, will have a suspension 

 containing 1,000,000,000 bacilli per cc. Each of these tubes is then inocu- 

 lated with an equal quantity of a very dilute culture of the bacteriophage 

 filtered through a bougie, about 0.000005 cc. We have then a series of 10 

 tubes containing a more and more concentrated suspension of B. dysenteriae 

 Shiga in the ratio of 1 : 2: 3: 4: 5: 6: 7:8: 9: 10, and an equal amount of bacterio- 

 phage culture. The tubes are carefully shaken, and 0.02 cc. from each tube 

 is planted upon agar slants. After incubation at 37C., each of the 10 

 tubes of agar shows a culture of B. dysenteriae spotted with plaques, and 

 the number of these plaques is practically the same for all the tubes. 



(B) Take 10 tubes, each containing 10 cc. of a suspension containing 

 100,000,000 B. dysenteriae per cc., that is, a like suspension in all 10 tubes. 

 To the first add 1/100,000 cc. of filtered bacteriophage culture, to the 

 second 1/200,000 cc., to the third 1/300,000 cc., 1/400,000 cc. to the fourth, 

 and so on, each tube receiving a smaller and smaller amount of bacterio- 

 phage culture, so that the tenth tube will contain only 1/1,000,000 cc. 

 Thus, we have a series of 10 tubes, all containing an equal number of dysen- 

 tery bacilli and an amount of bacteriophage culture varying according to 

 the ratio 10:9:8:7:6:5:4:3:2:1. Shake the tubes thoroughly and plant 

 0. 02 cc. from each of the 10 suspensions on to agar slants. After incubation 

 at 37C. each of the agar tubes will show a covering growth of B. dysenteriae 

 studded with plaques, but the number of plaques in the tubes varies with 

 the proportion of bacteriophage culture which has been added. Prac- 



