BACTERIOPHAGOUS ULTRAMICROBE 117 



Experiment XXXII. In the following experiments the culture of bac- 

 teriophage under test, previously filtered through a bougie, is taken up in 

 capillary pipettes, sealed at both ends, and completely submerged in a 

 water-bath maintained at the temperatures indicated in each experiment. 

 In each series of experiments 8 tubes with culture are maintained for 

 thirty minutes at temperatures of 60, 62, 64, 66, 68, 70, 72, and 75 C. 



Anti-Shiga bacteriophage 



Two drops of the culture from tubes maintained at 60, 62, 64, and 66C., 

 when introduced into suspensions of Shiga bacilli, cause complete lysis 

 in less than fourteen hours. The tests repeated with a second strain of 

 Shiga bacilli give identical results. The bacteriophage heated to 68 and 

 70C. causes lysis with one strain of Shiga bacilli but not with the other. 

 When heated to 72 and 75C. the bacteriophage fails to cause lysis. 



One drop of each of these suspensions, which had received the bacterio- 

 phage cultures previously maintained at 68, 70, 72 and 75 C., and which 

 had not been submitted to lysis, are planted on slant agar. After incuba- 

 tion, all of the cultures, except the last, which is normal, show plaques 

 characteristic of the presence of the bacteriophage. 



Serial passages may be effected, thus permitting the enhancement in 

 virulence of the bacteriophage attenuated by the action of temperature. 

 After two such passages, with the ultramicrobe heated to 68 and 70C., 

 and after three passages with that which was heated to 72C., lysis in 

 liquid media is obtained. 



Comparable experiments have demonstrated that the bacteriophagous 

 ultramicrobes active for B. dysenteriae Flexner, B. dysenteriae Hiss, B. coli, 

 and B. paratyphosus B, act in a quite similar manner. With the bacterio- 

 phage active for B. paratyphosus A attenuation begins at about 64C. (at 

 least with the strain tested). With that virulent for B. typhosus attenua- 

 tion is already apparent at about 62C. In all cases, when heated to 75C. 

 the bacteriophage is completely inactive, either actually destroyed or 

 attenuated to such an extent that its presence can no longer be detected. 

 In all these instances the bacteriophage shows a recuperative power, the 

 virulence being restored when the temperature to which the virus has been 

 subjected is not higher than 72C. 



Anti-staphylococcus bacteriophage 



Attenuation of this bacteriophage is already manifest after heating to 

 60C. Subcultures of suspensions which have not been lysed show that it 

 is a simple attenuation, for, even with suspensions inoculated with a bac- 

 teriophage previously held at 72C. for thirty minutes, plaques are obtained 

 characteristic of the presence of an active bacteriophage. Moreover, two 

 passages suffice to restore the original virulence to cultures heated to 62, 

 64, 66, and 68C. After heating at 70 and 72C. the attenuation of virulence 

 does not disappear until after six passages. When heated to 75C. the 

 bacteriophage is deprived of all activity. 



