PROPERTIES OF THE BACTERIOPHAGE 293 



in the water-bath, adjusted to maintain the temperature desired, as 

 indicated in each of the following experiments. In each part of the 

 experiment, 8 tubes containing the suspension are held for 30 minutes 

 at a constant temperature. The temperatures selected were 60, 62, 

 64, 66, 68, 70, 72, and 75°C. 



Shiga-bacteriophage 



From each of the tubes held at each of the temperatures 60, 62, 64, 

 and 66°C. two drops of suspension were inoculated into bouillon sus- 

 pensions of Shiga bacilli. In all instances a complete dissolution of the 

 bacilli took place within 14 hours. When these tests were repeated 

 with a second strain of susceptible Shiga organisms the results were 

 identical in every respect. With the suspensions which had been 

 heated to 68° and to 70°C. the material caused a complete dissolution 

 when added to one of the Shiga strains, but it failed to induce a dis- 

 solution of the other. The material heated to 72°C., as well as that 

 heated at 75°C. failed to cause dissolution with either strain. 



For control purposes, plantings on agar were made; one drop of the 

 bacterial suspensions receiving the bacteriophage corpuscles heated to 

 68, 70, 72, and 75°C. being spread over agar prior to the incubation. 

 Later examination of these cultures, after incubation, showed that all, 

 except those receiving the bacteriophage heated to 75°C. showed 

 characteristic plaques; the growths obtained from the 75°C. tubes were 

 normal bacterial cultures. 



When the virulence of a bacteriophage is attenuated by heating, the 

 activity may, within limits, be restored by effecting serial passages. 

 For example, with the corpuscles heated at 68 and at 70°C. two passages 

 were required, with those heated at 72°C. three passages were necessary 

 to restore the virulence to such an extent that a dissolution of the 

 bacteria would take place in a fluid medium. 



Experiments comparable to the above in every respect showed 

 that an entirely similar effect occurs with bacteriophage races active 

 for other organisms: B. dysenteriae Flexfier, B. dysenteriae Hiss, B. coli, 

 and B. paratyphosus B. It is interesting that with the strain of B. 

 paratyphosus A tested, the attenuation of virulence began at a tem- 

 perature of 64°C., while with the race virulent for B. typhosus an attenua- 

 tion was evident at a temperature as low as 62°C. However, despite 

 these individual racial variations, with all races exposure to 75°C. renders 

 the corpuscles inactive. Either they are actually destroyed, or their 

 virulence is attenuated to such a point that they can no longer be de- 



