RESISTANCE OF BACTERIA 197 



sages are continued in this same manner, allowing the filtrate which has 

 been in contact with the resistant bacteria to remain unfiltered in each 

 passage until secondary cultures form, it will be found that a gradual 

 attenuation of virulence takes place. 



Hadley-^^ has reported that a Shiga-bacteriophage (one which I sent 

 him) produced, at the beginning, plaques with a diameter of about G 

 mm. During a series of passages continued throughout a period of 

 2 years the diameter of the plaques became progressively less, up to the 

 point where they ceased to be larger than 1 mm. At this time, on going 

 back to the original suspension which had been preserved for 2 years in 

 a sealed tube, he found that the plaques formed were just as at the begin- 

 ning, i.e., about 6 mm. in diameter. And since the diameter of the 

 plaques, all conditions being equal, is directly proportional to the viru- 

 lence of the bacteriophage (d'Herelle^^^-^^^), Hadley has concluded that 

 by successive cultures the bacteriophage has degenerated.' 



But I have maintained this same race of the bacteriophage* and it 

 has undergone in my laboratory a great many passages and the plaques 

 still actually measure between 5 and 6 mm. in diameter. This would 

 seem to prove that the degeneration observed by Hadley is a result of 

 the conditions under which bacteriophagy took place throughout his 

 experiments. Whatever may have been the unfavorable conditions, 

 there was most certainly a formation of secondary cultures and these 

 must always be avoided if it is desired to maintain intact the virulence 

 of a bacteriophage. To accomplish this it is only necessary to filter the 

 material just as soon as bacteriophagy is complete. This is the best 

 means of avoiding, even from the beginning, the development of second- 

 ary cultures which macroscopically might pass undetected. 



A bacteriophage, then, becomes attenuated during the same process 

 which leads to an acquisition of resistance by the bacterium. Even a 

 bacteriophage of maximum virulence may be ''overcome." To bring 

 this about it is only necessary to inoculate it into a very concentrated 

 suspension of bacteria. f It can be shown that under these conditions 

 the virulence of the bacteriophage l^ecomes weakened. After a series 

 of passages is made in very heavy suspensions (8000 to 10,000 million 



* Intentionally every time that I have given out a Shiga-bacteriophage I have 

 always selected this same race. 



t We have seen that if a suspension contains more than 700 to 800,000,000 

 bacteria per cubic centimeter the dissolution of the bacteria is incomplete, even 

 under the action of a bacteriophage of maximum virulence. With a bacteriophage 

 of less virulence the number of bacteria capable of being dissolved is less. 



