198 THE BACTERIOPHAGE AND ITS BEHAVIOR 



per cubic centimeter) the virulence is attenuated to such a degree that 

 after a few passages the bacteriophage is lost. 



In a word, each time that a secondary culture develops the virulence 

 of the bacteriophage concerned becomes attenuated and this attenua- 

 tion is the more pronounced when, on the one hand, the initial virulence 

 was low and when, on the other, the number of resistant bacteria in 

 proportion to the number of bacteriophage corpuscles present was high. 



This is precisely the experiment performed by Bordet and Ciuca^'^ 

 who inoculated a relatively large amount (a twenty-millionth of a cubic 

 centimeter) of a Coli-bacteriophage suspension of average virulence* 

 into a tube of bouillon which they then seeded with a culture of B. coli. 

 After a few days in the incubator at 37°C., that is to say, after a long 

 contact of the corpuscles with a heavy culture of bacilli which had 

 acquired a resistance, they heated the mixture at 57°C. in order to kill 

 the bacilli (a further cause of the attenuation of the virulence). They 

 demonstrated that the fluid contained a bacteriophage attenuated both 

 quantitatively and qualitatively. 



These authors have attempted to explain this attenuation by assum- 

 ing that a degeneration of the "lytic principle" occurred. As a result 

 of its low concentration each bacterium in the suspension could fix but 

 a very small amount and being thus but very slightly stimulated it 

 could regenerate only a weak "lytic principle." 



Such an explanation is not admissible for it does not take into account 

 the fact that the bacteriophage exists in the form of corpuscles. Fol- 

 lowing this publication of Bordet and Ciuca, I offered to give a demon- 

 stration of the experiment proving the corpuscular nature of the 

 principle,^''^ but the offer was not accepted. As a matter of fact, shortly 

 after Bordet had published his experiment one of his collaborators. 

 Gratia, published an experiment confirming the fact that the "concen- 

 tration" of the bacteriophage principle plays but a secondary role.^^-f 

 It is very evident that the explanation of Bordet can not be correct since 

 if one inoculates the smallest active dilution of a suspension of bacterio- 

 phage into 1, 10, 100, or 1000 cc. of bacterial suspension bacteriophagy 

 takes place in the 4 suspensions in the same manner. 



As a final proof let us prepare from a young agar culture of the staphy- 

 lococcus 2 cc. of suspension each cubic centimeter containing 10,000 

 million cocci. Remove 1 cc. and introduce it into 99 cc. of sterile bou- 



* As indicated by their experiments. 



t The experiment of Gratia and DeKruif has been referred to in the section 

 dealing with "Evaluation of Virulence." 



