Dissociation of Microbic Species. 



4i 



fections, is possessed of powerful invasive properties, while its 

 mutant form G is characterized by very low virulence. 



The fixity of the character of high virulence for type D is 

 demonstrated by the following experiment. Strain R-19, type D, 

 was tested a few days after its isolation from a rabbit dead of 

 broncho-pneumonia. The test was carried out by injecting high 

 dilutions of a six-hour serum broth culture intrapleurally into 

 young rabbits of 600 grams weight. The strain proved itself 

 fatal in io~ 8 c.c. of the serum broth culture. This culture was 

 transplanted every seven days on serum agar. Tests made one 

 and three months after the first experiment indicated its virulence 

 to be still of the same titer. 



The individuals of a given strain of type D appear to differ very 

 little in the characteristic of virulence. Six pure-line strains, 

 isolated by the Barber method from stock strain R-15, were tested 

 for virulence by the method just described. All were fatal in 

 dose of io -6 c.c. 



The virulence of type D not only remains constant during 

 passage on serum agar, but persists under conditions that may be 

 considered as distinctly unfavorable. For example, a pure-line 

 strain of type D was planted in plain broth. It was allowed to 

 remain at 37 0 C. for 9 days and 12 hours without further trans- 

 plantation. At the end of this time a culture was streaked on a 

 serum agar plate. Marked D — * G mutation had occurred, counts 

 showing D = 40, G = 60. A colony of each type was fished into 

 serum broth tubes. These were incubated for the usual time, 

 diluted appropriately, and injected into two series of rabbits of 

 600 grams weight. The D culture, injected over a range from 

 io~ 7 to io -1 c.c, proved fatal in every case. The G culture, on 

 the other hand, failed to provoke a noticeable effect, even when 

 0.5 c.c. of whole culture was injected. 



It has been remarked in the second paper of this series that 

 pure-line strains of microbe D may mutate during daily passage 

 in plain broth. A pure culture of type D, virulence io~ 6 , was 

 transplanted daily in plain broth for 25 passages. Its virulence 

 remained constant during this time. At the 30th passage a few 

 G colonies were observed on the serum agar sub-plates. In two 

 months, sub-cultures on serum showed a large preponderance of G 



