Dissociation of Microbic Species. 



35 



organisms, it would be unjustifiable to conclude that the original 

 type D had changed into the microbe of the G variety. Con- 

 sequently, 8 pure-line strains were isolated from a D culture by 

 the Barber method. Single cells, removed from three-hour cul- 

 tures, were planted in undiluted rabbit serum. The percentage of 

 positive cultures obtained by performing the entire operation in 

 serum was much higher than when broth was employed. 



The resulting pure-line strains were planted daily in undiluted 

 rabbit serum. Tests were carried out to determine the conditions 

 under which the low-virulent type G makes its appearance. The 

 method of detection of this type consisted in streaking the test 

 material upon the surface of 10 per cent, rabbit serum agar. The 

 G type colonies can be readily distinguished from those of the 

 strongly fluorescent type D. With proper attention to the 

 technique of streaking, quantitative estimates of the propor- 

 tionality between the D and G varieties can be made. 



In undiluted rabbit serum, with daily transplant, type D 

 breeds true for long periods of time. The G variety has seldom 

 been observed to arise under these conditions. In plain broth, 

 transplanted daily, a few G colonies have been detected after 25 

 passages. On the other hand, when 3 or 4 days are allowed to 

 elapse between transplants in this -medium, many colonies of this 

 type make their appearance on the serum agar sub-plates. 



This observation led to the following experiment. 0.05 c.c. of 

 a pure-line strain, type D, was seeded into tubes of plain broth and 

 of undiluted rabbit serum. The tubes were placed at 37 0 C. and 

 a loopful of the material from each tube was streaked at 12 -hour 

 intervals on rabbit serum agar plates. In the sub-plates from un- 

 diluted rabbit serum no G colonies were detected during incuba- 

 tion for 109 hours at 37 0 C. In the plain broth, G colonies began 

 to appear at 48 hours, and had reached a concentration of 50 per 

 cent, of the total organisms in 109 hours. These G colonies, 

 fished from serum agar plates, remained true to type for over 50 

 passages, showing no tendency to revert to the parent D form, 

 even when returned to undiluted rabbit serum. All of the pure- 

 line strains under study have been found to undergo this mutation 

 when allowed to stand in plain broth, but do so with varying 

 degrees of rapidity and completeness. 



