22 MASS. EXPERIMENT STATION BULLETIN 319 



Six months after the last set of broth tubes was inoculated three additional 

 variants, of Strains XII and XIII, were isolated from the broth culture. One 

 variant of Strain XIII was placed on liver infusion agar, one type of variant 

 (XH-a) of Strain XII was cultivated on meat extract agar, and the other (XH-b) 

 was grown on liver infusion agar. The different variants will be described more 

 fully in another part of this section. (See Table 4.) 



Experiment III. According to the results obtained thus tar, the author was 

 inclined to believe that, while only six of fifteen strains had yielded variants, the 

 remaining strains would also produce variants as a result of transfer and aging 

 in beef broth, if conditions were suitable and if the platings were made at the 

 proper time. With this view in mind, 13 of the strains employed in Experiment II 

 (I, II, III, IV, V, VII, VIII, X, XI, XII, XIII, XIV, andXV)and three addi- 

 tional strains (XVI, XVII, and XVIII), were subjected to the same treatment 

 that was employed in the second experiment. The history of the three new 

 strains is as follows: — 



Strain XVI — Isolated from the ovary of a hen in 1911 



by Doctor Rettger 

 Strain XVII — Isolated from the liver of a chick in 1917 



by Doctor Rettger 

 Strain XVIII — Isolated from the liver of a chick in 1916 

 by Doctor Rettger 



During the process of transferring from day to day it was observed that Strain 

 XIV did not appear as turbid as the other cultures. The same characteristic 

 had been noted in Experiment II. Ten days after the last transfer in broth three 

 sets of the broth cultures were examined and plated on plain agar. The three 

 sets included the first, seventh, and fourteenth broth culture transfers. 



An examination of the bouillon cultures in the three sets revealed that Strains 

 III, IV, V, VII, X, XIII, and XV produced a ring on the side of the tube at the 

 surface of the broth, and also formed a heavy flaky sediment in the bottom of 

 the tube. The remaining strains did not show these characteristics. 



The agar plates that were streaked with the broth culture sets revealed definite 

 colony variants of Strains III, V, X, XV, XVI, and XVIII. Three colony types of 

 one strain were observed. In a few instances the colonies varied slightly in their 

 appearance, but not sufficiently to designate them as definite types. Such colonies 

 apparently had deviated from the normal type, and when placed under suitable 

 environment the original characteristics re-appeared. This may also be true of 

 certain types that seem rather remote from the normal in colonial features. 

 Colony selection may be of no avail in stabilizing such types. The different variants 

 isolated in this experiment will be discussed in the latter part of this section. 

 The broth cultures were placed at room temperature and retained for further 

 examination. The pH was determined approximately two months after the 

 cultures had been placed in storage. Several cultures in different series were 

 examined, and in every instance a pH of 8.8 was observed, which meant that the 

 medium had become more alkaline (7.0 to 8.8). Uninoculated control broth tubes 

 possessed a pH of 7.0. 



Influence of Frequent Transfer and Aging in Liver Infusion Broth 



Experiment IV. Since beef infusion broth appeared to be a suitable medium 

 for producing variants by rapid transfer and aging, it seemed plausible that a 

 more complex and favorable medium might exert an even greater influence. 

 The liver infusion broth was prepared as follows: — 



