VARIATION OF SALMONELLA PULLORUM 25 



year. During this period the colony type varied but slightly for Strains I and XII • 

 Strain IV-a reverted partly to the normal type. The original variant type of 

 colony was observed for four months before it reverted to the normal. Occa- 

 sionally, definite rough features were observed. At the time of reversion to the 

 normal, the original parent Strain IV-a was again placed on liver infusion agar. 

 The growth which resulted contained colonies that were identical with those 

 observed in the first instance. However, this last variant also exhibited a marked 

 tendency to revert to the normal type. After eight months of frequent transfer, 

 colony types appeared that did not resemble typical S. pullorum. Strain XIV 

 also yielded a type of colony that did not remain stable. While fluctuation in 

 colony type was observed it never approached the normal type. The different 

 variants isolated in this experiment are designated as follows: — 

 C — Variant derived from Strain I 

 D — Variant derived from Strain IV-a (first trial) 

 D-l — Variant derived from Strain IV-a (second trial) 

 E —Variant derived from Strain XIV 

 Strain XII remained typical in its colonial features; variation was observed 

 at no time in this strain (See Table 4). 



Miscellaneous Attempts to Induce Variation 



When this investigation was undertaken, little knowledge was available as 

 to what methods would be most effective in bringing about variation in 5. pul- 

 lorum. Among some of the successful methods applied to other microorganisms 

 are (a) alternate transfer in liquid and solid media, (b) cultivation in medium 

 with increased concentration of peptone, (c) growing in deep liquid medium, 

 (d) cultivation in meat extract solution. The author was especially interested 

 in the influences that such methods might exert on S. pullorum, since similar 

 conditions are often found in the isolation and study of this organism. 



Experiment VII. An effort was made to determine the effect of alternate 

 transfer from liver infusion broth onto beef infusion agar. Four strains employed 

 in Experiment I were selected. The liyer infusion broth was prepared in the 

 same manner as in Experiment IV. The beef infusion agar contained 1 per cent 

 peptone and 1.5 per cent agar (pH 7.2). The organisms were incubated in the 

 broth for 24 hours at 37° C. and then streaked on agar plates. After incubation 

 for 24 hours at 37° C, the roughest colonies were selected for transfer to fresh 

 liver infusion broth. Each strain was cultivated in broth and on agar plates six 

 times. At the termination of the transfer period no definite variation was observed 

 in the strains treated. After a period of seven weeks several broth cultures were 

 streaked on plates. Only two strains revealed growth but no variation. This 

 experiment was not repeated since some of the previously mentioned experiments 

 proved more successful in bringing about variation. It is interesting to note that 

 the organisms in this experiment, as well as in those previously reported, grew 

 with difficulty in liver infusion broth, and in the majority of cases died within 

 two months. 



Experiment VIII. In this experiment a study was made of the influence of 

 peptone concentration on colony form. Five per cent peptone solution, pH 7.2, 

 was placed in flasks in 250 cc. quantities. Meat extract broth (1 per cent peptone, 

 0.3 per cent meat extract, pH 7.2) was also prepared in 250 cc. quantities. Strains 

 I, II, and IV employed in Experiment I were selected. Each strain was inoculated 

 into a flask of peptone solution and of meat extract peptone broth. The flasks 



