VARIATION OF SALMONELLA PULLORUM 23 



Beef liver, 500 grams 



Peptone (Difco) 10 " 



Tap water 1000 cc. 



(pH 7.0—7.2) 



The beef liver was ground fine and added to the desired quantity of water. 

 This was placed in the refrigerator over-night and then slowly brought to a boil. 

 The extract was decanted and filtered off, then adjusted to pH 7.2. After the 

 peptone was added the pH was checked before and after heating at 15 pounds 

 steam pressure. The broth was tubed in 10 cc. quantities and sterilized. 



The cultures inoculated into the liver infusion broth were the same as those 

 which were employed in beef broth in Experiment II. The strains were trans- 

 ferred daily for a period of two weeks, and at different intervals liver infusion 

 agar plates were streaked to detect any colony variation. An examination of the 

 broth tubes from day to day revealed that a slight amount of gas was being 

 liberated. All tubes appeared uniformly turbid except those which were inoculated 

 with Strain XIV. This strain settled out as it did in the beef infusion broth. 

 Liver infusion agar plates were streaked with broth cultures transferred on the 

 first, seventh, and fourteenth days. 



No definite colony variation was observed on the plates inoculated with the 

 broth cultures. Two months after the last transfer in broth, liver infusion agar 

 plates were inoculated with ten of the fourteen broth sets. Only a few strains in 

 the last three inoculated sets showed growth on the plates. The hydrogen-ion 

 concentration of the medium had changed from 7.0-7.2 to 4.5-5.8. This marked 

 acid production, in the presence of other metabolic products, may have been 

 responsible for the sterility of most of the cultures. Among the strains that 

 survived none exhibited definite variation in colony form. Six strains (II, III, 

 VIII, IX, and two cultures of Strain X, designated b and c) were transferred at 

 three- to four-day intervals on liver infusion agar. Strains III, VIII, and IX 

 died off after four weeks of frequent transfer. It was observed that certain strains, 

 including all roughs and certain so-called typical strains, appeared to be rather 

 sensitive to liver infusion agar, and that they required frequent transfer in order 

 to maintain their viability. If the cultures are placed at a lower temperature, the 

 viability period may be increased, but even then such cultures do not remain 

 alive as long as on plain meat extract agar. Strain X-b during the course of fre- 

 quent transfer revealed a temporary change in colony appearance which resembled 

 some of the more stable forms. This variation in colony type reverted to the normal 

 after two transfers. The temporary alteration can not be attributed to any known 

 cause, since this strain had been subjected to different environments. However, 

 it appears that the conditions in the liver infusion agar were particularly suitable 

 for stimulating a certain type of colony which had not been observed before in 

 this strain. Failure to stabilize this type cannot be accounted for in this instance. 

 Unfortunately, this strain also failed to survive after it had been transferred for a 

 period of four months. Strains II and X-c will be discussed later with the variants 

 previously reported. (See Table 4.) 



Influence of Frequent Transfer and Colony Selection on 

 the Enhancement of Variation 



Selection of variant colonies on solid medium has been demonstrated to be a 

 valuable means of isolating types that differ from the normal parent type. Baerth- 

 lein (1912, 1918), Hadley (1927) and Arkwright (1930) have reported that selec- 

 tion of variants by means of colony appearance may lead to the stabilization of 



