28 MASS. EXPERIMENT STATION BULLETIN 319 



Experiment IX. In this experiment the influence of the quantity of medium 

 on colony and cellular form was studied. Meat infusion broth prepared as men- 

 tioned above was used in flasks in 300 cc. quantities and in tubes in 10 cc. amounts. 

 Strains I, II, and IV, employed in Experiment I, and Strain VII, used in Experi- 

 ment II, were studied. The flasks and tubes, after being inoculated, were in- 

 cubated at 37° C. Meat infusion peptone agar plates were streaked at frequent 

 intervals. As in the previous experiments, a heavy scum formation was observed 

 in all strains, especially I and II. The turbidity of the broth was approximately 

 the same in every case, except in the flask and tube inoculated with Strain I. 

 The cultures were examined for a period of one month, with the result that no 

 definite colony variants were detected. While Strains I and II did reveal tenden- 

 cies toward colony variation, no definite variants were established. The larger 

 quantity of broth apparently had no different effect on the strains than did the 

 smaller quantity. 



Experiment X. An attempt was made to determine the influence of meat 

 extract solution on colony and cellular form. A 3 per cent meat extract (Difco) 

 solution (pH 7.2) was prepared. The medium was flasked in 125 cc. quantities. 

 Strains I, II, and IV listed in Experiment I were studied. Ordinary meat extract 

 broth, as employed in Experiment VIII, was used as a control for determining 

 the influence on the growth characteristics of 5. pidlorum. In the meat extract 

 flasks it was difficult to ascertain the presence of growth by macroscopic examina- 

 tion. At the end of the first week of incubation agar plates were streaked from both 

 the meat extract solution flasks and meat extract broth flasks. No growth was 

 obtained from the meat extract solution. Apparently a 3 per cent solution of meat 

 extract exerted a toxic effect that was detrimental to the growth of 5. pullorum. 

 Further investigation of the influence of meat extract as such was not continued. 

 Lower concentrations of meat extract would probably permit growth of this 

 organism. The flasks containing the meat extract peptone broth revealed growth, 

 but no definite colony variants were isolated. 



Summary of Results 



While variants of 5. pullorum have been reported by a few investigators to 

 occur spontaneously, the foregoing data show that variants of this organism may 

 be induced by artificial conditions. These results should be of much interest, since 

 most investigators and diagnosticians in animal diseases regard 5. pullorum as 

 exceedingly stable in its various characteristics. Like other species of micro- 

 organisms, it will respond to certain environmental influences. Under some condi- 

 tions extreme variation may be observed in which the variant does not resemble 

 the parent strain in any of its colonial and cellular features. It will change also 

 in other characters, as will be brought out later. Different degrees of variation 

 may be observed in cultures that are subjected to certain influences. These 

 changes may be temporary or the new types may be stabilized by various methods, 

 such as frequent transfer, colony selection, and growing on special media. In this 

 section 28 variants are reported. Several different types are included in this 

 number, as may be seen in Table 4. 



The extremely rough colony type which was stabilized on plain meat extract 

 agar has undergone a marked change in colony appearance. As will be pointed 

 out later, profound alterations were also observed in virulence, antigenicity, and 

 ability to remain in suspension in saline solution. The cellular morphology of 

 this type, while exhibiting slight pleomorphism at times, appeared on the whole 

 to be quite stable and typical. 



