28 Walter H. Burkholder 



in the two cases, however, was distinct. The organism under considera- 

 tion produced a deep color, which began to be formed on the second day 

 of incubation. Color did not appear in cultures of Phyt. phaseoli until 

 after three weeks. 



A further experiment, similar to the one just described, was conducted, 

 using, in place of Conn's medium, the synthetic medium suggested by 

 Ayers, Rupp, and Johnson and modified in the Manual of Methods of the 

 Society of American Bacteriologists (1923-29). Dextrose was used as the 

 sugar. There was no substitution in the nitrogen source, but to one lot 

 of media 0.1 per cent of tyrosine was added. In no case, however, did 

 a brown color develop on the agar. It is possible that there was an 

 inhibiting factor present. Just what this inhibition is, was not deter- 

 mined, although it has been noticed that in beef-extract agar to which 

 dextrose has been added, the color is somewhat slow in developing, and 

 the amount is never great. This sugar may be accountable for the failure 

 of the brown color to appear in the above-named synthetic medium. 



That this brown water-soluble pigment in the various media is due to 

 the action of an enzyme or enzymes upon tyrosine seems evident, however, 

 from the following facts: First, the color appears in media containing 

 peptone, potato, or some other substance, where the tyrosine would prob- 

 ably be present, and in milk where it could be formed. Secondly, no 

 brown color develops in a synthetic medium in the absence of tyrosine. 

 Thirdly, such a pigment develops in Conn's medium when tyrosine is 

 substituted for sodium asparaginate. Fourthly, in milk cultures that 

 have begun to show a brown pigment, masses of tyrosine crystals may be 

 found which are partly digested. It is probable that the pathogene, 

 acting upon casein, produced tyrosine, which in turn is acted upon by 

 a further enzyme or enzymes. 

 Longevity. No carefully planned experiments have been conducted on this 

 phase of the problem, but certain observations are of interest. On 

 beef-extract agar and broth, many of the strains die within a month. 

 Cultures usually are transferred every two to three weeks to insure 

 their remaining viable. The browning and clearing of broth cultures 

 often is accomplished by death. On beef-infusion agar, where the brown 

 pigment appears early and becomes intense, many cultures die within 

 a week. There appears to be a correlation between the production of 

 the brown color and death of the culture. Old potato cultures, however, 

 have revealed viable organisms. 



Nomenclature 



Although the production of this brown pigment in various media is a 

 very striking character of the pathogene, it is the only cultural character- 

 istic so far noted which separates the organism from Phyt. phaseoli. The 

 formation of filaments is a slight, but distinct, morphological character, 



