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J Bacteriol. 1965 October; 90(4): 1026-1031
Copyright © 1965 American Society for Microbiology. All Rights Reserved.

Microbiological Method for the Determination of L-Tryptophan

Department of Microbiology, The Upjohn Company, Kalamazoo, Michigan

ABSTRACT

SEBEK, OLDRICH K. (The Upjohn Company, Kalamazoo, Mich.). Microbiological method for the determination of L-tryptophan. J. Bacteriol. 90:1026–1031. 1965.—The ability of Chrombacterium violaceum to utilize L-tryptophan for the synthesis of a purple pigment, violacein, served as a basis for the development of a quantitative estimation of this amino acid. The method consists of suspending washed colorless cells of the organism in an agar layer, placing a paper disc impregnated with a tryptophan solution on top of the layer, and allowing the system to incubate. As tryptophan diffuses into the agar, it is converted into violacein, and appears as a zone of striking purple color. Since the diameter of the zone is a function of the amount of tryptophan applied, the amino acid can be quantitatively estimated within the range of 10 to 320 µg per sample with 5.6% standard deviation. The method is fairly specific for free tryptophan, since only indole, indole-3-pyruvic acid, and, to a small degree, anthranilic acid interfere. Other amino acids, tissue homogenates, tryptophan in peptide linkage, or compounds related to this amino acid do not affect its determination. The bacterium does not utilize tryptophan for the synthesis of cellular material unless its growth has been initiated by another substrate.