Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.