Simple detection of xylosidase activity in single colonies, using 1-naphthyl-beta-D-xylopyranoside

Since the xylosidase of Bacillus pumilus hydrolyzed 1-naphthyl-beta-D-xylopyranoside (naphthyl-X) to produce xylose and 1-naphthol and a chromogenic azo compound is produced by coupling 1-naphthol and Fast Blue Salt B, a simple method for detection of xylosidase activity in single colonies was studied. Escherichia coli JM109 carrying the xylosidase gene of B. pumilus was cultivated at 37 degrees C for 18 h on a LB plate containing 0.5 mg/ml naphthyl-X, and then the plate was overlaid with 3 ml of a top layer containing 24 mg of agar and 6 mg of Fast Blue Salt B. After incubation of the plate at 37 degrees C for 1 h, each colony became reddish-brown. Even a small colony with xylosidase on the plate was easily distinguished from colonies without the enzyme.