Fluorometric Method for Enumeration of Escherichia coli in Molluscan Shellfish

Abstract

A novel procedure for enumerating Escherichia coli in molluscan shellfish has been developed and evaluated. The method, a singular modification of the APHA most probable number (MPN) procedure, incorporates a fluorogenic molecule, 4-methylumbelliferyl-β-D-glucuronide (MUG), into the confirmed medium (EC) at a concentration of 50 μg/ml. The substrate could not be incorporated into the presumptive medium (LST) because of fluorescence resulting from the shellfish tissues themselves. After incubation, tubes were examined for fluorescence under long-wave ultraviolet light. Fluorescence indicated presence of E. coli. Of 1151 EC tubes examined for E. coli, both positive and negative for fluorescence, the confirmed positive rate was 91% and the false-negative rate was 11%. Certain problems inherent to the classical MPN procedure itself may understate and overstate these rates, respectively. Of more than 500 E. coli and non-E. coli isolates examined for β-glucuronidase activity, 95% of the E. coli isolates were positive for this enzyme, and only one non-E. coli isolate (a Shigella species) produced the enzyme. The MUG method, which requires only 48 h for enumeration of E. coli in shellfish, provides a considerable saving in time and labor over the standard APHA MPN procedure.