Evaluation of the β-Glucuronidase Substrate 5-Bromo-4-Chloro-3-Indolyl-β-D-Glucuronide (X-GLUC) in a 24-Hour Direct Plating Method for Escherichia coli

Abstract

A 24-h direct plating method for Escherichia coli using Peptone-Tergitol agar was used to compare the effectiveness of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-[}-D-glucu-ronide (X-GLUC) with the fluorogenic substrate 4-methylumbel-liferyl-P-D-glucuronide (MUG) for P-glucuronidase activity. Values obtained for enumeration of two strains of E. coli recovered from artificially inoculated raw minced chicken (i.e., plating efficiencies on the inoculum, cells per g, and recovery percentages related to those on Plate Count Agar) indicate that X-GLUC at 50 |i.g/ml was as effective as MUG in an agar medium. Unlike MUG, X-GLUC does not require ultraviolet light illumination, and the color reaction produced remains localized in the positive colonies. p-Glucuronidase activity is used as a rapid and relatively specific indicator of Escherichia coli in many foods and environmental samples. The fluorogenic substrate 4-mefhy-lumbelliferyl-p-D-glucuronide (MUG) (5) is an efficient indicator of enzyme activity. The addition of MUG to lauryl tryptose broth by Feng and Hartman (2) improved both the speed and sensitivity of the standard most probable number (MPN) method. Recently, Damare et al. (1) developed a 24-h direct plating medium containing MUG and also Tergitol 7 to inhibit gram-positive bacteria. In this study we have evaluated the new chromogenic substrate 5-bromo-4-chloro-3-indolyl-p-D-glucuronide (X-GLUC) (3) as a substitute for MUG in a 24-h plating assay. When added to the basal medium of Damard et al. (l),v/e found that X-GLUC was as effective as MUG in recovering E. coli from artificially inoculated minced chicken, and in addition, had certain advantages over MUG in the enumeration of colonies on agar surfaces. METHODS AND MATERIALS Stationary phase cultures of E. coli (two strains isolated from ground beef-Fl 113 andFl 114) were grown for 24 h in Brain Heart Infusion Broth (Difco, Detroit, MI) at 37°C before resuspension in an equal volume of 0.85% saline solution. After 454 g of thawed minced chicken were inoculated with 10 ml of a 1:10 dilution of the resuspended cells in saline solution, three 25-g samples were removed and each was blended in 225 ml of 0.1 % peptone water for 2 min. All dilutions for spread-plating in triplicate were made in 0.1% peptone water. Peptone-Tergitol Agar (PTA) (1) was used as the base for preparation of both the MUG-containing agar (PTG) (7) and the X-GLUC-containing agar (PTX). A stock of X-GLUC (Research Organics, Cleveland, OH) prepared by dissolving 20 mg in 1 ml of dimethylformamide (DMF) (Sigma Chemical Co., St. Louis, MO) was added without sterilization to the tempered agar base at a concentration of 50 u,g/ml. All plates were incubated at 35°C and observed 16-24 h after inoculation. In addition to PTG and PTX media, appropriate dilutions of both the inoculum and the inoculated chicken were also plated on Plate Count Agar (PCA) (Difco, Detroit, MI) and added to Lauryl Sulfate Broth (LSB) (BBL, Cockeysville, MD) for a 3-tube MPN determination at 35°C. Positive MPN tubes were inoculated into EC Broth (Difco, Detroit, MI) and incubated at 44°C for 48 h. RESULTS AND DISCUSSION To determine the optimal concentration of X-GLUC in PTA, a series of plates containing X-GLUC at concentrations up to 50 Lig/ml were spread with E. coli and observed after 24 and 48 h. Maximal color intensity was obtained in 24 h at 50 LLg of X-GLUC/ml, thus agreeing with a previous report of its use in a different medium (3). Color intensity was almost as vivid at 40 Lig/ml; however, at lower concentrations (20 or 30 Lig/ml) the intensity was reduced perceivably and color development was not detectable as early. Neither X-GLUC nor its solvent DMF produced any discernible inhibition of colony size or number at the levels used in this study-or even at DMF levels over 6 times greater (data not presented). All dilutions, therefore, were plated on PTX agar containing 50 LLg of X-GLUC/ml. Enumeration of the two strains of E. coli recovered from artificially inoculated raw minced chicken using PTG agar and PTX agar is summarized in Table 1. Counts obtained on a nonselective medium, PCA, and from the MPN assay on one strain are also included as well as plating efficiencies and recovery percentages. These data show very similar recovery values on all the media used, with good agreement between the PTX and PTG media