The rapid unambiguous identification of Escherichia coli in water samples is a matter of concern to water utilities and public health authorities in the United ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2758-2759
Vol. 59, No. 8
0099-2240/93/082758-02$02.00/0
Copyright X 1993, American Society for Microbiology
Evaluation of Indoxyl-,3-D-Glucuronide as a Chromogen in Media Specific for Escherichia coli JOHN R.
HAINES,"*
TERRY C. COVERT,2 AND CLIFFORD C. RANKIN2
Risk Reduction Engineering Laboratory' and Environmental Monitoing and Systems Laboratory, 2 U.S. Environmental Protection Agency, 26 West Martin Luther King Dnive, Cincinnati, Ohio 45268 Received 21 December 1992/Accepted 11 May 1993
Indoxyl-f3-D-glucuronide (indoxyl) was evaluated as a specific chromogen for detection of Escherichia coli by the membrane filter method. In all, 413 colonies were tested from the indoxyl-supplemented media, yielding 93.3% confirmation, as E. coli. Compared with the indoxyl medium, other media gave either much lower recovery with high verification or equal recovery with poor verification.
river samples were collected below treatment plant effluent discharge points. All samples were collected in sterile 2- or 4-liter polypropylene bottles. All samples were returned to the laboratory and refrigerated within 1 h. Water samples were either diluted or used without dilution to inoculate M-TECI, ECI, and M-TECM by membrane filtration. All samples and dilutions were filtered in duplicate through Gelman GN-6 filters (Gelman Products, Ann Arbor, Mich.). After filtration, the filters were transferred to ECI, M-TECI, and M-TECM and placed in a 44.5 + 0.5°C waterbath for 24 + 2 h. At the end of incubation, apparent E. coli colonies were counted. Up to 25 colonies were transferred to Simmon's citrate agar and EC plus MUG for verification. Other colonies not typical of E. coli were also transferred to confirmation media to estimate the proportion of false-negative colonies. Up to 15 nontypical colonies were picked for verification. The EC broth plus MUG and citrate agar were prepared and dispensed into sterile 24-well tissue culture plates, 2 ml per well. Cells from colonies were transferred to both media simultaneously with sterile hardwood sticks. Positive MUG reactions were detected by exposing the tissue culture plates to 366-nm UV light and observing fluoresence. Citrate reactions were scored by color change from green to blue. Positive and negative controls were included with each set of confirmation media. Isolates from each sample were prepared on Levine's EMB agar (Difco) by streaking for isolation and identified with the API 20E system (Analytab Products, Plainview, N.Y.). Table 1 shows the results of eight samples analyzed on M-TECI and M-TECM agar. ECI agar results were not included because colony recovery on ECI was consistently poorer than on the other media. Totals of 134, 358, and 413 apparent E. coli colonies were recovered on ECI, M-TECM, and M-TECI, respectively. The colonies recovered from ECI were found to be 84.4% E. coli with the citrate and MUG reactions as confirmatory tests. Colonies recovered from M-TECM were 79.3% E. coli, and M-TECI had 93.3% E. coli. The fraction of false-negative colonies found on M-TECM was 10%, and it was less than 2% on M-TECI. ECI was not evaluated further because the recovery of apparent E. coli colonies was so low. Statistical analysis was performed on the confirmed colony counts from M-TECM and M-TECI membrane filter plates. Two-way analysis of variance was performed on both confirmed colony counts and verification percentages (9). No significant difference was found between colony verification percentages. Verifi-
The rapid unambiguous identification of Escherichia coli in water samples is a matter of concern to water utilities and public health authorities in the United States. The presence of E. coli is evidence for fecal contamination of water. Different methods exist for recovering E. coli from water. Most methods rely on the fermentation of lactose to acidic products at elevated temperatures. E. coli, a gram-negative rod-shaped (bacillus) bacterium, grows well at 35 and 44.5°C. E. coli also produces indole from tryptophane and is citrate negative, urease negative, and oxidase negative but ortho-nitrophenyl-pi-galactoside positive. Examples of recovery methods include M-TEC (2), EC broth plus methylumbelliferyl-pi-D-glucuronide (MUG) (6, 8), and the Colilert test (3). A new chromogen proposed for differentiation of E. coli is indoxyl-p-D-glucuronide (IBG) (1, 7). E. coli metabolizes both MUG and IBG to produce chromophores. The IBG metabolite indigo does not require UV exposure for visualization. Metabolism of IBG by E. coli via glucuronidase activity releases a characteristic blue color, easily detectable visually. The MUG test for E. coli also uses glucuronidase activity to produce 4-methylumbelliferone, which fluoresces under UV light (366 nm). This study was designed to evaluate the utility of IBG as a chromogen for detection of E. coli by the membrane filter procedure. Drinking-water regulations address the allowable limits for fecal coliforms and the methods used to measure them (4, 5). Media. The effectiveness of IBG as a chromogen in E. coli-specific media was evaluated by comparing recovery of E. coli from several natural water samples on different media. The media were M-TECI with glycerol (10 ml/liter) and IBG (250 mg/liter) substituted for lactose and bromcresol purple, respectively; M-TECM, with indoxyl-P-Dglucoside (250 mg/liter) added to M-TEC agar; and EC broth plus MUG (50 mg/liter). In some cases. EC broth was used as a solid medium containing IBG (250 mg/liter) and agar (15 g/liter) (ECI). The media used for verification of colonies were Simmon's citrate agar and EC broth plus MUG (Difco Laboratories, Detroit, Mich.). Sample sources. Samples known to be positive for E. coli were collected from two wastewater treatment plants and two sites along the Ohio River on different dates. The treatment plant samples were untreated effluent, and the *
Corresponding author. 2758
VOL. 59, 1993
2759 TABLE 1. Recovery and verification of apparent E. coli colonies from two media
Sample
site" Anderson Ferry (R) Little Miami (W) Muddy Creek (W) Schmidt Park (R) Anderson Ferry (R) Muddy Creek (W) Anderson Ferry (R) Muddy Creek (W) Total (%)
No. of colonies on
M-TECMb 13, 13 18, 17 5, 1 21, 10 7, 6 16, 24 51, 49 36, 71
Verified E. coli coloniesc
11/15
No. of colonies on M-TECI
16, 11 22, 18 10, 5 20, 22 9, 5 36, 37 37, 45 60, 60
13/19 4/5 11/21 13/15
15/15 10/11
15/15 92/116 (79.3)
Verified E. coli colonies
16/16 16/18 10/10 18/20 12/15 14/15 11/11
15/15 112/120 (93.3)
a All samples were collected on different dates. R, river water samples; W, wastewater treatment plant samples. b Values for individual sample replicates are shown. c Numbers are the number of positive reactions/total colonies tested.
cation percentages are important to consider because each medium performs differently in recovering apparent E. coli colonies. Adjusting raw colony counts for false-positive and false-negative results allows comparison of medium performance in terms of confirmed E. coli counts. Recovery of confirmed E. coli colonies by M-TECI agar was found to be significantly higher than recovery of E. coli colonies by M-TECM agar at P = 0.001. The efficacy of these tests was further evaluated by selecting 33 isolates from M-TECM (7) and M-TECI (2, 6) and identifying them by the API 20E system. All of the isolates were citrate negative. Thirteen isolates were MUG negative, and 19 were MUG positive. Seven MUG-negative isolates came from M-TECM agar, and six came from M-TECI agar. Three of the six M-TECI isolates had previously given positive MUG reactions. All of the isolates were oxidase negative. The API 20E system identified all but one isolate as E. coli. No isolates from ECI agar were tested because of low recovery. Membrane filtration with M-TECI agar proved to be effective in recovering E. coli from contaminated water samples. Apparent E. coli colonies were blue on a white background. All other colonies were translucent or white. M-TECI recovered 24.5% more E. coli colonies than M-TECM agar. Caution must be advised with regard to the use of the MUG reaction, because several isolates that produced positive reactions upon original isolation failed to do so upon subsequent testing. Confirmation of apparent E. coli colonies with citrate and MUG proved to be effective and easily carried out with 24-well tissue culture plates. The tissue culture plates offer the advantage of allowing many tests per plate while using less EC broth with MUG. The small volumes of medium used in the wells conserves media that contain the high-cost MUG. The use of IBG as a chromogen for the detection of E. coli appears to be an acceptable technique. Large numbers of E. coli colonies were easily detected relative to detection in
other media selective for E. coli. More confirmed E. coli colonies were recovered from the M-TECI agar. M-TECIpositive colonies were often identified as E. coli when the MUG test was negative. Therefore, IBG in the correct medium base may be a more reliable chromogen for E. coli than MUG. REFERENCES
1. Delisle, G. J., and A. Ley. 1989. Rapid detection of Escherichia coli in urine samples by a new chromogenic p-glucuronidase assay. J. Clin. Microbiol. 27:778-779. 2. Dufour, A. P., E. R. Strickland, and V. J. Cabelli. 1981. Membrane filter method for enumerating Escherichia coli. Appl. Environ. Microbiol. 41:1152-1158. 3. Edberg, S. C., M. J. Allen, D. B. Smith, and The National Collaborative Study. 1988. National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Eschenichia coli from drinking water: comparison with the standard multiple fermentation tube method. Appl. Environ. Microbiol. 54:1595-1601. 4. Federal Register. 1989. National primary drinking water regulations, total coliforms (including fecal coliforms and E. coli): final rule. Fed. Regis. 54:27544-27568. 5. Federal Register. 1989. National primary drinking water regulations. Fed. Regis. 54:29998-30007. 6. Koburger, J. A., and M. L. Miller. 1985. Evaluation of a fluorogenic MPN procedure for determining Escherichia coli in oysters. J. Food Protect. 48:244-245. 7. Ley, A. N., R. J. Bowers, and S. Wolfe. 1988. Indoxyl-,3-Dglucuronide, a novel chromogenic reagent for the specific detection and enumeration of Escherichia coli in environmental samples. Can. J. Microbiol. 34:690-693. 8. Shadix, L. C., and E. W. Rice. 1991. Evaluation of 3-glucuronidase assay for the detection of Escherichia coli from environmental waters. Can. J. Microbiol. 37:908-911. 9. Sokal, R. R., and F. J. Rohlf. 1981. Biometry: the principles and practice of statistics in biological research, 2nd ed. W. H. Freeman and Co., San Francisco.
