## 9223 ENZYME SUBSTRATE COLIFORM TEST*

9223 A. Introduction

The enzyme substrate test utilizes hydrolyzable substrates for the simultaneous detection of total coliform bacteria and Escherichia coli enzymes. When the enzyme technique is used, the total coliform group is defined as all bacteria possessing the enzyme 

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### -galactosidase, which cleaves the chromogenic substrate, resulting in release of the chromogen. Escherichia coli are defined as bacteria giving a positive total coliform response and possessing the enzyme -glucuronidase, which cleaves a fluorogenic substrate, resulting in the release of the fluorogen. The test can be used in a multiple-tube, multi-well, or a presence-absence (single 100-mL sample) format.

1. Principle



### a. Total coliform bacteria: Chromogenic substrates, such as ortho-nitrophenyl- 

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### -galactopyranoside (ONPG) or chlorophenol red- 

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### -galactopyranoside (CPRG), are used to detect the enzyme 

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### -galactosidase, which is produced by total coliform bacteria. The 

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### -galactosidase enzyme hydrolyzes the substrate and produces a color change, which indicates a positive test for total coliforms at 18 and 24 h (ONPG) or 24 h (CPRG) without additional procedures. Noncoliform bacteria, such as Aeromonas, Flavobacterium, and Pseudomonas species, may produce small amounts of the enzyme 

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### -galactosidase, but are suppressed and generally will not produce a positive response within the incubation time unless more than 10 4 

### colony-forming units (CFU)/mL (10 6 

### CFU/100 mL) are present. b. Escherichia coli: A fluorogenic substrate, such as 4-methylumbelliferyl- 

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### -glucuronide (MUG), is used to detect the enzyme 

-glucuronidase, which is produced by E. coli. The  -glucuronidase enzyme hydrolyzes the substrate and produces a fluorescent product when viewed under long-wavelength (365-nm) ultraviolet (UV) light. The presence of fluorescence indicates a positive test for E. coli. Some strains of Shigella and Salmonella spp. also may produce a positive fluorescence response. Because Shigella and Salmonella spp. are overt human pathogens, this is not considered a detriment for testing the sanitary quality of water.

2. Applications



### The enzyme substrate coliform test is recommended for the analysis of drinking and source water samples. Formulations alsoare available for the analysis of marine waters. Initially, laboratories planning to use this procedure should conduct parallel quantitative testing (including seasonal variations) with one of the standard coliform tests to assess the effectiveness of the test for the specific water type being analyzed and to determine thecomparability of the two techniques. This is particularly important when testing source waters.

Water samples containing humic or other material may be colored. If there is background color, compare inoculated tubes to a control tube containing only water sample. In certain waters, high calcium salt content can cause precipitation but this should not affect the reaction.

Do not use the enzyme substrate test to verify presumptive coliform cultures or membrane filter colonies, because the substrate may be overloaded by the heavy inoculum of weak 

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### -galactosidaseproducing noncoliforms, causing false-positive results.



## 9223 B. Enzyme Substrate Test

1. Substrate Media



### Formulations are available commercially* in premeasured packets for presence-absence or quantification † 

### and disposabletubes for the multiple-tube procedure.* The need for good quality assurance and uniformity requires the use of a commercial substrate medium. Avoid prolonged exposure of the substrate to direct sunlight. Store media according to directions and use before expiration date. Discard colored media. 2. Procedure



### a. Multiple-tube procedure: Select the appropriate number of tubes per sample with predispensed media for the multiple-tube test and label. Follow manufacturer’s instructions for preparing serial dilutions for various formulations. Aseptically add 10 mL sample to each tube, cap tightly, and mix vigorously to dissolve. The mixture remains colorless with ONPG-based tests and turns yellow with the CPRG format. Some particles may remain undissolved throughout the test; this will not affect test performance. Incubate at 35  0.5°C for period specified by substrate manufacturer.

The procedure also can be performed by adding appropriateamounts of the substrate media to the sample, mixing thoroughly, and dispensing into five 20-mL or ten 10-mL sterile tubes. Incubate as stated for multiple-tube procedure. * Approved by Standard Methods Committee, 2004. Joint Task Group: Carol Palmer (chair), Terry C. Covert, Robert E. Grant, Nancy H. Hall, Eugene W. Rice, Bruce M. Roll, Helena Solo-Gabriele.

* Colilert® and Colilert 18® and ColisureTM for multi-tube, P/A, and tray formats available from IDEXX Laboratories, Inc., Westbrook, ME. † Quanti-Tray® or Quanti-Tray®/2000, available from IDEXX Laboratories, Inc., Westbrook, ME.

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### b. Multi-well procedure: The multi-well procedure is performed with sterilized disposable packets. Add enzyme substrate to a 100-mL sample in a container, shake vigorously, and pour into tray. The tray sealer dispenses the sample into the wells and seals the package. Incubate at 35  0.5°C for period specified by substrate manufacturer. The MPN value is obtained from the table provided by the manufacturer.

c. Presence-absence procedure (P/A): Aseptically add preweighed enzyme medium to 100-mL sample in a sterile, transparent, nonfluorescent borosilicate glass or equivalent bottle or container. Optionally, add the enzyme substrate to a 100-mL sample in a sterile nonfluorescent container that is purchased commercially. Aseptically cap and mix thoroughly to dissolve. Incubate as specified in manufacturer’s instructions.

3. Interpretation



### a. Total coliform bacteria: After the minimum proper incubation period, examine tubes or containers for the appropriate color change (Table 9223:I). ONPG is hydrolyzed by the bacterial enzyme to yield a yellow color. CPRG is hydrolyzed by the bacterial enzyme to yield a red or magenta color. If the color response is not uniform throughout the sample, mix by inversionbefore reading. Read manufacturer’s instructions for interpretation guidelines. Some manufacturers suggest comparing sample against a color comparator available through the manufacturer. Samples are negative for total coliforms if no color is observed in ONPG tests or if the tube is yellow when CPRG is used. If a chromogenic response is questionable after 18 or 24 h for ONPG, incubate up to an additional 4 h. If response is negative after 24 h for CPRG, incubate up to an additional 24 h. If the chromogen intensifies, the sample is total-coliform positive; if it does not, the sample is negative.

b. Escherichia coli: Examine positive total coliform tubes or containers for fluorescence using a long-wavelength (365-nm) ultraviolet lamp (6-W bulb). Compare each tube against the reference comparator available from a commercial source of the substrate. The presence of fluorescence is a positive test for E. coli . If fluorescence is questionable, incubate for an additional 4 h for ONPG tests and up to an additional 24 h for CPRG tests; intensified fluorescence is a positive test result.

4. Reporting



### If performing an MPN procedure, calculate the MPN value for total coliforms and E. coli from the number of positive tubes asdescribed in Section 9221C. If using the presence-absence procedure, report results as total coliform and E. coli present or absent in 100-mL sample. 5. Quality Control



### Test each lot of media purchased for performance by inoculation with three control bacteria: Escherichia coli, a total coliform other than E. coli (e.g., Enterobacter cloacae ), and a noncoliform. Also add a sterile water control. If the sterile water control exhibits faint fluorescence or faint positive coliform result, discard and use a new batch of substrate. Avoid using a heavy inoculum. If Pseudomonas is used as the representativenoncoliform, select a nonfluorescent species. Incubate these controls at 35  0.5°C as indicated above. Read and record results. Other quality-control guidelines are included in Section 9020. 6. Bibliography

EDBERG , S.C., M.J. ALLEN , D.B. S MITH & T HE N ATIONAL C OLLABORATIVE S TUDY . 1988. National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: Comparison with the standard multiple tube fermentation method. Appl. Environ. Microbiol. 54:1595. EDBERG , S.C. & M.M. EDBERG . 1988. A defined substrate technology for the enumeration of microbial indicators of environmental pollution. Yale J. Biol. Med. 61:389. C OVERT , T.C., L.C. S HADIX , E.W. RICE , J.R. H AINES & R.W. FREYBERG

. 1989. Evaluation of the Autoanalysis Colilert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55:2443. E DBERG , S.C. & D.B. S MITH . 1989. Absence of association between totalheterotrophic and total coliform bacteria from a public water supply. Appl. Environ. Microbiol. 55:380. E DBERG , S.C., M.J. ALLEN , D.B. S MITH & T HE N ATIONAL C OLLABORATIVE S TUDY . 1989. National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escherichia coli from drinking water: Comparison with presence-absence techniques. Appl. Environ. Microbiol. 55:1003. EDBERG , S.C., M.J. ALLEN , D.B. S MITH & N.J. KRIZ . 1990. Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology. Appl. Environ. Microbiol. 56:366. RICE , E.W., M.J. ALLEN & S.C. EDBERG . 1990. Efficacy of -glucuronidase assay for identification of Escherichia coli by the definedsubstrate technology. Appl. Environ. Microbiol. 56:1203. RICE , E.W., M.J. ALLEN , D.J. B RENNER & S.S. EDBERG . 1991. Assay for 

-glucuronidase in species of the genus Escherichia and its application for drinking water analysis. Appl. Environ. Microbiol. 57:592. S HADIX , L.C. & E.W. RICE . 1991. Evaluation of  -glucuronidase assay for the detection of Escherichia coli from environmental waters. Can. J. Microbiol. 37:908. EDBERG , S.C., M.J. ALLEN & D.B. S MITH . 1991. Defined substrate technology method for rapid and simultaneous enumeration of total coliforms and Escherichia coli from water: Collaborative study. J. Assoc. Offic. Anal. Chem. 74:526. EDBERG , S.C., F. LUDWIG & D.B. S MITH . 1991. The Colilert® System for Total Coliforms and Escherichia coli. American Water Works Association Research Foundation, Denver, Colo. C OVERT , T.C., E.W. RICE , S.A. J OHNSON , D. B ERMAN , C.H. J OHNSON & P.M. M ASON . 1992. Comparing defined-substrate coliform tests for the detection of Escherichia coli in water. J. Amer. Water Works Assoc. 84(5):98. M CCARTY , S.C., J.H. S TANDRIDGE & M.C. S TASIAK . 1992. Evaluating acommercially available defined-substrate test for recovery of chlorinetreated Escherichia coli. J. Amer. Water Works Assoc. 84(5):91. PALMER , C.J., Y. T SAI , A.L. LANG & L.R. S ANGERMANO . 1993. Evaluation of Colilert-marine water for detection of total coliforms and Escherichia coli in the marine environment. Appl. Environ. Microbiol. 59:786. T ABLE 9223:I. C OLOR C HANGES FOR V ARIOUS M EDIA

Substrate Total Coliform Positive E. coli Positive Negative Result

ONPG-MUG Yellow Blue fluorescence Colorless/no fluorescence CPRG-MUG Red or magenta Blue fluorescence Yellow/no fluorescence ENZYME SUBSTRATE COLIFORM TEST (9223)/Enzyme Substrate Test

2C LARK , J.A. & A.H. S HAARAWI . 1993. Evaluation of commercial presence-absence test kits for detection of total coliforms, Escherichia coli , and other indicator bacteria. Appl. Environ. Microbiol. 59:380. U.S. ENVIRONMENTAL P ROTECTION A GENCY . 1994. National Primary andSecondary Drinking Water Regulation: Analytical methods for regulated drinking water contaminants; Final Rule. 40 CFR Parts 141 & 143; Federal Register 59:62456. M C FETERS , G.A., S.C. B ROADWAY , B.H. P YLE , M. P ICKETT & Y. E GOZY

.1995. Comparative performance of ColisureTM and accepted methods in the detection of chlorine-injured total coliforms and E. coli. Water Sci. Technol. 31:259. ENZYME SUBSTRATE COLIFORM TEST (9223)/Enzyme Substrate Test

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