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 -D-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--D-galactopyranoside (ONPG) or chlorophenol red--D-galactopyranoside (CPRG), are used to detect the

enzyme -D-galactosidase, which is produced by total coliform

bacteria. The -D-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 -D-galactosidase, but are suppressed and generally will not produce a positive response within

the incubation time unless more than 104 colony-forming units

(CFU)/mL (106 CFU/100 mL) are present.

b. Escherichia coli: A fluorogenic substrate, such as 4-methylumbelliferyl--D-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 also

are 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 the

comparability 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 -D-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 disposable

tubes 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 350.5°C

for period specified by substrate manufacturer.

The procedure also can be performed by adding appropriate

amounts 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 350.5°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 inversion

before 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 as

described 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 representative

noncoliform, 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. SMITH & THE NATIONAL COLLABORATIVE

STUDY. 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.

COVERT, T.C., L.C. SHADIX, E.W. RICE, J.R. HAINES & R.W. FREYBERG.

1989. Evaluation of the Autoanalysis Colilert test for detection and

enumeration of total coliforms. Appl. Environ. Microbiol. 55:2443.

EDBERG, S.C. & D.B. SMITH. 1989. Absence of association between total

heterotrophic and total coliform bacteria from a public water supply. Appl. Environ. Microbiol. 55:380.

EDBERG, S.C., M.J. ALLEN, D.B. SMITH & THE NATIONAL COLLABORATIVE

STUDY. 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. SMITH & 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. BRENNER & 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.

SHADIX, 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. SMITH. 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. SMITH. 1991. The Colilert® System for

Total Coliforms and Escherichia coli. American Water Works

Association Research Foundation, Denver, Colo.

COVERT, T.C., E.W. RICE, S.A. JOHNSON, D. BERMAN, C.H. JOHNSON &

P.M. MASON. 1992. Comparing defined-substrate coliform tests for

the detection of Escherichia coli in water. J. Amer. Water Works

Assoc. 84(5):98.

MCCARTY, S.C., J.H. STANDRIDGE & M.C. STASIAK. 1992. Evaluating a

commercially available defined-substrate test for recovery of chlorinetreated Escherichia coli. J. Amer. Water Works Assoc. 84(5):91.

PALMER, C.J., Y. TSAI, A.L. LANG & L.R. SANGERMANO. 1993. Evaluation

of Colilert-marine water for detection of total coliforms and Escherichia coli in the marine environment. Appl. Environ. Microbiol.

59:786.

TABLE 9223:I. COLOR CHANGES FOR VARIOUS MEDIA

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

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CLARK, J.A. & A.H. SHAARAWI. 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 PROTECTION AGENCY. 1994. National Primary and

Secondary Drinking Water Regulation: Analytical methods for regulated drinking water contaminants; Final Rule. 40 CFR Parts 141

& 143; Federal Register 59:62456.

MCFETERS, G.A., S.C. BROADWAY, B.H. PYLE, M. PICKETT & Y. EGOZY.

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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