Colilert Test Kit Procedure

Introduction and Product Use further sample manipulation or testing is necessary. Field and in-house
Colilert* is used for the simultaneous detection, specific identifica- data show Colilert to be sensitive and specific for the detection of total
tion, and confirmation of total coliforms and E. coli in water. coliforms and E. coli at the 1 CFU/100 ml level in water samples with as
DO NOT USE FOR MARINE WATER. For Marine Water use Colilert many as 20,000 heterotrophic bacteria present per ml.
Cat No. MW 200. Materials
Principle W100 -100 tubes each containing Colilert reagent.
Colilert is based on the Defined Substrate Technology ® (DSTTM). W200 - 200 tubes each containing Colilert reagent.
DST utilizes indicator nutrients which cause target microbes Materials required but not provided:
contained in the sample and incubated in the DST reagent system to
produce a color change (or another signal, ie., fluorescence), both 1. Sterile 10 ml or 20 ml transfer pipets
indicating and confirming their presence. The indicator-nutrient is 2. 35° ± 0.5°C Incubator Cat. No. W1300
cleaved by the target microbe which metabolizes the nutrient and 3. Long wavelength (365nm) ultraviolet lamp, i.e., Cat. No. WL160
frees the indicator to express a specific color. The growth and 4. Color and fluorescence comparator - Cat. No. W102
reproduction process of the target microbe is fueled by the nutrient. (All items listed above are available from IDEXX Laboratories, Inc.
Please refer to back cover.)
Colilert is a specially designed reagent formulation of salts, nitrogen,
and carbon sources that are specific to total coliforms. It provides
Storage and Shelf Life
specific indicator nutrients: ONPG (O-Nitrophenyl-ß-d- Store at 4°-30°C, away from light. Colilert is stable under these
galactopyranoside) and MUG (4-Methylumbelliferyl-ß-d- conditions through the expiration date provided on the label.
glucuronide) for the target microbes, total coliforms and Escherichia Sample Collection
coli. As these nutrients are metabolized, yellow color (from ONPG)
Aseptically collect water samples as described in the 17th Edition,
and fluorescence (from MUG) are released confirming the presence
Standard Methods for the Examination of Water and Wastewater.1
of total coliforms and E. coli, respectively. Non-coliform bacteria are
suppressed and cannot metabolize the indicator nutrients. Procedural Notes
Consequently, they do not interfere with the specific identification of 1. Adhere to good laboratory practice throughout the test
the target microbes during the test incubation period. procedure. Avoid touching the reagent or the inside of the
* Colilert is referred to as MMO-MUG by the EPA in the Federal Register and as tubes or caps.
Chromogenic Substrate by Standard Methods. 2. Colilert is for analytical testing only.
3. Do not pipet by mouth.
Performance Characteristics 4. Thoroughly mix all samples immediately before inoculating.
Total coliforms and E. coli are specifically and simultaneously detected 5. Never autoclave Colilert prior to use. This process will destroy
and identified at 1 CFU/100 ml of sample, in 24 hours or less, by the reagent which is heat labile.
inoculating the reagent with the water sample and incubating it. No 6. Avoid prolonged exposure of the inoculated Colilert system to
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 direct sunlight. The indicator compounds may be hydrolyzed, become trapped in the filter, restricting their access to the
creating a false-positive ( yellow) result. indicator-nutrients in the Colilert reagent and their
7. After inoculation, Colilert should be incubated for 24 hours at subsequent growth and detection.
35° ± 0.5°C. Avoid incubation at this temperature beyond 28 9. Do not dilute the sample in buffered water for
hours because non-coliform heterotrophic bacteria present addition to Colilert. Colilert is already buffered and
may overcome the suppressant systems after this time, additional buffer compounds can adversely affect the growth of
yielding a false positive result. Yellow color after the 28 hour the target microbes and test performance.
incubation period should be verified or the sample repeated. 10. If additional confirmation is desired after incubating 24 to 28
8. Colilert is a primary water test. Colilert performance hours and reading results, transfer 0.1 ml with a pipet to EC +
characteristics do not apply to samples altered by MUG or other confirmation media.
any form of pre-enrichment or concentration. This 11. Upon mixing of Colilert reagent with the sample, a transient
includes any method such as growth on a membrane filter or blue color may appear in samples containing an excessive
growth in lactose based broth in which there is a non-specific amount of free chlorine. The sample should be considered
growth enhancing step, or any pre-filtration method such as invalid and testing discontinued.
filtering the sample through a membrane filter and then using 12. As with any coliform test method, if large numbers of
the filter to inoculate Colilert. refrigerated samples are prepared for incubation simulta-
a. Do not transfer colonies or cultures pre-grown in any neously, they should be warmed to room temperature before
enrichment media to Colilert. Colonies grown in such being placed in the incubator to avoid chilling of the incubator
non-specific media may or may not be coliforms. Colilert's contents, especially when using smaller low wattage
suppressant reagents may be overloaded by transferring such incubators.
heavy inocula of certain very weak b-galactosidase containing
non-coliforms (e.g., some Aeromonas and Pseudomonas), Test Procedure
causing a false positive total coliform (yellow) result. 1. Select the appropriate number of tubes per sample for your
Similarly, transfer of high numbers of other heterotrophs (for MPN test (5,10, etc.).
example, Flavobacterium) can cause a false positive b- 2. Aseptically fill each Colilert tube with 10 ml of a well mixed
glucuronidase fluorescence and an inaccurate indication that water sample.
E. coli is present.2 While one would not normally expect to 3. Cap the tubes tightly.
encounter such extremely high levels of heterotrophs in a 4. Mix vigorously to dissolve the reagent by repeated inversion.
water sample, pre-enrichment could produce them. Some particles may remain undissolved. Dissolution will
b. Do not pre-filter a sample and then place that filter in continue during incubation.
Colilert. The filtration step can concentrate coliforms but 5. Incubate inoculated reagent tubes at 35° ± 0.5°C for 24 hours.
also non-coliform heterotrophs, particulates, and certain 6. Read tubes at 24 hours. If yellow color is seen, check for
chemicals (divalent cations, heavy metals, etc.) which can fluorescence. Color should be uniform throughout the tube. If
overlay and suppress coliforms adversely affecting the not, mix by inversion before reading.
sensitivity of the test.3 Furthermore, coliform bacteria can
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Test Results and Interpretation MPN Index and 95% Confidence Limits for Various
At 24 hours, compare each tube against the color comparator. If no Combinations of Positive and Negative Results When
yellow is observed, the test is negative for total coliforms and E. coli. Five-10 ml Portions are Used1
If any tube has a yellow color greater or equal to the comparator, the No. of Tubes 95% Confidence
presence of total coliforms is confirmed. Giving Positive MPN Limits
If yellow is observed at 24 hours, check each tube for fluorescence by Reaction Out of Index/ (Approximate)
placing the U.V. lamp three-five inches in front of the tube and 5 of 10 ml Each 100 ml Lower Upper
making sure it is facing away from your eyes and toward the tube. 0 < 2.2 0 6.0
**Observe for fluorescence in a dark environment. If fluorescence of 1 2.2 0.1 12.6
tube(s) is greater or equal to fluorescence of the comparator, the 2 5.1 0.5 19.2
presence of E. coli is specifically confirmed. 3 9.2 1.6 29.4
4 16.0 3.3 52.9
The comparator is the lowest level of yellow and fluoresence which 5 > 16.0 8.0 Infinite
can be considered positive. A typical positive test is much more
intense than the comparator.
MPN Index and 95% Confidence Limits for Various
If a sample is yellow after 24 hours of incubation, but slightly less Combinations of Positive and Negative Results When
than the positive comparator or indeterminate, it may be incubated up Ten-10 ml Portions are Used1
to an additional 4 hours (but no more than 28 hours total). If the
sample is coliform positive, the color will intensify. If it does not No. of Tubes 95% Confidence
intensify, consider the sample negative. If the sample color remains Giving Positive MPN Limits
indeterminate, the laboratory should consider the sample invalid and Reaction Out of Index/ (Approximate)
request another sample from the same site. Some water samples 10 of 10 ml Each 100 ml Lower Upper
containing humic material may have an innate color. If a water 0 < 1.1 0 3.0
sample has background color, compare inoculated Colilert tubes to a 1 1.1 0.03 5.9
control blank of the same water sample. 2 2.2 0.26 8.1
3 3.6 0.69 10.6
If an inoculated Colilert tube is inadvertently incubated over 28 hours, 4 5.1 1.3 13.4
the following guidelines apply: No yellow color is a valid NEGATIVE 5 6.9 2.1 16.8
TEST. A yellow color after this incubation period should be verified 6 9.2 3.1 21.1
or test repeated. 7 12.0 4.3 27.1
To find the concentration of total coliforms or E. coli per 100 ml, 8 16.1 5.9 36.8
compare the number of positive tubes per sample set to the standard 9 23.0 8.1 59.5
MPN (Most Probable Number) probability chart as shown. 10 > 23.0 13.5 Infinite

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 Quality Control Procedures 4. Transfer the inoculum to the appropriately labeled Colilert tube.
Routine quality control should be conducted on each lot of Colilert 5. Repeat steps 3 and 4 for the two remaining control organisms.
received to ensure integrity and proper product performance. 6. Incubate the inoculated Colilert tubes at 35° ± 0.5°C for 24
hours.
Recommended Procedure
1. Prepare one set of Quanti-CultTM cultures (Cat. # WKIT 1001)
according to the Quanti-Cult directional insert. Each set Results should be observed within 24 hours as follows
contains the following organisms which are pre-quantitated E. coli - Yellow and fluorescent (ATCC #25922, 11775 or equivalent)
and ready to use:
K. pneumoniae - Yellow, no fluorescence (ATCC #13883 or
Pseudomonas aeruginosa
equivalent)
Klebsiella Pneumoniae
Escherichia coli Pseudomonas aeruginosa - No color, no fluorescence (ATCC
#10145, 27853 or equivalent
2. Reconstitute the contents of each of three Colilert tubes with
10 ml of sterile water (distilled or deionized). Mix thoroughly
to aid dissolution.
ATCC (American Type Culture Collection, 1-800-638-6597)
3. Add the entire contents of each Quanti-Cult vial to seperate
tubes of reconstituted Colilert.
4. Incubate the inoculated Colilert vessels at 35° ± 0.5°C for 24
If the results listed above are not obtained, repeat the test on
hours. Results should be observed within 24 hours as follows:
additional aliquots from the same lot. If again the proper results are
Pseudomonas aeruginosa - no color, no fluorescence not obtained, please call IDEXX Laboratories, Inc.
Klebsiella pneumoniae - yellow, no fluorescence
Escherichia coli - yellow, fluorescent
Alternatively
1. Reconstitute each of three Colilert tubes with 10 ml of sterile,
water (distilled or deionized). Mix thoroughly to aid
dissolution.
2. Label the tubes “Escherichia coli,” “Klebsiella pneumoniae,”
and “Pseudomonas aeruginosa,” respectively.
3. Touch a sterile inoculating loop or needle to an 18-24 hr. pure
culture slant of one of the bacteria listed. (Alternatively, ATCC
strains or equivalent may be used as the source of the inoculum.)

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References
1 Standard Methods for the Examination of Water and Wastewater.
17th edition. APHA • AWWA • WPCF • Denver, Colorado
(1989)
2 Covert, T.C., L.C. Shadix, E.W. Rice, J.R. Haines, and R.W.
Freyberg. 1989. Evaluation of the autoanalysis Colilert test for
detection and enumeration of total coliforms. Appl. Environ.
Microbiol. 55: 2443-2447.
3 Brenner, K.P., C.C. Rankin. 1990. New screening test to
determine the acceptability of 0.45 mm membrane filters for
analysis of water. Appl. Environ. Microbiol. 56(1): 54-64.
4 Edberg, S.C., Allen, M.J., Smith, D.B., and 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(6): 1595-1601.

IDEXX®, Colilert® and Defined Substrate Technology® are registered trademarks and
DSTTM is a trademark of IDEXX Laboratories, Inc. Quanti-Cult is a trademark of Chrisope Technologies.
U.S. Patent No. 4925789. OTHER PATENTS PENDING

For technical assistance call:

IDEXX Technical Service 800-321-0207
IDEXX Laboratories, Inc. Tel: 207-856-0300 Fax: 207-856-0630
IDEXX GmbH Tel: 06732-4038 Fax: 49-6732-64349
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IDEXX France S.A. Tél: 1-34-30-02-00 Fax: 1-34-30-02-08
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