Detection and enumeration of coliforms in drinking

water: current methods and emerging approaches

Annie Rompréa, Pierre Servaisb, ,
, Julia Baudarta, Marie-Renée de-Roubinc and Patrick Laurenta
a
NSERC Industrial Chair on Drinking Water, Civil, Geological and Mining Engineering, Ecole Polytechnique of Montreal, PO Box
b
Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Boulevard du Triomphe, Campus Plaine, CP 221, 1050, Bruss
c
Anjou Recherche, 1 Place de Turenne, 94417 Saint Maurice Cedex, France
Accepted 10 September 2001.
Available online 19 October 2001.

Abstract
The coliform group has been used extensively as an indicator of water quality and has historically led to
the public health protection concept. The aim of this review is to examine methods currently in use or which
can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid,
sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are
discussed, as are methods which have emerged from recent research developments.
Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique
and the membrane filter (MF) technique using different specific media and incubation conditions. These
methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack
of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms.
Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for
routine enumeration of coliforms in drinking water.
The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these
methods. The enzymes β- galactosidase and β- glucuronidase are widely used for the detection and
enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic
substrates exist for the specific detection of these enzymatic activities, and various commercial tests based
on these substrates are available. Numerous comparisons have shown these tests may be a suitable
alternative to the classical techniques. They are, however, more expensive, and the incubation time, even
though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid
phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic
activities, with a low detection threshold.
Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and
rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here:
the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the
immunological approach, various antibodies against coliform bacteria have been produced, but the
application of this technique often showed low antibody specificity. PCR can be used to detect coliform
bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (β-galactosidase gene)
and the uidA gene (β- glucuronidase gene) has been used to detect total coliforms and E. coli,
respectively. However, quantification with PCR is still lacking in precision and necessitates extensive
laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary
sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA
molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water
samples. FISH should be an interesting viable alternative to the conventional culture methods for the
detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8
h), but still requires research effort.
This review shows that even though many innovative bacterial detection methods have been developed, few
have the potential for becoming a standardized method for the detection of coliforms in drinking water
samples.

Author Keywords: Coliforms; Detection; Drinking water ; Cultural methods; Enzymatic methods; Molecular
tools

Article Outline

1. Introduction
1.1. What are coliforms?

2. Objectives
3. Classical methods
3.1. Multiple-tube fermentation technique
3.2. Membrane filter technique

4. Enzymatic methods
4.1. General principles
4.2. Presence/absence techniques and enumeration by multi-tube techniques using enzymatic
methods
4.3. MF technique conjugated to enzymatic detection of coliforms
4.4. Direct determination of enzymatic activity by fluorimetry
4.5. Detection of coliforms by enzymatic methods using solid-phase cytometry
4.6. Conclusion on enzymatic methods

5. Molecular methods
5.1. Immunological methods
5.2. Nucleic acid-based methods
5.2.1. Polymerase chain reaction methods
5.2.2. In situ hybridization techniques

6. Conclusions
Acknowledgements
References

Table 1. Some existing bacterial contamination regulations and guidelines for

drinking water
 Table 2. Use of PCR amplification for the detection of microorganisms in
water

Table 3. Oligonucleotide probes used for identification of Enterobacteriaceae

and E. coli in the environment

Corresponding author. Tel.: +32-2-650-5995; fax: +32-2-650-5993; email:

pservais@ulb.ac.be

Journal of Microbiological Methods

Volume 49, Issue 1, March 2002, Pages 31-54

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You are Here: Home Page > Drinking Water Protection Program > Coliform Bacteria in
Drinking Water Supplies

Coliform Bacteria in Drinking Water Supplies

A copy of the Coliform Bacteria in Drinking Water Supplies is available in Adobe
Portable Document Format (PDF, 762KB, 2pg.).

What are Coliforms?

Coliforms are bacteria that are always present in the digestive tracts of animals, including
humans, and are found in their wastes. They are also found in plant and soil material.

"Indicator" Organisms

Water pollution caused by fecal contamination is a serious problem due to the potential
for contracting diseases from pathogens (diseasecausing organisms). Frequently,
concentrations of pathogens from fecal contamination are small, and the number of
different possible pathogens is large. As a result, it is not practical to test for pathogens in
every water sample collected. Instead, the presence of pathogens is determined with
indirect evidence by testing for an "indicator" organism such as coliform bacteria.
Coliforms come from the same sources as pathogenic organisms. Coliforms are relatively
easy to identify, are usually present in larger numbers than more dangerous pathogens,
and respond to the environment, wastewater treatment, and water treatment similarly to
many pathogens. As a result, testing for coliform bacteria can be a reasonable
indication of whether other pathogenic bacteria are present.
Total Coliforms, Fecal Coliforms, and E. Coli

The most basic test for bacterial contamination of a water supply is the test for total
coliform bacteria. Total coliform counts give a general indication of the sanitary
condition of a water supply.

A. Total coliforms include bacteria that are found in the soil, in water that has been
influenced by surface water, and in human or animal waste.
B. Fecal coliforms are the group of the total coliforms that are considered to be present
specifically in the gut and feces of warm-blooded animals. Because the origins of fecal
coliforms are more specific than the origins of the more general total coliform group of
bacteria, fecal coliforms are considered a more accurate indication of animal or human
waste than the total coliforms.
C. Escherichia coli (E. coli) is the major species in the fecal coliform group. Of the five
general groups of bacteria that comprise the total coliforms, only E. coli is generally
not found growing and reproducing in the environment. Consequently, E. coli is
considered to be the species of coliform bacteria that is the best indicator of fecal
pollution and the possible presence of pathogens.

Are Coliform Bacteria Harmful?

Most coliform bacteria do not cause disease. However, some rare strains of E. coli,
particularly the strain 0157:H7, can cause serious illness. Recent outbreaks of disease
caused by E. coli 0157:H7 have generated much public concern about this organism. E.
coli 0157:H7 has been found in cattle, chickens, pigs, and sheep. Most of the reported
human cases have been due to eating under cooked hamburger. Cases of E. coli 0157:H7
caused by contaminated drinking water supplies are rare.

Coliform Testing

Testing for bacteria is the only reliable way to know if your water is safe. You cannot tell
by the look, taste, or smell of the water if disease-causing organisms are in it. The New
York State Department of Health recommends that well owners test their water for
coliform bacteria at least once a year. If you have experienced bacteria problems in the
past, it is recommended that you test your well more frequently.
When Should I Test?

Late spring or early summer are the best times to test your well, since coliform
contamination is most likely to show up during wet weather. Whether your test results are
positive or negative, understand that the sample you collected is just a "snapshot" of your
well?s water quality. The more samples you have tested, the more confident you can be
about the quality of the water you are drinking.

What do the Results Mean?

If coliform bacteria are present in your drinking water, your risk of contracting a water-
borne illness is increased. Although total coliforms can come from sources other than
fecal matter, a positive total coliform sample should be considered an indication of
pollution in your well. Positive fecal coliform results, especially positive E. Coli results,
should be considered indication of fecal pollution in your well.

What Should be done if Coliform Bacteria are Detected in a Well?

When coliforms have been detected, repairs or modifications of the water system may be
required. Boiling the water is advised until disinfection and retesting can confirm that
contamination has been eliminated. A defective well is often the cause when coliform
bacteria are found in well water.

What Kinds of Defects can Allow Contamination?

• a missing or defective well cap - seals around wires, pipes, and where the cap
meets the casing may be cracked, letting in contaminants
• contaminant seepage through the well casing - cracks or holes in the well casing
allow water that has not been filtered through the soil to enter the well. This seepage
is common in the wells made of concrete, clay tile, or brick
• contaminant seeping along the outside of the well casing - many older wells
were not sealed with grout when they were constructed
• well flooding - a common problem for wellheads located below the ground in frost
pits that frequently flood during wet weather.

Long-Term Options for Dealing with Bacterial Contamination of a

Well
• Connecting to the regional public water system, if possible
• Inspecting wells for defects and repairing them if possible
• Constructing a new well
 • Installing continuous disinfection equipment
• Using bottled water for drinking and food preparation

For further information contact

New York State Department of Health,

Center for Environmental Health
at 1-800-458-1158 ext. 2-7650
or bpwsp@health.state.ny.us

Questions or comments: bpwsp@health.state.ny.us

Revised: March 2005

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