&EPA
www.epa.gov
TECH
BRIEF
n o
E
E. coll and Total Coliform Detection in Drinking Water
Human waterborne diseases often result from exposure to
water that is contaminated with the feces of warm-blooded
animals, including humans. Public water systems (PWS) are
required to produce and distribute drinking water that is free
from fecal contamination. A Federal regulation, the Total
Coliform Rule (TCR), requires PWS to monitor for the
presence of specific bacterial organisms that serve as
indicators of fecal contamination. These indicator organisms
are total coliforms (TC) and Escherichia coli (E. coli). In
2010 and 2011, the U.S. EPA Environmental Technology
Verification (ETV) Program's Advanced Monitoring
Systems (AMS) Center, operated by Battelle under a
cooperative agreement with EPA, evaluated the performance
of two detection technologies for TC and E. coli. Both
technologies were designed to detect the presence or absence
of TC and E. coli in water. Technology descriptions are
provided in Table 1.
Technology Description and Verification Testing
The TCR specifies the approved methods to detect these
indicator organisms in water. The TCR also allows for
entities to apply for Agency permission to use an alternate
test procedure in place of an EPA-approved method. The
Alternate Testing Procedures (ATP) program uses a protocol
in which 200 water samples, roughly half of which are
positive and half are negative, are analyzed with a reference
method and the proposed new method. Such a protocol
design is considered adequate for evaluation of methods
proposed for low-level organism detection. Due to
constraints in the number of samples that could be generated
for this study, a modified ATP protocol was used in which
raw sewage was collected and chlorinated to achieve a 2-4
log reduction in bacterial concentration. The resulting
chlorine-stressed bacteria were then diluted with de-
chlorinated tap water to achieve low organism density in the
fractional positive range to spike drinking water dilution sets
that yielded a 50 ± 25% positive result for both the TC and E.
coli.
Standard Method (SM) 922IB for TC and SM9221F for E.
coli were the reference methods of choice used for domestic
verification. One vendor also chose to have E. coli results
compared to the Colilerf-18 reference method for possible
verification in Denmark. The verified detection technologies
utilize fluorogenic enzyme substrate compounds to detect the
presence of enzymes specific to TC and E. coli.
Environmental and Public Health Regulatory
Background of Total Coliform and E. coli at a Glance
Total Coliform bacteria (TC) are a group of bacteria that
are regularly present in environmental waters. Fecal
coliforms (FC) and E. coli are a sub-group of TC that are
more associated with the feces of people and warm-
blooded animals. FC or E. coli presence can indicate
contamination of water supplies resulting in an increased
risk of the presence of waterborne pathogens. Bacterial
indicators such as TC and E. coli are also valuable
indicators of the performance of drinking water treatment
processes and distribution system integrity.
On June 29, 1989, U.S. EPA issued the Total Coliform
Rule (TCR) which sets both maximum contaminant limit
goals (MCLGs) and maximum contaminant limits (MCLs)
for TC and E. coli in drinking water (DW) supplies. The
TCR requires all public water systems (PWS) to monitor
for the presence of TC and E. coli in the distribution
system. Distribution systems must collect and test
samples on a monthly basis, with no more than 5% of
those DW samples being TC-positive. If a sample result
is TC-positive, then the sample must be further tested for
the presence of FC or E. coli. The distribution system
population and/or the number of service connections in
the system serve as the basis for the number of samples
tested per month.
Positive (left) and negative (right) results of an ETV-verified technology.
Contact Information
ETV Advanced Monitoring Systems Center
John McKernan, EPA Project Officer
Phone:(513)569-7415
Email: mckernan.john@epa.gov
Amy Dindal, Battelle
Phone:(561)422-0113
Email: dindala@battelle.org
The ETV Program operates largely as a public-private partnership through competitive cooperative agreements with non-profit research institutes.
The program provides objective quality-assured data on the performance of commercial-ready technologies.
Verification does not imply product approval or effectiveness. EPA does not endorse the purchase or sale of any
products and services mentioned in this document.
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Table 1. Description of Total Coliform and £ coli Detection Technologies
Vendor and
Technology Name
Type of
Result
Technology Description
Colifast
ALARM At-Line
Automated Remote
Monitor
Presence/
absence
The technology is a system that uses the substrate 4-methylumbelliferyl (MU)-p-D-galactoside that is hydrolyzed by the (3-D-
galactosidase enzyme for TC detection and the substrate 4-MU-p-D-glucuronide that is hydrolyzed by p-D-glucuronidase enzyme for
E. coli detection. An increase in the amount of TC or E. coli leads to an increase in the fluorescent product MU, which is detected at
specific wavelengths by an internal spectrophotometer. Can be operated in both manual or continuous detection modes.
Pathogen Detection
Systems (PDS)
Automated Microbi-
ology Platform
(AMP)
Presence/
absence
The technology is a bench top incubator/analyzer/data logger system that utilizes an enzyme substrate ((3-galactosidase enzyme for
TC and (3-glucuronidase enzyme for £ coli) to detect the presence of TC and £. coli. Enzymes produced by TC and £. coli cleave
the substrate, resulting in the release of fluorescent products. These products accumulate in an optical sensor detected by an ultra-
violet light source/charge-coupled device optical detection system. Can be operated in both manual or continuous modes.
ETV AMS initiated verification testing in the summer of 2010 when raw sewage samples were taken from Southerly Wastewater
Treatment Plant (SWTP) in Columbus, Ohio. Technicians evaluated a total of 20 samples by comparing the reference method
proportion of positive and negative results to resulting proportions produced by the tested technologies. From this comparison,
the false positive (specificity) and false negative (sensitivity) rates were calculated. Technologies were also evaluated for opera-
tional factors such as ease of use, analysis time, laboratory space and equipment required. Table 2 provides selected perform-
ance data for the two ETV-verified detection technologies. Additional information is available in the verification reports and
statements on the ETV website at http://www.epa.gov/nrmrl/std/etv/vt-ams.htmltfecoli.
Potential Outcomes of Verified Total Coliform and E. coli Detection Technologies
Verification results show that these two detection technologies can detect both TC and E. coli in water samples. Operators can
run both of these technologies in an automated or a continuous detection mode following sample setup, obtaining results in as
little as 14 to 24 hours compared to the conventional standard methods that range from 48 to 72 hours.
Table 2. Selected Verification Results for Total Coliform and £ coli Detection Technologies
Technology Company1
Colifast
PDS18h2
PDS 24h
Colifast
PDS18h
PDS 24h
Organism
TC
TC
TC
£ coli
E. coli
E. coli
Sensitivity3
100%
65%
91%
75%
67%
67%
Specificity4
100%
100%
82%
100%
100%
100%
False Positive Rate
0%
0%
18%
0%
0%
0%
False Negative Rate
0%
35%
9%
25%
33%
33%
1 . Sewage samples were collected, chlorine-stressed, and spiked to a 10 organism/1 OOmL dilution, except the Colifast test for £ coli used a 50 org/100ml_ dilution.
2. For the PDS TC sample, a dilution of 1 org/1 OOmL also resulted in the targeted 50 +- 25% ratio but is not included in this table.
3. Sensitivity is defined as the percent of positive samples correctly identified as positive.
4. Specificity is defined as the percent of negative samples correctly identified as negative.
References:
Total Coliform Rule, United States Federal Register, 54 FR 27544-27568, June 29, 1989, Vol.54, No. 124
Revisions to the Total Coliform Rule, Proposed Rule, United States Federal Register, 75 FR 40926-41016, July 14, 2010 Vol. 75
No. 134
U.S. EPA Office of Ground Water and Drinking Water, http://water.epa.gov/drink/index.cfm
U.S. EPA ETV Program, http://www.epa.gov/etv
EPA/600/S/12/512
April 2012
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