By Kelly A. Reynolds, MSPH, Ph.D.

Internationally, bacterial indicators are used to monitor and predict drinking water quality as well as compliance reporting. Guidelines and standards have been developed by the U.S. Environmental Protection Agency (USEPA), European Union (EU) and others. The use of bacteria to ensure the safety of drinking water is questioned, however, because of its ability—or lack thereof—to accurately predict the presence of viral and protozoan pathogens.

What’s an indicator?
Typical indicators used in water quality monitoring include nitrogen and phosphorous (markers for the presence of nutrients); chlorophyll-a (a marker for algal blooms); suspended solids and turbidity (water clarity indicator); dissolved oxygen (an indicator of the oxygen available to aquatic organisms); pH (a measure of the acidity or alkalinity of the water); conductivity (a measure of the salinity of water), and coliform bacteria, among others. Monitoring the physical, chemical and biological markers of a particular water source provides a means to determine the overall quality of the source water without directly monitoring the infinite number of potential toxicants that may be present.

The total coliform group of bacteria includes species such as Enterobacter, Klebsiella, Citrobacter and Escherichia. They’re gram negative, non-spore forming rods. Metabolically, total coliforms are defined as a group of closely related bacteria with the ability to ferment lactose, producing both acid and gas by-products when incubated at 35 degrees C for 48 hours. Fecal coliforms are a sub-group of the total coliform group of bacteria, differentiated by their thermotolerance, i.e., their ability to grow at 44.5 degrees C.

Pathogens enter water via fecal contamination that can lead to severe and widespread human illness from drinking, swimming and bathing waters. Sources of fecal contamination in surface waters include wastewater treatment plants, septic systems, domestic and wild animal manure, and storm runoff. Because direct testing of individual pathogens isn’t cost-effective or practical, scientists have identified indicator organisms to indicate the presence of more harmful pathogens. Fecal and total coliforms are widely used to assess suitability of water for drinking and recreational purposes.

While bacterial indicators have been highly effective for indicating presence of disease-causing bacteria–such as those associated with typhoid, dysentery and cholera—they’re known to be far less effective for determining presence of viral and protozoan pathogens. This may be due to a number of factors including different survival, transport and growth characteristics of viruses, bacteria and protozoa.

Although total coliforms include species that often inhabit intestines of warm-blooded animals, they can also occur naturally in the environment and even multiply in certain environments. They’re usually not pathogenic themselves, although their presence in drinking water often indicates a treatment failure or contamination event: thus, they’re often associated with disease outbreaks.

Coliform bacteria have been used primarily by drinking water laboratories for microbial analysis of potable water, but are also used to evaluate food, pharmaceuticals, distribution lines, treated effluents, bottled water, groundwater, marine water and other environmental samples. In June 1989, the USEPA published the Total Coliform Rule (TCR), requiring all public water systems to monitor for presence of coliforms in their distribution systems. Total coli-forms are regulated by USEPA standards where zero is considered the maximum contaminant level goal (MCLG). The enforced maximum contaminant level (MCL) requires that no more than 5 percent of samples be coliform-positive in a month. Every sample that’s positive for total coliforms must be analyzed for fecal coliforms. No fecal coliforms are permitted in any sample.

Quality indicators
Waterborne viruses of public health concern include hepatitis A, rotaviruses, Norwalk virus and other caliciviruses. Viruses and protozoa are regulated by the USEPA via the treatment technology used to render the source water free of 99.99 percent and 99.9 percent, respectively, of the pathogens. Given that direct measurement of specific viral and protozoan pathogens is deemed unfeasible, finding an appropriate bacterial indicator to represent contamination of other pathogens is still desired.

A good indicator organism is:4

  • Universally present in the feces of humans and warm-blooded animals and in large numbers,
  • Readily detected by simple methods,
  • Doesn’t grow in natural waters, and
  • Persistent in water and susceptibility to treatment is similar to waterborne pathogens.

Most drinking water guidelines also refer to total bacterial counts (i.e., total heterotrophic plate count, or HPC) in water as a means to monitor the changes in a drinking water system over time, but these bacteria haven’t proven useful for health-based monitoring or compliance measures. Their use as water quality indicators has come under much scrutiny and debate over recent years.4 The performance of a system is determined by the frequency of positive coliforms detected in routine drinking water samples over a 12-month period.

History of water indicators
Used since the 1920s, total and fecal coliforms are the standard microbial indicators of water quality. In the mid-’80s, however, research began to show that fecal coliforms didn’t correlate with swimming-associated gastrointestinal illness.

Monitoring for bacterial indicators had a clear impact on incidence of bacterial disease caused by organisms such as Vibrio cholerae (cholera), Yersinia entero-colitica (gastroenteritis), Shigella (gastroenteritis), Listeria (flu-like symptoms), Salmonella (gastroenteritis, typhoid) and Campylobacter (gastroenteritis).

In 1948, methods were developed to distinguish thermotolerant (fecal) coliforms from total coliforms and it was discovered that E. coli represents the majority (>90 percent) of the population of total coliform bacteria in human and animal feces, which can number around 109 bacteria per gram.

The concept of indicator organisms was suggested as early as 1880 after observation that Klebsiella was present in human feces and water.2 By the early 1900s, methods were developed to distinguish E. coli from other intestinal bacteria. The technology of these methods is still applicable today.

Coastal water quality
No indicator has proven perfect. For example, the total coliform group lacks specificity with regard to health risk and fecal coliforms don’t necessarily indicate recent fecal contamination or correlate with the majority of enteric pathogens. Although more specific and indicative of fecal contamination, E. coli is a poor indicator of viruses and protozoa able to survive for much longer periods of time in the environment. Interpreting results of indicator tests isn’t an easy task. Some coliform bacteria can come from sources other than warm-blooded animals (i.e., plants or soil) and overestimate health risk. Another problem is some fecal indicators can grow in the environment, increasing in numbers to falsely suggest an increase in contamination.

History shows no indicator can represent presence (or absence) of all pathogens, all the time. Currently, however, all indicators are bacteria, which have different life cycles, transport mechanisms and survival characteristics than viruses and protozoa. Many scientists are calling for more direct monitoring methods.

Alternative indicators
In highly purified water, the presence of indicator bacteria can result from a serious failure in the system. This may not mean, however, there’s a human health risk. Epidemiological studies suggest a positive relationship between high concentrations of E. coli and enterococci in ambient waters and incidents of gastrointestinal illnesses associated with recreational activities. Research supports use of E. coli and enterococci rather than the broader group of fecal coliforms as indicators of microbiological pollution.

E. coli is a member of the total coliform group and is always found in feces, providing a more direct indicator of fecal contamination and possible presence of enteric pathogens (i.e., viral, protozoan and bacterial pathogens of the gastrointestinal route). Certain strains of E. coli are directly pathogenic themselves—particularly, the serotype E. coli O:157-H7.

Enterococci are a subgroup of fecal streptococci, which are bacteria primarily found in the gut of warm-blooded animals. They’re unrelated to coliform bacteria. They have the ability to survive in salt water and closely mimic survival of pathogens in recreational environments while being closely related to recreational water contaminants.

Likewise, fecal streptococci are used as indicators of fecal contamination. They’re generally not harmful themselves but are present, along with human pathogens, in the gut of humans and animals. Other bacteria under investigation as better indicators include Campylobacter and Clostridia.

Current research, future directions for testing
Future indications are that water quality criteria will be developed for Cryptosporidium and Giardia—protozoan parasites associated with large and severe waterborne outbreaks when present in source waters. Numerous techniques have been developed for direct detection of viral and protozoan pathogens. No technique, however, has proven to be reliable, reproducible or cheap enough to replace bacterial indicators.

Because microbial outbreaks are often acute and short-lived, it’s not useful to labor over direct detection methods of pathogens that can frequently require days to weeks of analysis. Over 100 types of viruses are known to be transmitted by the fecal-oral route. For many, methods haven’t been developed for isolation and identification. Most agree total coliforms are a useful marker for non-health-related operational monitoring. In addition, turbidity of filtered drinking water and measures of disinfection such as CT—contact time–values are being increasingly used as indicators of microbial quality. Therefore, water quality may be monitored by direct detection of chemical and microbial contaminants, evaluation of the multiple treatment barriers (i.e., filtration, disinfection), or by analysis of indicator organisms.

The TCR’s importance
Implementation of the TCR has resulted in reduction in risk of illness due to enteric organisms found in sewage and animal wastes. Symptoms of enteric infections include nausea, diarrhea, cramps, jaundice, headaches and fatigue. In addition, many enteric organisms are associated with a multitude of long-term, chronic effects such as arthritis and diabetes. They may pose a significant risk to infants, young children, the elderly and severely immunocompromised people.

Under the TCR, total coliform samples must be collected at regular time intervals throughout the month and at various locations representative of water quality throughout the distribution system. The number of actual samples required depends on the population served. For example, utilities serving 10,000 persons or more are required to collect a minimum of 10 samples per month while those serving 100,000 or more are required to sample 100 samples per month.

Systems positive for total coliforms must be further tested for the presence of fecal coliforms or E. coli. If any routine sample is total coliform positive, it must be sampled at least three consecutive times from the same tap and within five service connections upstream and downstream of the original sample. A violation of the TCR means either high levels of coliforms were found in the water or that fecal coliforms or E. coli were found.

Looking back on the Total Coliform Rule and the use of indicator bacteria for predicting water quality, we find that not much has changed. Even with the invention of rapid and cost effective approaches for direct monitoring of drinking water supplies for the presence of viral and protozoan pathogens, the technical difficulty of applying these methods for routine monitoring has prevented their widespread application. For effective water quality monitoring in the future, new indicators and improved methods for direct pathogen detection continue to evolve.


  1. Federal Register, “Guidelines Establishing Test Procedures for the Analysis of Pollutants; Analytical Methods for Biological Pollutants in Ambient Water,” Vol. 68, No. 139,:, July 21, 2003
  2. Hendricks, C.W., et al., “Exceptions to the coliform and fecal coliform tests,” In Berg G., Ed. Indicators of Viruses in Water and Food, p. 99, Ann Arbor Science, Ann Arbor, Mich., 1978.
  3. Stevens, M., D. Ashbolt and D. Cunliffe, “Microbial Indicators of Water Quality,” National Health and Medical Research Council, Canberra, Australia, September 2001:, September 2001.
  4. World Health Organization, Guidelines for Drinking Water Quality: Second Edition, “Vol. 2. Health Criteria and Other Supporting Information,” WHO, Geneva, 1996: www.who. int/water_sanitation_health/dwq/gdwq2v1/en/index1.html, 1996

About the author
Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on development of rapid methods for detecting human pathogenic viruses in drinking water. She holds a master of science degree in public health (MSPH) from the University of South Florida and doctorate in microbiology from the University of Arizona. Reynolds has also been a member of the WC&P Technical Review Committee since 1997.


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