Water Quality Monitoring: Estimation of Fecal Indicator Bacteria

Fecal Indicator Bacteria

Fecal indicator bacteria are organisms whose presence in water signals contamination by faecal material from warm-blooded animals. These indicators are used to infer the possible presence of waterborne pathogens (viruses, bacteria and protozoa) and to assess the microbiological quality of water.

Why indicator organisms are used

• Faecal material from warm-blooded animals may contain a variety of intestinal microorganisms, including pathogenic bacteria (for example, species of Salmonella, Shigella and Vibrio) that can cause gastroenteritis, bacillary dysentery, typhoid fever, cholera and other illnesses.
• The direct detection of every possible pathogen in routine monitoring is impractical; therefore indicator organisms are used as proxies to indicate faecal contamination and potential pathogen risk.
• The presence of Escherichia coli (E. coli) in water is generally regarded as direct evidence of recent faecal contamination from warm-blooded animals.
• Most strains of E. coli are not pathogenic, but some strains (for example E. coli O157:H7) are pathogenic; presence of E. coli indicates contamination and potential risk, not necessarily disease-causing organisms.
• Densities of other indicator groups (for example total coliforms, faecal coliforms and faecal streptococci) may be associated with faecal contamination but are not uniquely specific to it.
• Despite limitations, indicators such as total coliforms are widely used to indicate groundwater vulnerability to faecal contamination; faecal coliforms and E. coli are commonly used to judge safety for drinking and body-contact recreation.

Common faecal indicator organisms - definitions and significance

• Total coliform bacteria: A broad group of Gram-negative, lactose-fermenting, rod-shaped bacteria found in soil, vegetation and faeces; used as a general indicator of water and treatment performance.
• Faecal coliform bacteria: A subgroup of coliforms that grow at elevated temperatures; more closely associated with faecal origin than total coliforms.
• Escherichia coli (E. coli): A species within the faecal coliform group; its presence is taken as a direct indication of recent faecal pollution from warm-blooded animals.
• Fecal streptococci (including enterococci): Gram-positive cocci used to indicate faecal pollution, and in some cases to distinguish human from animal sources when ratios are used.
• Enterococci: A subset of fecal streptococci important as indicators for marine and recreational water quality due to their persistence in saltwater.

Sampling and sample handling (practical guidance)

• Collect samples in sterile, labelled glass or plastic bottles dedicated to microbiological sampling.
• Avoid contamination of the sample by hands or unsanitised surfaces; use aseptic technique.
• If residual disinfectant (chlorine) is present in the source, add a suitable neutraliser (for example sodium thiosulphate) to the sample bottle before collection.
• Transport samples chilled (approximately 4°C) to the laboratory and process them as soon as possible; samples should be analysed promptly - preferably within a few hours and not later than 24 hours.
• Record field observations: temperature, turbidity, sample source, time and date, and any unusual findings that may affect interpretation.

Laboratory Methods For Enumeration of Indicator Bacteria

Total bacterial count (plate count method)

• This method estimates the total number of viable heterotrophic bacteria in 1 mL of sample.
• Procedure (typical): dilute the sample; for example, add 1 mL of sample to 99 mL sterile diluent (a 1:100 dilution).
• Mix a known volume of diluted sample with molten agar or pour the diluted sample on/in culture medium and incubate.
• Incubate plates at an appropriate temperature (for example 37°C for 24 hours or 20°C for 48 hours, depending on the test objective).
• After incubation count the colony forming units (CFU) on the plate using a colony counter or microscope where required.
• Multiply the counted colonies by the dilution factor to obtain the number of viable bacteria per mL of the undiluted sample.
• Notes: this method counts only culturable bacteria under the chosen incubation conditions and does not enumerate organisms that are viable but non-culturable.

Membrane filtration method

• In membrane filtration, a measured volume of water is passed through a sterile membrane filter of known pore size that retains bacteria on its surface.
• The membrane is then placed onto a selective culture medium and incubated for a defined period (often 24 hours) at an appropriate temperature (commonly around 35-37°C for many coliform tests).
• Colonies that develop on the membrane are counted directly and reported as counts per volume filtered (for example colonies per 100 mL).
• Advantages: suitable for low-concentration samples (drinking water, recreational waters); allows direct enumeration and isolation of colonies for further identification.

Liquid broth methods: presence-absence and Most Probable Number (MPN)

• Liquid enrichment tests use selective lactose broth or other media to detect the presence of coliforms or E. coli in tubes or wells inoculated with measured volumes or dilutions of a sample.
• Presence-absence format: a specified volume (for example 100 mL) is added to a growth medium and incubated; the presence of gas production, turbidity, or a colour change indicates probable coliform presence.
• Most Probable Number (MPN) format: several tubes at different dilutions are inoculated; after incubation the pattern of positive and negative tubes is compared with statistical tables (for example Maccardy's tables as referenced in the literature) to estimate the MPN per 100 mL.
• Typical detection signs: acid production or carbon dioxide (gas) in lactose broth often indicate the presence of coliform/E. coli; confirmation by further biochemical tests may be required.
• Advantages: useful for turbid or particulate samples, or when membrane filtration is impractical; widely used where laboratory resources limit direct plate counting.

Comparison of the common methods

• Plate counts give a direct count of culturable cells as CFU per unit volume but require that organisms grow on the chosen medium.
• Membrane filtration is quantitative and sensitive for low concentrations but requires clear samples that can be filtered.
• MPN methods are statistical estimates expressed as MPN per unit volume (commonly per 100 mL); they are flexible with turbid samples but less precise for single samples than direct counts.

Interpreting results and regulatory benchmarks

• Results are commonly reported as CFU per mL or per 100 mL (plate and membrane methods) or as MPN per 100 mL (MPN method).
• The presence of E. coli is considered evidence of recent faecal contamination and a potential health risk.
• National and international guidelines commonly set targets or limits for indicator organisms; for example, many drinking water standards require the absence of coliforms in 100 mL of processed drinking water (see national standard documents such as IS 10500 for country-specific limits).
• For recreational waters, specific criteria based on E. coli or enterococci densities are used to evaluate suitability for swimming and other body-contact activities.

Limitations of indicator organisms

• Indicator bacteria are not perfect substitutes for all pathogens - viruses and protozoan pathogens may be present even when indicator counts are low or non-detectable.
• Different organisms have different survival and transport characteristics in the environment; some indicators may survive longer or regrow under environmental conditions, producing false impressions of contamination age or source.
• Environmental sources (soil, vegetation) may contain organisms that can give positive results for some indicators even in the absence of faecal pollution.
• Interpretation must consider hydrology, recent weather (rainfall can mobilise faecal material), upstream discharges, and land use.

Applications of faecal indicator monitoring

• Assessment of drinking water safety and verification of treatment processes.
• Surveillance of recreational waters (beaches, lakes, rivers) to protect public health during body-contact activities.
• Monitoring of wastewater effluents and assessment of treatment plant performance.
• Groundwater vulnerability studies and source protection planning.
• Outbreak investigations and sanitary surveys when faecal contamination is suspected.

Complete Assessment of the Quality of the Aquatic Environment

• Chemical analyses of water and aquatic organisms.
• Biological tests such as toxicity tests and measurements of enzyme activities.
• Descriptions of aquatic organisms including their occurrence, density, biomass, physiology and diversity.
• Physical measurements of water temperature, pH, conductivity, light penetration, particle size of suspended and dissolved material, flow velocity and hydrological balance.

Water quality parameters that are commonly determined

Monitoring systems used to determine the quality of water in water bodies and liquid effluents