Coliform Testing As An Andicator Of Sewage Contamination

Coliform testing is routinely used as an indicator of sewage contamination. Sewage contamination is a concern because it may contain potentially infectious organisms such as such as Escherichia coli, Salmonella, and Shigella. Exposure to sewage contamination increases the risk of gastrointestinal infections and other related illnesses. Sources of sewage contamination may include raw sewage overflows, severe flooding and leaking sewer lines or septic tanks.

To determine the potential health risks from sewage contamination swab samples could be collected and tested for sewage contamination indicators. Indicator organisms include Total Coliform, Fecal Coliform and Enterococcus.

Total Coliform

Surface Sampling Method for Total Coliform, Fecal Coliform and Enterococcus.

Sampling Procedure

  1. Measure the area of the surface to be sampled (both in the suspected area and a control in an area where low or no contamination is expected) with a tape measure and record the area in square centimetres (a 100 cm2 is recommended);
  2. Collect swab samples by removing a sterile, rayon (non-cotton) swab from a sterile tube, moisten it by inserting it into a second tube which contains a sponge soaked with sterile 1.5 mL of phosphate-buffered saline (PBS) at pH 7.2;
  3. Swab the selected surface by moving the swab back and forth across the surface with several horizontal strokes, then several vertical strokes. Rotate the swab during sampling to ensure that the entire surface of the swab was used.
  4. After sampling, return the swab to the sterile tube (with the sponge);
  5. Before collecting another sample, label the sample appropriately;
  6. On the chain of custody clearly indicate what indicator organisms you want tested, i.e., total coliforms, fecal coliforms, E. Coli and/or Enterococcus. Submit the samples to Mold & Bacteria Laboratories for testing.

Results Interpretation

Total Coliform

Coliform are found in the intestines of warm-blooded animals including human beings and hence they end up in sewage. However, they are also present in the environment as part of the natural microflora. These naturally occurring coliforms can potentially cause false positive results when testing environmental samples. For environmental samples, positive tests for total coliforms must be interpreted with caution and always be confirmed by more specific testing.

Fecal Coliform

Fecal coliform include bacteria such as E. coli, Klebsiella, Enterobacter, and Citrobacter. The fecal coliforms test is more specific than the total coliform test in indicating potential sewage contamination. However, this test is also subject to giving false negatives since these bacteria with the exception of E. coli could also be associated with plants. Therefore, as with the total coliform test, the results of the fecal coliform assay must be interpreted with caution, and positive results should be confirmed.

Escherichia coli (E. coli)

Presence of E. coli has been demonstrated to be a very specific indication of fecal or sewage contamination. E. coli has also been isolated from soil suggesting that it may not be 100% reliable indicator of fecal or sewage contamination.


Alternative indicator organisms are the enterococci. The enterococci, belong to the genus Enterococcus. They are less ubiquitous than the coliform in the environment and are always present in the feces of warm-blooded animals. Although research has indicated that enterococci might be a more stable indicator of sewage contamination than E. coli and fecal coliform, some species of Enterococcus are also associated with Plants.


No single indicator organism on it’s own is 100% reliable to demonstrated sewage or fecal contamination. Some experts therefore recommend use of 3 or more indicators, i.e., total coliforms, fecal coliforms, E. coli and/or enterococci.

For additional information or if you have any questions please contact us by phone at 905-290-9101.

MBL is Proficient For Bacteria Testing

The Canadian Association for Laboratory Accreditation Inc.(CALA) has rated MBL proficient for the following bacteria testing:

Test Parameter Method Of Analysis Status
Escherichia coli (E. coli) Membrane Filtration (DC-Agar) Proficient
Fecal (Thermotolerant) Coliforms Membrane Filtration (mFC) Proficient.
Heterotrophic Plate Count (HPC) Membrane Filtration (PCA) Proficient
Total Coliforms Membrane Filtration (DC-Agar) Proficient.
Escherichia coli (E. coli) Presence/Absence Proficient
Total Coliforms Presence/Absence Proficient.

Coliform Bacteria

The coliform bacteria are used as indicators of potential health risk for water. Coliforms are divided into 2 groups; total coliforms, i.e., all the coliform bacteria; and the fecal coliforms. The fecal coliforms are dominated by Escherichia coli (E. coli). E. coli are common in human intestines and they are generally harmless. However, some strains such as 0157 can cause serious infections.

Presence of fecal coliforms in water is widely accepted as indicator of potential contamination of water with fecal material. Contamination of water with fecal material presents greater risk of infectious microorganism such as viruses, other bacteria, protozoa and even worms.

Microbial Contamination Of Wastewater

Microbial Contamination Of Wastewater And Associated Health Risks

Wastewater consists of water that carries wastes (dissolved or suspended solids) from homes, businesses, and industries and also stormwater discharges. This water is rich in chemical and biological pollutants. Therefore, wastewater effluents, if not properly treated, can cause a number of ecological, economical, and human health problems such as:

  • algal blooms from nutrient enrichment;
  • depletion of dissolved oxygen – sometimes resulting in fish death;
  • destruction of habitats from sedimentation;
  • health risks from drinking water contaminated with pathogens or toxic chemical substances;
  • loss of revenue from closures of shellfish growing areas; and
  • loss of tourism and recreational opportunities as a result of beach closures and restrictions of beneficial uses of water.

Decontamination of wastewater before it’s released into the environment is therefore very important. This article will focus on microbial contamination of wastewater and sludge and associated health risks.

Health Risks Associated With Wastewater And Sewage Sludge

Microbial contamination of wastewater and sludge is monitored to determine the degree of pollution and potential exposure to harmful bacteria, fungi and enteric viruses. Many of the microorganisms found in sewage sludge are potentially pathogenic. These microorganisms include bacteria, fungi, parasites, and viruses.

The most common type of bacteria found in sewage sludge are of enteric origin (i.e., from the intestines) since sewage contains human waste. Also found are members of the genera Streptococcus, Clostridium, Mycobacterium, and Listeria. The presence and concentration of a given pathogenic microorganism depend, however, on the presence of healthy or sick carriers in a population. The sewage sludge can contain as high as 108 to 109 total coliforms, 106 to 107 of fecal coliforms, and 102 to 103 salmonellae bacteria per gram of dry matter. Generally, bacteria can survive in the soil for a few months. Some are partially or completely rendered inactive by extreme temperatures below the freezing point or by summer dry spells. Before using farmland to which sludge has been applied, a waiting period of a full year, or at least one summer is recommended.

Sludge may also contain a number of pathogenic fungi, such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Since human infection by these fungi is through direct contact or inhalation spores and hyphal fragments, workers who handle sludge, and especially those who are immunocompromised, are at the greatest risk.

Parasites pose health risks primarily to workers at sites where sludge has been applied. The important parasites are protozoa (e.g., amoebae, Toxoplasma gondii, Giardia lamblia, and Cryptosporidium sp.) and helminthes (such as Ascaris sp., Trichuris sp., and Taenia sp). These parasites are often present in sludge in the form of cysts or eggs. The eggs and cysts are very resistant and can be destroyed only by heat treatment at temperatures of at least 70 °C. The number of egg and cysts in a kilogram of dry matter range from several hundred to several thousand. Such numbers are very high, given that only a few parasites are required to cause infection. If present in the soil after application of sludge on land, eggs or cysts can survive several years.

Viruses may be present in raw sludge at concentrations as high as 1000 virus particles per gram of dry matter. Most of the viruses are those that attack the digestive system, i.e., enteric viruses including the hepatitis A virus. In the soil, some viruses can survive for several months in the first 5-15 cm of soil thus posing a health risk to humans.

Monitoring Microbial Contamination Of Wastewater

filter membrane with colonies

Since it’s not practical to test for every potentially pathogenic organism in wastewater, the degree of pollution of wastewater is determined by use of indicator organisms. Two groups of organisms used as indicators of pollution are the total coliforms and fecal coliforms. The fecal coliform group, however, is considered the most significant by majority of laboratories.

The coliform group of organisms consist of primarily the genera Escherichia, Enterobacter, Citrobacter, and Klebsiella. The coliforms are widely distributed in nature, and many are in the gut of humans and warm-blooded animals.

A more rigorous test to assess the extent of fecal contamination may involve testing a suite of microbial indicators (e.g., total and fecal coliforms, enterococci, Escherichia coli, coliphage, Clostridium perfringens, and human enteric viruses).

Collection Of Samples

Sampling is a critical part of sanitary water testing. As often stated, the laboratory results are only as good as the sample collected. Therefore, it’s not only important that the sample accurately represents the mass of water being tested but also to prevent deterioration and contamination of the sample before analysis.

Generally there are two methods for sampling wastewater. The grab sampling and composite sampling. Grab sampling refers to a sample collected at one time. The disadvantage of a grab sample is that it reflects the condition of the water only at the point in time that the sample was collected. Composite sampling, on the other hand consists of a collection of numerous individual discrete samples (about 100 mL each) taken at regular intervals, usually of 1 hour over a period of 24 hours. The collected samples are poured into a larger bottle and kept refrigerated at around 4 oC over the sampling period. The analysis of this composite sample, collected over a period of time, represent the average condition of the wastewater during the collection period.

counting coliform colonies

Sample containers

Two types of sample containers may be used: a wide-mouth, 120 mL borosilicate glass bottle with glass stopper or screw-cap closure or an autoclavable, nontoxic polypropylene bottle. The containers must be sterilized before use.

Sample Treatment And Handling

If the sample to be tested contains residual chlorine, it has to be dechlorinated. This is achieved by adding appropriate amount of sodium thiosulphate dechlorination agent to the bottle before sample collection. This amount is typically 1.0 mL of 1% solution of sodium thiosulphate for a 120-mL bottle and is typically added before sterilization of the bottle.

Ideally, the sample should be processed within 1 hour of collection or refrigerated below 10 °C and then processed within 6 hours. When collecting samples, sufficient space (approximately 2.5 cm) in the bottle should be left to allow mixing of the sample by shaking. Contaminating the mouth of the bottle with hands or other non-sterile objects should be avoided.

Testing For Total Coliforms And Fecal Coliforms In Wastewater

The two accepted methods used to test for total coliforms and fecal coliforms are the membrane filter procedure and the most probable number (MPN) method. The former is the most commonly used today because it’s easy to use, accurate, inexpensive, and results are obtained within 24 hours.

On M-Endo medium coliform colonies appear golden-green sheen at 10x magnification under a fluorescent light source. The coliform count of the sample size tested is expressed as the number of colony forming units (CFU) per 100 mL of water. Although each colony of organisms recovered on the membrane filter typically represents one organism, occasionally, more than one organism will merge to form a larger than normal colony.

To be considered valid, the number of coliform colonies on the filter surface should not exceed 80 CFUs, and the total number of all colonies should not exceed 200 CFUs. If these limits are exceeded, the sample should be diluted appropriately to give between 20 to 80 coliform colonies and less than 200 of total number of all colonies.

Fecal Coliforms ferment lactose at elevated temperatures as well as at 35 °C. The incubation temperature required is 44.5 plus or minus 0.2 °C. When grown on M-FC medium, they appear as blue colonies. No more than 60 fecal coliforms, or more than 200 of total colonies should be present on the membrane for a valid count.

coliform colonies on filter membrane

Results Interpretation

The recovery of fecal coliform organisms in wastewater is an indication of possible presence of enteric pathogens.

Jackson Kung’u is the Principal Microbiologist, Mold & Bacteria Consulting Laboratories (MBL) Inc.