Publication No. 11164
Bacterial Pollution (Coliform)
Super Value Laboratory Kit
Materials Included In Kit
Lactose broth/indicator tubes, 75
Pipets, graduated, sterile, 75
Additional Materials Required
Collecting bottle, sterile (one per site)
Field testing should be carried out under the supervision of an instructor. By the very nature of coliform testing, the potential exists for the culturing of pathogenic microorganisms to hazardous levels. Students should never be permitted to open the tubes after they have been inoculated. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines. Please review current Safety Data Sheets for additional safety, handling and disposal information.
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The culture tubes may be disposed of according to Flinn Suggested Biological Waste Disposal Procedure, Type 1—Potentially Harmful Wastes Due to Microorganism-Type Contamination.
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering PracticesUsing mathematics and computational thinking
Developing and using models
Asking questions and defining problems
Planning and carrying out investigations
Constructing explanations and designing solutions
Analyzing and interpreting data
Disciplinary Core IdeasMS-LS1.C: Organization for Matter and Energy Flow in Organisms
MS-LS2.A: Interdependent Relationships in Ecosystems
MS-ESS2.C: The Roles of Water in Earth’s Surface Processes
HS-LS1.C: Organization for Matter and Energy Flow in Organisms
HS-LS2.A: Interdependent Relationships in Ecosystems
HS-LS2.B: Cycle of Matter and Energy Transfer in Ecosystems
HS-ESS3.C: Human Impacts on Earth Systems
Crosscutting ConceptsCause and effect
Structure and function
Systems and system models
Stability and change
MS-LS2-1: Analyze and interpret data to provide evidence for the effects of resource availability on organisms and populations of organisms in an ecosystem.
American Public Health Association. Standard Methods for the Examination of Water and Wastewater; 16th ed. American Public Health Association: Washington, D.C., 1990.
Bacterial Pollution (Coliform)
Conduct a simple, proven test for the presence of coliform bacteria in water. Depending on which of two suggested procedures are followed, results can be reported as either a qualitative (presence/absence) or semi-quantitative (estimated numerical density) value.
In water quality testing, coliform bacteria are used to indicate the presence of untreated (or under treated) sewage, or fecal contamination. Not necessarily pathogenic (disease causing) themselves, fecal coliforms are nevertheless valuable indicators for two reasons: (1) They are relatively easy to test for, and (2) Their presence typically coincides with numerous potentially pathogenic microbes. If coliforms are found to be present above a designated level (dependent on water use, see the Discussion section), it is assumed that potential pathogens are present, and the water is judged unfit. It would be prohibitive, in time and expense, to conduct individual tests for all potential pathogens.
Coliforms are common in the digestive tracts of cold- and warm-blooded animals, and are passed with feces. Animal wastes, agricultural runoff, and untreated sewage effluent are all likely sources of fecal coliforms in lakes and streams. Untreated sewage effluent may originate from illegal discharge sites, or, frequently, it is the result of heavy storm runoff overwhelming wastewater treatment plants that handle combined storm and sanitary input. Check with your local treatment plant to see if it handles combined input, or if storm runoff and sanitary sewage are handled by separate systems. If combined, ask plant officials if storm runoff ever overwhelms plant capacity, how they handle it, and whether or not they issue warnings or notices when that occurs.
The culture medium in the supplied tubes is lactose broth with chlorphenol red indicator. Most coliform bacteria (particularly those from the guts of warm-blooded animals) are capable of fermenting the lactose present in the medium. Lactose fermentation leads to the production of carbon dioxide (CO2) gas. The CO2 dissolves in the water-based medium and drives the pH (initially around 7) down into the acid range. The chlorphenol red indicator is red at pH 6.8 and above, and turns to yellow below 5.2. Following the incubation period, any tubes that have turned yellow (or greenish yellow) should be considered positive for coliform bacteria.
An MPN (coliform density) can be calculated using the following formula:
A sample calculation for a case with 3 positive tubes out of 5 total tubes, each with 1 mL of sample is shown:
A limitation of this simplification of the MPN procedure is that coliform concentrations higher than 180/100 mL should cause all five tubes to turn. In the event that all five tubes do show positive, results should be recorded as >180/100 mL.
Coliform density standards are established at state and regional levels for several categories of water use (check your local regulations). Typical ranges appear in Table 1.
A typical standard for treated effluent (discharged from a wastewater treatment plant) is on the order of ≤200 coliforms/100 mL. This suggests one potential sampling site: downstream from the discharge point of a nearby treatment plant. The opportunity may exist to visit a treatment facility to discuss their standards and the results of any testing you’ve already conducted.
Collecting bottles or flasks, sterile, one per site
Lactose broth/indicator tube
Pipet, sampling, sterile, disposable
Field testing should be carried out under the supervision of an instructor. By the very nature of coliform testing, the potential exists for the culturing of pathogenic microorganisms to hazardous levels. Never open the tubes after they have been inoculated. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.