Bacterial Pollution (Coliform)

Introduction

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.

Concepts

• Coliform bacteria
• Indicator
• Most probable number (MPN)

Background

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 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.

This kit is designed so that 25 separate sites may be sampled (one tube per site), or a simplified form of the multiple tube fermentation procedure can be conducted at five separate sites (five tubes per site). Inoculating several tubes per site results in a much more reliable estimate of bacterial density and reduces the chances of a false negative result. The multiple tube fermentation procedure allows the derivation of a Most Probable Number (MPN)—a statistical probability providing an estimate of the mean density of coliforms present.

Safety Precautions

Field testing should be carried out under the supervision of a teacher or other qualified adult. 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 tubes after the tubes have been inoculated. This activity requires the use of hazardous components and/or has the potential for hazardous reactions. Please review current Safety Data Sheets for additional safety, handling and disposal information before beginning this activity.

Disposal

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. Dispose of tubes by: (1) Autoclaving and discarding with regular solid waste, or (2) Filling a lab sink with a mixture of ½ cup commercial chlorine bleach to one gallon of water, fully immersing tubes and uncapping them while submerged, and soaking them for one to two hours. Rinse lab sink contents down the drain with excess water. Wash hands thoroughly.

Prelab Preparation

Sampling is best done on-site to avoid sample contamination and resulting inaccuracy. If on-site testing is not possible, samples should be brought to the lab and tested within one or two hours. Samples can be held up to six hours if packed in ice. To calculate a most probable number (MPN) value, five tubes should be inoculated from each sampling site.

Collect samples in small (50 to 100 mL), sterile bottles or flasks. For lake or pond testing, sample away from shore and below the water surface (from a dock, bridge or small boat if possible). For rivers and streams, the optimum sampling location is midstream and a few inches below the surface.

Procedure

1. Uncap the collecting container, immerse it mouth first several inches below the water surface, and turn the bottle mouth into the current. Recap the filled bottle.
2. Unwrap a sterile pipet and, using sterile technique, transfer a 1 mL sample from the collecting bottle to a lactose broth tube. If several tubes are being inoculated from a single sample, the same pipet may be used. After filling each tube, any water remaining in the pipet should be discarded, and a fresh sample drawn from the bottle.
3. Label inoculated tubes with the collection site, site characteristics, date and time.
4. Incubate tubes at 35 °C for 48 hours (72 hours at room temperature).
5. Check the tubes every 24 hours during the incubation period, and note and record any color changes.

Discussion

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 the following table. 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.

References

American Public Health Association. Standard Methods for the Examination of Water and Wastewater; 16th ed. American Public Health Association: Washington, D.C., 1990.

Campbell, G.; Wildberger, S. The Monitor’s Handbook; LaMotte Co.: Chestertown, MD, 1992.

Jacobsen, C. Water, Water Everywhere: Water Quality Factors Reference Unit; Hach Co.: Loveland, CO, 1991.

Mitchell, M. K.; Stapp, W. B. Field Manual for Water Quality Monitoring; 9th ed. Thomson-Shore, Dexter, MI, 1995.