Methods for Testing Water for Coliform Contaminants

The feces of humans and other warm-blooded animals contain bacteria, called coliforms, that naturally occur in the digestive tract, where they aid in the digestion of food. Although coliform bacteria do not cause disease, their presence in water, soil and vegetation may indicate the presence of other bacteria, viruses and parasites capable of causing illness. Testing for the presence and quantity of coliforms in a water sample determines the water's usability. Methods used to detect and estimate coliform numbers include the multiple-tube fermentation technique, the membrane filtration technique and the chromogenic substrate coliform test.

Multiple-Tube Fermentation

• When placed in a growth medium containing lactose, coliform bacteria ferment the sugar and produce gas. Using the multiple-tube fermentation technique, tubes of broth are inoculated with various dilutions of a water sample and incubated at 35 degrees Celsius to grow bacteria. The analysis requires five tubes each of at least three serial dilutions of the sample, such as 1 ml, 0.1 ml and 0.01 ml. Gas production or visible growth within 48 hours indicates a positive result. The number of tubes showing positive and negative results makes it possible to estimate the number of coliform bacteria present in the sample. A table, called the Most Probable Number (MPN) Index, is used to calculate coliform density.

Membrane Filter Technique

• In testing for fecal coliforms with the membrane filtration technique, a sample of water is passed through a membrane filter containing pores measuring 0.45 microns, a diameter small enough to retain any bacteria. The filter is then placed on a broth specific for growing fecal coliforms and incubated at 44.5 degrees Celsius for 24 hours. After the incubation period, bacterial colonies are counted, and final results for coliform density are calculated.

Chromogenic Substrate Test

• The chromogenic substrate coliform test employs substances that change color during a chemical reaction. This is used to detect certain proteins, or enzymes, specific to coliform bacteria. The method can be used to detect the presence or absence of bacteria as well as estimate their numbers, a process similar to the multiple-tube fermentation technique. Substrate media is commercially available in disposable tubes, wells or in containers capable of holding 100 ml samples. Inoculated media is incubated at 35 degrees Celsius for a period of time specified by the substrate manufacturer and examined for evidence of color changes. Coliform density is then determined using a table provided by the manufacturer.

Sample Size

• The intended use of the water being tested dictates the sample size needed for testing. Drinking water requires a 100 ml sample; river water, a 5 ml sample; storm water runoff, a 1 ml sample, and raw sewage, a sample of 1 ml or less.