Public health risk monitoring In waters of the U.S.,
Canada and other countries,
water quality is monitored to protect the health of the general public. Bacteria contamination is one monitored pollutant. In the U.S., fecal coliform testing is one of the nine tests of water quality that form the overall water-quality rating in a process used by U.S. EPA. The fecal coliform assay should only be used to assess the presence of fecal matter in situations where fecal coliforms of non-fecal origin are not commonly encountered.
Analysis Bacteria reproduce rapidly if conditions are right for growth. Most bacteria grow best in dark, warm, moist environments with food. When grown on solid media, some bacteria form
colonies as they multiply, and they may grow large enough to be seen. By growing and counting colonies of fecal coliform bacteria from a sample of water, the amount of bacteria originally present can be determined.
Membrane filtration is the method of choice for the analysis of fecal coliforms in water. Samples to be tested are passed through a filter of a particular pore size (generally 0.45
micrometre). The microorganisms present in the water remain on the filter surface. The filter is placed in a sterile
Petri dish with a selective
medium; growth of the desired organisms is encouraged, while other non-target organisms are suppressed. Each cell develops into a separate colony, which can be counted directly, and the initial inoculum size can be determined. Typically, sample volumes of 100 ml will be used for water testing and filtered to achieve a final desirable colony density range of 20 to 60 colonies per filter. Contaminated sources may require dilution to achieve a "countable" membrane. The filter is placed on a Petri dish containing M-FC
agar and incubated for 24 hours at 44.5 °C (112.1 degrees F). This elevated temperature heat shocks non-fecal bacteria and suppresses their growth. As the fecal coliform colonies grow, they produce an acid (through fermenting lactose) that reacts with the aniline dye in the agar, thus giving the colonies their blue color. Newer methods for coliform detection are based on specific enzyme substrates as indicators of coliforms. These assays use a sugar linked to a dye which, when acted on by the enzyme
beta-galactosidase, produces a characteristic color. The enzyme beta-galactosidase is a marker for coliforms generally and may be assayed by hydrolysis of enzyme-specific glycosides such as o-nitrophenyl-beta-D-galactose. Assays typically include a second sugar linked to a different dye, which produces a fluorescent product when acted on by the enzyme beta-glucuronidase. Because
E. coli produces both beta-galactosidase and
beta-glucuronidase, combining two dyes makes it possible to differentiate and quantify coliforms and E. coli in the same pot. More recently, the chemistry behind enzymatic detection compounds has been updated so that the indicating component is redox active, as opposed to the more usual chromogenic format, allowing fecal indicator bacteria such as
E. coli and
E. faecalis to be detected electrochemically without any sample pre-treatment. Since the colour of the detection compound is of no consequence, this allows detection in deeply coloured matrices.
US EPA testing requirements In 1989, the
U.S. Environmental Protection Agency (EPA) published its
Total Coliform Rule (TCR), which imposed major monitoring changes for
public water systems nationwide. The testing requirements under the 1989 TCR were more thorough than the previous requirements. The number of routine coliform tests was increased, especially for smaller water utilities. The regulation also required automatic repeat testing from all sources that show a total coliform positive (known as triggered source water monitoring). In 2013, EPA revised the TCR, with minor corrections in 2014. ==See also==