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To evaluate the general microbiological safety of waters, testing for the presence of coliforms is conducted.  While results may not indicate precisely what types of organisms are present, it will indicate whether the water has been exposed to human or animal sewage, and help dictate the types of treatment(s) required.

Surface, spring, and well waters are often tested for total coliforms, fecal coliforms, and Escherichia coli prior to conventional treatment.  The following is a comparison of the Multiple Tube Fermentation (MTF), Membrane Filtration (MF), and Chromagenic Substrate Test procedures for the enumeration of these organisms.  Working conditions, level of laboratory expertise and resources available, and end use of the results may dictate the best test method for given circumstances.

Multiple Tube Fermentation (MTF) Method:

MTF utilizes the results of replicate tubes and dilutions reported in terms of the most probable number (MPN).  These numbers are based on certain probability formulas, and are an estimate of the mean density of coliforms in the sample.  Precision is based on the number of tubes used, and the density is estimated by the number of positive tubes in the multiple dilutions.

Preliminary Testing

Lauryl tryptose broth is used as the presumptive medium.  Production of gas or an acidic reaction within 48 hours constitutes a positive presumptive reaction.

Confirmation Testing

Inoculation of positive presumptive tubes into brilliant green bile (gas confirms total coliforms), and E. coli medium (gas confirms fecal coliforms) is normally used for confirmation.

In addition, EC-MUG medium as a tube procedure can be used to confirm E. coli by release of a fluorogen.

These secondary steps after presumptive testing could add another 48 hours to the test procedures.  The MPN calculation yields coliform density.

The MTF method requires several different mediums, many pieces of glassware and tubes, and the inoculation of positive results to confirm steps beyond presumption.  It also requires proper interpretation of an acidic reaction and/or gas production.  If timely results are required, this is probably not the best method for water analyses.

Membrane Filtration (MF) Method:

The membrane filtration method involves drawing sample water through a specific sized membrane (such as 0.45 micron) capable of complete retention of coliform bacteria, and inoculating the filter with an Endo-type medium containing lactose.  Typically, microorganisms from the coliform group will produce a variety of red colonies with a metallic sheen on the filter.  There are some coliforms that may not produce a metallic sheen. Verification of the colonies may be required.  The number of confirmed organisms per sample volume tested will yield an enumeration.  A similar test, using M-FC medium is used to identify and enumerate fecal coliforms.  E. coli can be enumerated by transferring the membrane from the total or fecal coliform positive sample to a nutrient agar substrate.  Blue fluorescence is considered a positive response for E. coli.

This MF method will have limitations in waters turbid -- because of algae, particles, or other materials -- that will not permit the filtration of volumes sufficient to yield significant results.  High heterotrophic bacteria densities in untreated waters may interfere with the growth of coliform bacteria on the membrane.  However, this method is valuable in testing high volumes of non-turbid waters, and produces numerical results more rapidly than the MTF method.  Using this method, test results are usually highly reproducible.  This may not be the best method for surface water analyses because of the mentioned limitations regarding turbidity.

Chromagenic Substrate Test Method:

The chromagenic substrate test procedure relies on the detection of an enzyme produced by total coliform bacteria.  When the enzyme hydrolyzes a specific substrate, a color change occurs.  Additionally, a fluorogenic substrate is used to detect a specific enzyme produced by E. coli, resulting in a fluorescent product when viewed under long-wavelength UV light.  Generally, results are available within 24 hours.  A multi-well procedure can be used to obtain MPN values, as well as disposable tubes for multi-tube procedures.

The time required to set up samples and perform the testing is less in this method than with the MF or MTF methods.  However, the cost for media and testing containers may be more expensive than the other two methods. High heterotrophic plate counts (HPC) and slight turbidity will not compromise the test.  The laboratory technician must be able to distinguish colors and intensities for accurate results.  Since this method does not require individual interpretation of individual colonies as with the MF method, nor further confirmation as in the MTF method, it yields the fastest results. Generally, surface water is more likely to have turbidity and microbiological loads, which makes this method a good choice.

Summary:

Determination of microbiological quality of water is important not only for safety and health reasons, but also in determining what is the best type of treatment for the source water.  Most types of microorganisms thrive in environments created in reverse osmosis and carbon filter units.  Heavy bacteriological loads will typically require pretreatment before using these types of units.

There are other microorganisms present in water, such as giardia, cryptosporidium, and viruses that are threats to human health.  However, coliforms have been used for decades as indicators of general bacteriological quality.