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Project: Evaluation of Microbial Source Tracking Methods using Mixed Fecal Sources in Aqueous Test Samples

Background and Objectives

Fecal contamination of surface waters can come from numerous sources, including human sewage, manure from livestock operations, indigenous wildlife and urban runoff. Effective water quality management requires identification of the dominant source(s) of fecal contamination in the watershed and targeted mitigative action. Several microbiological source tracking (MST) methods have been developed to meet these needs. MST methods are intended to discriminate between human and non-human sources of fecal contamination, while some methods even differentiate among fecal contamination from individual animal species.

Despite some initial success using MST techniques to disentangle sources of fecal contamination, most of these methods are still experimental. They have been tested in only a limited number of locations with a limited number of possible fecal sources, often within a single watershed. They have not yet been subjected to standardized comparative testing. In addition, most have not been tested in marine waters. Public agencies may spend millions of dollars on MST applications with the hope of identifying sources of recreational water contamination. Without comparative studies, though, water quality managers do not have the necessary information to make logical, cost-effective choices regarding which source tracking method to utilize. The extent to which these results are reliable is also unknown. To address these issues, SCCWRP conducted comparative testing to determine the usefulness of different methods for identifying sources of fecal pollution to California beaches.


This study was conducted in 2001.


Four basic types of MST methods were evaluated: (1) genotypic library-based methods, such as ribotyping, pulsed-field gel electrophoresis (PFGE) and box-PCR; (2) library-based phenotypic methods, such as antibiotic resistance analysis (ARA) or carbon source utilization (CSU); and (3) genetic methods, which include host-specific PCR, t-RFLP and toxin-gene biomarkers. Library independent methods operate at the population rather than the isolate level. Genetic methods differentiate between sources by identifying the presence of genetic markers unique to the fecal bacteria of the targeted host species. The fourth class is direct measurement of human or bacterial viruses. Methods in this class target viruses that occur in human fecal material, but not in that of other animals and include those that detect human enteroviruses and adenoviruses or F+ coliphage, a virus that infects E. coli.

In this study, 22 researchers employing 12 different methods processed sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull, or sewage). Researchers were also provided portions of the fecal material used to inoculate the blind water samples for their use as a library material.


• No MST method tested predicted the source material in the blind samples perfectly.

• Host-specific PCR performed best at differentiating between human and non-human sources.

• Virus and F+ coliphage methods reliably identified sewage, but were not able to identify fecal contamination from individual humans since the target organisms for these methods occur infrequently in healthy individuals.

• Library-based isolate methods were able to identify the dominant fecal source in most samples, but had difficulty excluding fecal sources that were not in the samples.

• Among the library-based methods, genotypic methods generally performed better than phenotypic methods.

The percentage of samples in which the dominant fecal source of contamination was correctly identified for several of the tested methods.
For more information on Evaluation of Microbial Source Tracking Methods using Mixed Fecal Sources in Aqueous Test Samples, contact John Griffith at (714) 755-3228.
This page was last updated on: 7/2/2014