Theses and Dissertations

Defense Date

1987

Document Type

Dissertation

Degree Name

Ph.D. Oceanography/Marine Biology

Department

Oceanographic Center

First Advisor

Curtis M. Burney

Second Advisor

Gary L. Hitchcock

Third Advisor

Murray M. Streitfeld

Fourth Advisor

Richard E. Dodge

Abstract

Escherichia coli, the preferred bacterial indicator for fecal pollution in fresh waters, does not conform to the concept of an indicator microorganism because it is rapidly killed or inactivated by seawater. This series of papers investigated the value of coliphage, a virus which infects E. coli, as an indicator of pollution in saline waters. In order to be an accurate indicator an organism must (1) be ubiquitous in wastewater, (2) survive and be detectable at least as long as the harmful organisms, and (3) be easy to isolate and identify.

A review of the literature determined that coliphage were more resistant than the common bacterial indicators to physico-chemical factors such as inorganic ions, temperature, heavy metals, nutrients, and antibiotics. Coliphage correlation with their bacterial hosts and similarities in behavior to the pathogenic viruses make them both bacterial and viral indicators.

Various culture media and host culture strains were investigated for maximum plaque forming unit (pfu) production. Two way analysis of variance showed that selection of a suitable host was of paramount importance. While selection of the culture medium was significant, it was of lesser importance. Host strain ATCC 13706 and tryptic soy agar gave the highest recovery of pfu's.

One ml log phase E.coli host culture, five ml of water sample or dilution, and five ml of culture media (maintained at 44.5 C) were combined in a sterile screw cap tube, mixed, poured into a sterile 100 X 15 mm petri dish, and incubated at 35 C. Plaque forming units were counted after 24 hrs. and expressed per 100 ml of sample.

The method proved repeatable; the titer of frozen phage aliquots declined slightly over 77 days but, the slope of the trend was not significantly different from zero at the 0.10 level (r = 0.55). These repeated analyses were done with different batches of media and hosts and represent a test of total method repeatability. Bench studies utilizing a decimal dilution series of sewage contaminated freshwater and uncontaminated seawater showed that both coliform and coliphage closely follow a theoretical dilution curve immediately after dilution with seawater. However, coliform bacteria die off at a higher rate than coliphage at higher salinities over time.

Field validation studies in fresh and brackish water (<10 >ppt) compared coliphage with total and fecal coliforms (n = 53) and gave correlation coefficients of 0.98 and 0.91 respectively. The regression equation for these samples was:

log coliphage = 0.983 (log total coliform) -1.001

The combined total coliform/coliphage relationship at 68 saltwater (>10 ppt) stations yielded a correlation coefficient of 0. 45.

Coliphage are a logical choice for a fecal indicator in marine waters since their titers are closely related to total and fecal coliform in freshwater, survive much better than coliforms in seawater, and they can be enumerated by a simple method which is not subject to salinity artifacts. The constant relation of coliphage and coliforms in freshwater indicate a possible link to current water quality standards based on total or fecal coliforms. Since coliphage pfu are a rather constant 8 - 10 % of total coliform cfu in low salinity waters where coliform inactivation is less severe, a coliphage titer of 80 – 100 pfu per 100 ml in seawater may indicate water quality equivalent to that indicated by a coliform count of 1000 cfu per 100 mI. This could aid in the interpretation of coliphage data relative to current coliform-based water quality codes.

Monitoring of sanitary water quality in Bell Channel Bay, Bahamas, during repair of a sewer plant showed that following chlorination and diversion of the effluent to a deep well, total coliform declined rapidly below detection limits. Coliphage remained easily detectable ten days later. Two canals and two marinas on Biscayne Bay were assayed for coliphage to compare sanitary water quality related to point and non-point source pollution. The Biscayne Canal was impacted by periodic upstream sewage spills, while the Little River displayed chronic contamination along its length by liveaboard boats or sewer leaks. Coliphage were shown to persist six days longer than coliform after a sewage spil l was tracked in the Canal. The liveaboard Dinner Key marina displayed low-level, spotty contamination with no seasonal pattern. King's Bay marina was free of detectable fecal contamination during the study. The use of coliphage allowed the assessment and monitoring of fecal contamination in marine waters where coliform bacteria were not suitable.

Comments

This research was supported by Contracts from the Department of Environmental Resource Management, Dade County, Florida.

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