Water Pollution and Control

While care must be taken to ensure sewage, storm water and agricultural runoff are controlled in a manner safe to public heath, recreational waters may become readily contaminated by anthropogenic factors.  In fact, “many epidemiological studies have identified gastrointestinal and upper respiratory illnesses in bathers that were a result of such contamination.” [1]

The ideal indicator of fecal contamination of recreational waters would be one of the enteric pathogens, such as Salmonella or Norwalk virus, however, because these are usually present at low levels and are irregularly distributed, even during disease outbreaks, they are difficult to isolate and quantify. [1] Moreover, the absence of one enteric pathogen does not necessarily preclude the absence of another. Testing for every possible waterborne disease-causing microorganism would not be pragmatic and it is therefore common practice to monitor more plentiful but non-pathogenic surrogate bacteria present in human and animal feces.

The indicator organism most widely used is the bacterium Escherichia coli (E. coli); sometimes referred to as fecal coli forms, which are present at about 10^7 bacteria per gram of dry feces. A simple, accurate, nonlethal membrane filter technique [2] for the rapid enumeration of E. coli from marine, estuarine and fresh water samples was used to determine the presence of possible pathogens in a natural stream within 24 hours without requiring subculture and identification of isolates. m TEC Agar was used for isolating, differentiating and rapidly enumerating thermo-tolerant Escherichia coli from water by membrane filtration.

Results after 24 hr of incubation showed zero E. coli cfu (colony forming unit) as expected given the tendency for natural river systems (where the sample was taken) to self-purify. A sample collected from a source expected to have possible coli forms was also tested and showed the presence of E. coli in addition to other coli forming units.

Introduction

Currently the membrane filtration (MF) technique for enumerating fecal coli forms in water is the most widely used. Counts are expressed as colony forming units or CFU.  Since coli form density may be high, in for instance raw sewage, sample dilution will be necessary to get optimum plate counts of about 20 to 80 CFU per plate. Usually about 10 to 100 ml are filtered in triplicate for each dilution level.  In this experiment, natural water is used, and results are expected to show <200 CFU/100ml. A 500ml undiluted sample will be used.

Method and Procedure

Wearing protective clothing and working in a biological hood, method and procedures are prepared in accordance with FDA Method 1603. [2] A well-mixed water sample of known volume is passed through a sterile 0.45 urn pore size membrane filter, under vacuum. After filtration the membrane retains all the bacteria on its surface. The membrane is placed on a nutrient-media and incubated for 24 hours; which promotes the growth of E. coli. Each bacterium can give rise to a visible, characteristically stained, colony for counting.

Results and Discussion

As would be expected, samples collected along a natural stream showed zero E. coli and eight other colony forming units as reported by the lab technician after 24 hours of incubation. In Figure 1 (right), 6 small and 1 large E. coli (fucia) and several other counts (yellow and pink) are observed on samples collected from a dog’s water dish [3] and incubated for 48 hours.

Despite having aeration, circulation, and filtration using activated carbon, water samples collected from the dish after 4 days of continuous operation show evidence of possible pathogenic growth. Further identification by experienced practitioners to generate genetically pure cultures from a mixed culture of genetically different organisms, using a technique known as streaking [4] would be required to adequately determine their nature and risk. Upon further discussion, the lab technician pointed out experience has shown natural flora and bacteria would be present in the dog’s mouth and the presence of E. coli doesn’t preclude the counts from being non-lethal. As such, this information does provide a reliable measure concerning the effectiveness of the practices employed on the dog’s dish. Furthermore, the results show that chlorine is no longer persistent in the tap water used in this study after 4 days and that aeration in addition to other factors may be reducing chlorine.

The MF technique described above does provide a quick and elegant indicator for the presence of enteric pathogens in water.  Early detection is paramount.  Figure 2 (left) shows molds growing on the natural water sample after 7 days!

Although the MF test is relatively simple, there are many assumptions and potential errors behind extrapolating the mean coli form density from a water sample to a water body such as temporal and spatial coli form distribution variation, potential die-off after sampling due to chlorine damage, or subsequent re-growth in the water body, believed to be due to cellular recovery after chlorine damage. Also, since a colony may derive from one coli form, or a clump of more than one bacterium, the actual bacterial count may be greater. Nonetheless, this simple test, when performed properly in conjunction with a sound sampling regime, can protect large populations of humans from the disastrous consequences of water contamination.

References

[1] Health Canada, Guidelines for Canadian Recreational Water Quality, May 8, 2009 http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/guide_water-1992-guide_eau/section3-eng.php
[2] Method 1603: Escherichia coli (E.coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC), September 2002, http://www.epa.gov/nerlcwww/1603sp02.pdf
[3] Rush, D. , Help your pets make better choices, Jul. 2009, http://eco-eng.blogspot.com/2009/07/update-help-your-pets-make-better.html
[4] Wikipedia, Streaking (Microbiology), May 2009, http://en.wikipedia.org/wiki/Streaking_%28microbiology%29