Public Lake vs. Private Lake Water Quality
Alverno College, Milwaukee, WI
Independent Research Project
Is our Water Safe to Swim In?
The purpose of my research experiment conducted October 3, 1999 through October 22, 1999 was to determine the presence of coliform contamination in eight random water samples from two different lakes. I obtained water samples from Mudd Lake and Big Cedar Lake located in Washington County. My tested hypothesis was that a public lake would contain less fecal coliform bacteria than a private lake due to state regulations.
Key Words: Water Quality, Big Cedar Lake, Mudd Lake, Fecal Coliform, Water Contamination
Water contamination in our recreational waters is not a new problem and neither is Swimmer’s Itch. Waste materials getting into our water systems cause both of these problems. In this experiment I will not be testing drinking water but I will direct my concern toward the water we use for entertainment. In the summer months people enjoy swimming, fishing, or simply boating in lakes. I cannot recall the last time someone addressed the importance of clean swimming water. The condition of the swimming water should be a concern for swimmers or property owners who constantly use the lake.
According to J. Raloff (1996),"a study also links sewage derived wastes to flulike diseases and to ear problems in swimmers." People may be getting sick from the water and not really know the water is the culprit. David Kay (1994) reports, "…respiratory diseases in the swimmers was associated with their exposure to fecal streptococcus, a type of noncoliform bacterium." A coliform is a facultative anaerobe that produces gas. Escherichia coli and Enterobacter aerogenes are gram negative and nonspore-forming rods. These organisms are also not usually found in water or soil, but in intestines (Benson1998). If sewage is getting into our swimming water, I will be able to identify from my samples: E.coli or E.aerogenes. These types of coliform findings confirm the fact that sewage is present in the water samples that I obtained from the different lakes. The private lake should contain more coliform bacteria than the public lake.
I used sterile glass bottles to collect 4 samples from Mudd Lake and another 4 bottles to collect samples from Big Cedar Lake. I made sure that no precipitation occurred prior to collecting the samples. After I performed a bacterial examination of the water by a Multiple tube method, Mixed Acid Fermentation (Methyl Red), Butanediol Fermentation (Voges-Proskauer), Tryptophan Hydrolysis (Indole), Citrate, and BBl Enterotube 2 test it is confirmed that coliforms are present in both water samples. Mudd Lake is classified as turbid and shows a more positive amount of coliforms in the samples. For example, there was more gas formation present in the turbid tubes versus the clear water tubes (Big Cedar Lake Samples).
The multiple tube method, Mixed Acid Fermentation (Methyl Red), Butanediol Fermentation (Voges-Proskauer), Tryptophan Hydrolysis (Indole), Citrate, and BBl Enterotube 2 tests were performed on all of the water samples times two.
The 8 water samples were collected randomly from Mudd and Big Cedar Lake on October 3, 1999. I went to 4 random sites on each lake and collected the water with 8 different sterile glass bottles. All of the inoculating and pouring of the broths was conducted in the Microbiology Prep Lab Room at Alverno College in Milwaukee, Wisconsin.
For the completion of the Multiple Tube Method, the first step was to determine the amount of pollution in the water by simple observation (clear or turbid). To tell if the water is clear: the water appears to be clear while holding up a white piece of paper. I was able to tell the turbid water because it had a yellow tint and floating particles. The Big Cedar Lake sample was clear and the Mudd Lake sample was turbid. From now on I will refer to the samples by turbid 1-4 and clear 1-4. For each sample the same tests will be performed but the turbid water requires more tubes in this case. I will explain what I did to prepare the "turbid" tubes. I needed the following materials for all 4 samples (remembering that each sample has to be tested twice): 96 tubes with caps, 24 Durham tubes of DSLB (double strength lactose broth), 72 Durham tubes of SSLB (single strength lactose broth), 10ml pipettes a clean pipette is needed for each transfer of water sample, 1ml pipettes. In a test tube rack place the 12 tubes horizontally four times. For the "clear" tubes tests I set up: 24 DSLB, 48 SSLB, and I had enough pipettes available. All of the broths were made under the supervision of Cathy Simmerling. I needed to make 48 tubes of DSLB and 120 SSLB tubes. On the bottle of the lactose broth I followed the instructions and did conversion factors for the correct milliliter amount each tube gets of the broth. After setting up each of the tubes, I put small tubes inside the test tubes (for collecting gas), put the correct strength amount in every tube, capped, and autoclaved. After autoclaving we had to wait for the tubes to cool and then I pipetted the following amounts in to each tube that was labeled. For turbid: 10ml of each sample into 3 DSLB tubes, 1.0 ml in 3 SSLB tubes, .1 ml of turbid in 3 SSLB tubes and .01 ml of each sample into 3 SSLB tubes. For clear: 10ml of sample into 3 DSLB tubes, 1.0 ml into 3 SSLB tubes, and 0.1ml in 3 SSLB tubes. For example, for turbid sample 1 there was 12 tubes (going from left to right), 3 of DDLB and 9 of SSLB. After pipetting the water samples into each of the test tubes, all tubes were placed into the incubator for 24-48 hours. After the time had lapsed I had to evaluate all of the tubes and rate the MPN (the Most Probable Number) of coliforms present in 100ml of water using Appendix A of the Microbiological Applications Lab Manual. After recording all the gas present in the tubes, it was already assumed that the water was unsafe. With the tubes that I considered a positive result (more than 10% gas present) I made Levine EMB plates to confirm coliform presence. First I had to determine the number of positive tubes and make the Levine EMB agar. To make things easier I make 16 plates and streaked turbid 1-4 and clear 1-4 twice. After 24 hours of incubation I recorded the plates that had colonies with dark centers or a greenish metallic sheen. Then I had to prepare all of the different broths and slants for the following tests. Each of the concentrations were different and each bottle gives directions for the dilutions.
Mixed Acid Fermentation (Methyl Red) 32 tubes of MR-VP medium broth. After making the broth again everything must be autoclaved. Then I had to incubate the tube for 24-48 hours after inoculating. Then to each tube I had to add 4 drops of methyl red indicator. When the tubes turned red I knew it was a positive methyl red test and positive for E.coli.
Butanediol Fermentation (Voges-Proskauer) & Barritt’s Reagents: 32 tube of 3-5 day culture of water samples. After the time had past I added 18 drops of Barritt’s solution A to each tube and then add the same amount of Barritt’s solution B to each tube and shook the tubes vigorously for 20 seconds until the tubes turn pink and let the tube stand for one hour. If the color changed pink/red that meant a positive reaction for E.aerogenes.
Tryptophan Hydrolysis (Indole) tests required 32 tubes of Tryptone broth that I inoculated with my samples. Then I added 10 drops of Kovacs’ reagent to each tube. The red layer that formed at the top of the water sample indicated positive for E.coli.
For Simmons Citrate agar slants, I made the agar using Simmons citrate agar and I had to wait for the 32 tubes to solidify. After the slant was solid I inoculated each tube and incubated for 24 hours. The blue color change indicated positive for E.aerogenes.
BBl Enterotube 2 is a quick and easy way to run a number of tests by inoculating the pin, which then puts the sample through all of the different tests. Some of the different types of tests that the Enterotube contains are glucose, gas, lysine, ornithine, H2S, indole, lactose, citrate and so on.
The following graph is the Durham Tubes with Lactose broth results. The graph represents both lakes for the number of positive tubes containing gas formation. I was able to tell that tubes were positive for gas and coliform because of the yellow color change and the air that formed in the tube. Having more than 10% of gas indicates coliform. I ran each test a second time to have more results and to confirm data. I used Excel to get an average number of positive turbid tubes and the standard deviation. The average number of positive turbid tubes are 17 and the standard deviation is 7. The average number of positive clear tubes is 5 with a standard deviation of 2.
I was also able to determine the most probable number of colifoms present with my tubes by using an appendix in Microbiological Application Lab Manual.
This next chart represents the average 95% Confidence Limit of the number of coliforms that are in a 100 ml of the sample. These numbers are from the appendix in the lab manual that I used. The clear water did not even have a high number of coliforms. For example the highest amount of coliforms in a clear sample was 43. The number of clear tubes that contain colifroms is substantially different. As I compared the graphs the clear lake by far contains less coliforms.
I ran the following tests (as listed in the method section): indole, methyl red, voges-proskauser & citrate slant. The indole and methyl red tests are E.coli indicators. The Voges-Proskauer and citrate slant indicates E.aerogenes.
This next graph shows the number of tubes positive for E.coli. Series 1 is the turbid water sample, which is much higher than the clear water. Series 1 column 1 shows the results for the indole test. The column 2 shows the results for the methyl red test.
This chart shows the tubes positive for coliform based on the tested I performed. For example, the positive Voges-Proskauer test indicates samples containing E.aerogenes.
The last set of data that I can report is the results of the EMB plates. The purpose of the plates was to again support the growth of coliforms. Out of 8 plates for the turbid 5 were positive for coliforms. Of the 8 plates that were streaked with the clear water samples 2 were positive for coliform formation. The Enterotube was a method of just rechecking all of my results and it did indicate that my method were accurate. It supported that coliforms were present because of gas formation.
Even though the water samples from the different lakes tested positive for coliforms I cannot state what type of coliform was in the water samples. Human sewage or animal fecal matter could be contaminating the water. The "turbid" (Mudd Lake) samples reacted more with the broths and Levine plates than the "clear" (Big Cedar Lake) water. The tests I performed were positive indicating that coliforms are present. It is important to know that E.coli is a better sewage indicator and E.aerogenes is of non-sewage origin (Benson 1998). The reason why it is very important to test water is because people who swim in the water might be at risk for becoming infected with a disease. For example, there are waterborne pathogens that are commonly found in lakes such as, hepatitis A or bacterial gastroenteritis. Health hazards are an issue and people can become extremely ill from water contamination.
Benson, Harold. Microbiological Application. 7th ed. Boston: MA: McGraw-Hill, 1998.
Kentucky State Natural Resources 1999. Fecal Coliform and Water Quality <http://water.nr.state.ky.us/ww/ramp/rmfec.htm>
Kay, David. American Journal of Public Health. October 1994: 255.
Raloff, J. " Clean water may infect swimmers." Science News Sept. 1996: 1999.