In an extension to last year’s project, I hypothesize that bacteria cultured from deep aquifer water is less harmful than the bacteria from lake water.
Methods
Obtain samples from two water sources (Lake Sheen in Orlando, FL, and a deep aquifer).
Measure the biological activity of Pseudomonas fluorescens and aerobic bacteria using two different methods. BART™ test tubes and agar plate testing. Biological activity reaction tests, as available from the Hach Company, were used to evaluate the activity of each of these biological groups. Samples were poured into pre-prepared BART vials and observed daily for the next 25 days. Observations were recorded and, in conjunction with the manufacturer’s instructions, the approximate concentration of each biological group was determined.
Agar plate testing, done in the microbiology laboratory of Osceola Regional Medical Center, was used to classify which types of bacteria were present in the water. Using a premade Trypticase™ soy agar plate with 5% sheep blood, I tested for accurate colony counts. An automated computer analyzer allowed us to classify the type of bacteria growing on the plates.
The results of these tests were analyzed and interpreted to evaluate the biological activity of each water source and to prove or disprove the hypothesis. Distilled sterile water was used as the experimental control.
Background
As I walked by the lake dock, I admired the tranquility of the lake. The sun was slowly setting, and the fresh breeze ran across my face. I looked into the beautiful waters while fish swimming nearby caught my eye. I leaned in closer and became interested. I had always heard my teachers say that bacteria are in lakes even though you can’t see them. I did not give it too much thought until I grabbed a water bottle from my bag. As I gulped down the water, I realized that there might be some bacteria in the water I was drinking too. Hopefully, the amount of bacteria was less than the amount in the lake. I ambled away from the dock when some foul-smelling water splashed onto my face. The clubhouse sprinklers were running, and the bad odor made me wonder how much bacteria this water contained. These thoughts circled my head as I biked back home. I decided to do some research about bacteria.
Bacteria are found almost everywhere on Earth, especially the Pseudomonas family. Bacteria in water supplies are a serious issue throughout the world. Bacterial outbreaks have caused deaths not only among humans but also among other living organisms. However, not all bacteria are harmful. As a matter of fact, many bacteria are beneficial. Some bacteria produce by-products that kill pathogens. A pathogen is an infectious agent such as a virus, a bacterium or another microorganism. While many bacteria are beneficial in nature, they can cause severe illness or even death if consumed by humans. I wanted to see whether the bacteria in deep aquifers and lake water are harmful or not. My curiosity led me to come up with the following hypothesis:
As a sequel to my last year’s project, I hypothesized that bacteria cultured from deep aquifer water are less harmful than the bacteria cultured from lake water.
Water Samples
Over 130 million Americans drink groundwater (“Bacteria and Water Wells”). Groundwater is found in the pores between subsurface soil, sand and rock. When water-bearing rock transmits water upward to wells and springs, it is called an aquifer. Wells can be drilled from which the water is pumped. Serious groundwater contamination is the result of human activities on land such as agriculture, septic systems, industrial activities, gasoline storage and many more. (“What’s Our Water Worth?”). Since groundwater moves slowly, contamination is often widespread by the time it is detected.
Florida’s principal source of water supply comes from groundwater (“South Florida’s hydrologic systems—Aquifers”). While uncontaminated groundwater supplies are supposedly bacteria-free in properly constructed drilled wells, I wanted to check if that statement was true. As such, I included groundwater sources in my experiment.
Groundwater from an aquifer deeper than 500 feet is called a deep aquifer. They are usually located underneath rock and gravel. The deep aquifer water for this experiment was obtained from a well 2,800 feet deep in my neighborhood.
A common belief is that lake water contains significant concentrations of bacteria because lakes are functioning ecosystems. Also, it is believed that groundwater usually has fewer microorganisms than lake water because of its long travel time in the subsurface environment. To check the validity of these beliefs, I gathered my samples from the northeast tip of Lake Sheen in Orange County, FL.
In order to ensure the quality of the experimental results, it was necessary to have an experimental control. I decided to use distilled water because it is the purest form of water I could procure. The distillation process vaporizes regular tap water into sterilized steam and then condenses it back into its original state. This process removes most of the viruses, bacteria, organic and inorganic chemicals, heavy metals and other pollutants (“Distilled Water Facts”).
Pseudomonas fluorescens (FLOR)
The bacteria that I was testing for was Pseudomonas fluorescens. The genus Pseudomonas is the most diverse and significant group of bacteria on Earth. Pseudomonas fluorescens are the most studied group in this genus. They are gram-negative, rod-shaped bacteria that inhabit soil, plants and water surfaces. Pseudomonas fluorescens allows plants to obtain key nutrients, degrade pollutants and suppress pathogens via antibiotic production. These abundant bacteria are beneficial to water and plants.
BART Test Tubes
Figure 1
The activity of each biological group was evaluated for each water sample by BART kits. Colin Hobbs, Ph.D, P.E., an environmental engineer and a friend of my family, recommended the use of these test kits for this experiment. Dr. Hobbs assisted me in this experiment by providing the BART kits, the manufacturer’s instructions and the ultraviolet light required by some of the BART kits. The BART tests were a great way to detect the number of bacteria in a semi-quantitative manner. The tester was designed to provide a variety of environments for the bacteria. The base cone of the tube is reductive, and the area around and under the floating ball is oxidative, as shown in Figure 1. This creates a vertical gradient so that the tester can encourage many different types of bacteria to grow. For different communities of bacteria to grow, a crystallized pellet of chemicals containing selective nutrients is placed in the base cone. These nutrients diffuse upward and trigger only the bacteria being investigated to respond and grow. This is done by a combination of reaction and timing. BART tests were a quick and accurate way of determining the presence or absence of a bacterial group in a sample, as well as an estimate of its concentration.
Agar Plate Testing
Figure 2
Another method I used was the agar plate method. This method allowed me to get more qualitative and specific results. Mrs. Mary Lou Cole, a medical technologist at Osceola Regional Medical Center’s Microbiology laboratory, provided me with the materials necessary for this experiment. I needed a centrifuge, a calibrated disposable inoculating loop, an incubator, agar plates, analyzer plates, sterile containers and the computer analyzer. Agar is a phycocolloid extracted from a group of marine algae called Rhodophyceae. Agar is used as a culture medium for various microorganisms, especially bacteria. To test for Pseudomonas fluorescens, I used blood agar plates, and to test for aerobic bacteria, I used MacConkey agar plates. I had to centrifuge the water samples to suspend the bacteria at the bottom. Then, taking a 100 uL inoculating loop, I streaked the agar plates very gently, as shown in Figure 2. Lastly, I put the samples in an incubator overnight to ensure growth. The next day I observed and analyzed the results by seeing how each bacterium reacted with the agar. Agar plate testing was useful to separate and distinguish the different bacteria in the waters.
Materials
In order to get a semi-quantitative measurement of the amount of bacteria in each type of water, I used premade BART kits. These kits are used to get the approximate number of colony-forming units per milliliter (CFU/mL). BART provided ready-made tubes that have a specific reaction to bacteria over a certain period of time. I obtained a BART kit. For the FLOR tests, I needed a UV light. For the agar plates, I went to a microbiology lab at Osceola Regional Medical Center. There, I used a centrifuge, an incubator, agar plates, a loop and an automated analyzer. In order to compare different water samples, I used deep aquifer water, lake water and distilled sterile water as a control.
Procedure
Figure 3: Vrinda filling test tubes
To get the lake water, I went to my subdivision clubhouse dock. Using a sterile fluid collecting bottle, I collected two samples of water from approximately six inches below the surface.
I used sterile fluid collecting bottle and went over to my neighbor’s house (Orlando, FL). I could smell the hydrogen sulfide in the sprinkler water as I collected two samples of the deep aquifer water.
After getting two samples each of lake water and deep aquifer water, I started testing using two different methods. I used the BART test tubes and the agar plate tests to measure the biological activity of Pseudomonas flourescens and aerobic bacteria in both lake water and deep aquifer water.
For each BART tube, I used similar instructions:
Remove the inner tube from the outer tube.
Fill the inner tube with sample until the level reaches the fill line (15 mL). After removing the cap for the inner tube, set it down on a clean surface to avoid contamination, as shown in Figure 3.
Tightly screw the cap back on the inner tube. Return the inner tube to the outer tube and screw outer cap on tightly. Without shaking, let the medium dissolve slowly and the ball rise at its own speed.
Each test had a reaction that must occur in a certain number of days for the bacteria growth to be positive.
For the agar plates, I used similar instructions:
Figure 4
Centrifuge water samples at 1,759 rotations per minute for 5 minutes.
Decant and discard the top fluid.
Use sterile premade blood agar and McKonkey medium agar plates.
Use a sterile, calibrated, disposable loop to collect and streak the sediment of centrifuged samples across the plates.
Incubate overnight at 35ºC.
To get the results, I had to count the number of colonies and multiply them by 100 to get the CFU/mL. Using a sterile multi-pipette, I removed the colonies from the plates and moved them into a saline broth. This broth was then poured into an analyzer plate, as shown in Figure 4, and placed into a Siemens WalkAway Computer Analyzer overnight. It generated the report with antibiotic sensitivity.
Results
The results obtained from the BART kits and agar plates are presented in the following figures and tables.
Agar Plating Results
Figure 5
In Table 1, I graphed the results from the BART test tubes. The lake water had the most CFU/mL of Pseudomonas fluorescens, and the deep aquifer water had a bit less. In Figure 5, the distilled water turned blue while the others remained yellow. This happened because the distilled water had no trace of Pseudomonas fluorescens.
Table 1
In Tables 2-4, I display the results from agar plate testing. Once again, lake water had the most Pseudomonas fluorescens, and the deep aquifer water had fewer CFU/mL than the lake water. This time, however, the deep aquifer water had many fewer Pseudomonas fluorescens than the lake water. Using the BART test tubes, there was only a slight difference between the amount of bacteria in the lake and the ground water. However, I believe the agar plates were more accurate because the results for the BART test tubes were based more on observation than on quantitative measurements. The agar plate results showed three other aerobic bacteria found in the lake: Serratia marcescens, Enterobacter cloacae and Chromobacterium violaceum. All three of these bacteria can cause infections if they enter a body through an open wound or through aspiration (water getting into the lungs). If humans ingest these bacteria, they will not cause harm unless the person has a suppressed immune system. As expected, there were no bacteria found in distilled water.
Bacteria
Colony County (CFU/mL)
P.flourescens/putida
100
Deep Aquifer - Table 2
Bacteria
Colony Count (CFU/mL)
P. flourescens/putida
400
Chromobacterium violaceum
300
Serratia marcescens
200
Enterobacter cloacae
200
Lake Water - Table 3
Bacteria
Colony Count (CFU/mL)
P. flourescens/putida
0
Distilled Water - Table 4
Analysis
After searching online and discussing my findings with Dr. Sajid Chaudhary, an infectious disease specialist, I concluded the following:
A) Pseudomonas fluorescens is widespread in soil and water. Even at a depth of 2,800 feet, it was still present even though it wasn’t as prevalent as in lake water.
B) I could not isolate any aerobic bacteria in the deep aquifer water.
C) The bacteria identified in Lake Sheen will not cause a stomach infection (gastroenteritis) even if swallowed during swimming.
D) The isolated lake bacteria can infect an open wound; therefore, those with exposed cuts should avoid swimming in lakes.
E) If the lake water gets into the lungs of patients with chronic lung disease or immunosuppressive diseases like AIDS or cancer, it may cause pneumonia.
Limitations
There were many factors that might have caused inaccuracy in my results.
A) I collected the samples on different days, so there may have been changes in the bacterial flora.
B) I collected samples from just one part of the lake. Had I collected samples from a different parts of the lake, I may have gotten different results.
Conclusion
As I hypothesized, the lake water had a higher concentration of aerobic and Pseudomonas fluorescens bacteria than the deep aquifer water, which had no aerobic bacteria due to the lack of oxygen at the depth of 2,800 feet. Based upon this data, I concluded that Lake Sheen is safe for swimming for individuals without open wounds and with a good immune system. Lake water and deep aquifer water will not cause harm to healthy people who drink it. Long-term studies are necessary to validate my results, but I had a great time testing the waters.
Haman, Dorota. “CIR803/WI002: Water Wells for Florida Irrigation Systems.” Electronic Data Information Source—University of Florida/IFAS Extension. Retrieved from the World Wide Web on 6 Mar. 2012. http://edis.ifas.ufl.edu/wi002
“Introduction to Algae and Basic Types of Algae - Forms of Algal Strains - Oilgae - Oil from Algae.” Oilgae. Retrieved from the World Wide Web on 3 Mar. 2012. http://www.oilgae.com/algae/algae.html
