What’s In Our Water?

Author(s)

Joanna Blonigen

Introduction

This summer, I participated in Dr. Jeffrey Turner and Rachael Adriel Bruce’s research, along with Angela Prestero, Lauren Vasquez, and Martha Buendia, which aims to identify the origin of Enterococcus faecalis (a bacteria found in the intestines of humans, as well as other mammals) in the water of Corpus Christi Bay, specifically how it affects the water quality by Ropes Park and Cole Park. Since the E. faecalis in the water can originate from a variety of sources, they are hoping to use DNA typing to determine what mammal the bacteria is coming from, and how it is getting into the water of the bay. The origin of E. faecalis is important for environmental and safety reasons-it has the potential to cause serious health consequences in humans who are infected, and its presence in the bay is an indicator of overall water quality. By isolating, cultivating, and identifying strains (genetically different micro-organisms) of E. faecalis, it may be easier to prevent its spread and reduce its population in the water. It’s possible that at least some of the E. faecalis in the water has a human origin, and its ability to make humans ill is a reflection of how we affect our environment and it affects us in turn-a good reminder for students.

Methods & Procedures

This research has a heavy focus on procedure and being able to reproduce results. The cycle of research was the same each week. The first step is collecting water from Cole and Ropes Parks in four sterile one liter collection bottles-two samples from each location, at sites about 200 feet away from each other. The samples cannot be collected from the water’s edge, so the collector must go into the water, attempting to sample at a depth of two to three feet, near the bottom of the bay. Sampling must also be done earlier in the day, ideally before 11 AM. Any later in the day, the UV rays from the sun will begin to kill the E. faecalis in the water, compromising the ability to find it in the samples. The water samples are only useable for four hours, and are kept in a cooler with ice until they are used. Back in the lab, the work space must be clean and sterile. Everyone is required to wear long pants, closed toe shoes, a lab coat, and gloves. The counter where work is done is sprayed with 70% ethanol and wiped clean before anything is set up. On sampling day, the water is also filtered. The filter system

Filtration system with sterile funnel attached

Filtration system with sterile funnel attached

is made up of PVC pipe, and attached to a vacuum and flask with tubing. It has valves that can be used to open or close, and space for three filters to run at once, although we only used one at a time. Before beginning filtration, an open flame needs to be set up using a Bunsen burner, creating an optimal sterile environment for work. The flame will also be used to sterilize tools as needed. A small beaker of 100% ethanol also need to be available for sanitizing. Start by inserting the sterile funnel into the rubber stopper. It needs a foil “cap” to keep as much contamination from the air out as possible. The funnel has a magnetic opening (the line between clear and solid plastic), which is where a membrane filter will be placed. Before the filter is placed, a pair of tweezers must be dipped in ethanol and placed in the flame to sanitize them. Membrane filters vary in the size of their pores, depending on what is being filtered out. A filter is placed in the funnel, where the magnets will hold it in place. The membrane is then soaked with PBS (a saline solution), which will evenly wet the membrane and make it easier for the sample water to filter through. The vacuum is turned on to drain the PBS, then a sterile pipette is used to place 100 mL of the first sample water in the funnel. After the water is vacuumed out, there will be a residue left on the membrane of the particles too large to filter through.

Residue left on membrane after filtering

Residue left on membrane after filtering

This is also where, hopefully, the E. faecalis will be. At this point, the membrane should be removed using tweezers, and placed in a Petri dish that contains Slanetz & Bartley Medium (S&B). This is an agar (gelatin-like substance with nutrients that grow bacteria) which will specifically grow Enterococcus, not all the possible bacterias in the sample. Placing the membrane is a part of the process that needs special care to avoid contaminating the sample. It should

Completed S&B plates before incubation

Completed S&B plates before incubation

be done as close to the flame as possible, with careful care taken to avoid breathing on the Petri dish. Any bubbles between the membrane and the agar should be smoothed out if they can’t be avoided. Three membranes should be used per water sample, and the dishes should be labeled with the date and sample number. Tweezers should be sanitized with ethanol and flame between uses, and each time the water sample changes there should be a new sanitized funnel and pipette. The water collection bottles should also be kept closed between samples and shaken before use in case particles have settled. When all twelves dishes are done, they are sealed with Parafilm, a stretchy plastic wrap, and placed in the incubator for 24 hours at 41͒ Celsius.

After the incubation period, the S&B Petri dishes can be removed from the incubator. At this point, E. faecalis colonies will show up as red dots on the membrane, due to

S&B plates after incubation, with visible colonies (the red dots)

S&B plates after incubation, with visible colonies (the red dots)

the S&B agar (Fig. 3). Each dish will have colonies counted and recorded. If the dishes were contaminated, dots of other colors will show up as well. Colonies from uncontaminated samples can then be streaked onto a second set of plates to grow further. The Petri dishes used for streaking are larger and have a different agar-Brain Heart Infusion Broth (BHIB) is mixed with agar powder in order to make this agar, which will grow all bacterias, but is specifically used to cultivate the E. faecalis colonies on the S&B plates. On occasion, a plate will become contaminated

A contaminated plate: this is what the bacteria will look like when cultivated

A contaminated plate: this is what the bacteria will look like when cultivated

before it is used, and must be discarded at that point. Again, streaking needs to be carried out near an open flame for sanitary purposes. Each colony is picked up using a metal loop (Fig. 4), which should be sanitized before use by holding the loop in the flame until the metal glows. When the loop is cool, gently pick up the colony and “streak” the plate by dragging the loop in a series of three zigzags across the plate. If one of the S&B plates has more than one viable colony, each colony can have its own BHIB plate, but each plate must be labeled to identify the water sample location (C.P.1/C.P.2/R.P.1/R.P.2), which S&B plate the colony came from (1, 2, or 3), and then a letter for each colony used (A, B, C, etc.). When all plates have

Sterile loops in holder and prepared BHIB plates

Sterile loops in holder and prepared BHIB plates

been streaked, the BHIB plates are also Parafilmed and placed in the incubator for 24 hours at 41͒ C.
The next step is to remove colonies from the BHIB agar using a sterile loop and to place them in small capped tubes that have BHIB broth in them. These are mixed with a vortex mixer and then incubated 16-20 hours at 41͒ C. I was never able to be present for this part of the procedure, unfortunately.
The last step is to prepare the BHIB E. faecalis cultures (a culture is the bacteria in a nutrient broth) for freezing and DNA testing. 1:1 ratios of the BHIB culture and glycerol (a liquid commonly used as a sweetener) are placed into test tubes using a pipette and vortexed. The glycerol is added to prevent damage to the E. faecalis in the culture during the freezing process. It thickens the broth and prevents ice crystals from forming, which would damage the cultures. At this point, 0.75 mL of BHIB culture and 0.75 mL of the glycerol mix should be placed in a small tube, which will be capped and vortexed. When each culture has been prepared for freezing, they will be placed in a freezer at -80͒ Celsius. They are now ready to be stored for DNA typing, which will happen at some point in the future.
In addition to the procedures listed above, I was also able to participate in making S&B and BHIB agar to create plates, BHIB broth, and in making sure that the work area and equipment were kept clean and sterile. Bacteria is so common in our environment that precautions such as the open flame have to be kept in order to preserve the validity of the samples. Ethanol was used frequently to keep the counters and tools clean, and we even sprayed it on our gloves frequently to keep them clean as we touched different objects. Glassware was cleaned with both soap and in an acid bath, while plastic was washed with soap. Almost every container was also sanitized in the autoclave, which heats deionized water (ultrapure water, comparable to distilled water) to create steam at about 132͒ C, killing microorganisms.

Results

Most of the plates grew colonies of E. faecalis, although some plates were contaminated by other bacteria and therefore unusable, except for to see that it was present. E. faecalis was found at every sample site, meaning it is present in the water at popular parks here in Corpus Christi. Since it is not possible to do DNA typing here, the scientists working on this project will have the results of the typing this fall, hopefully. Research will continue and they should know the source of Corpus Christi’s E. faecalis strains within the year.

Discussion

When the results are in, it may lead to finding the source of contamination and the possibility of maintaining a higher water quality level with proactive measures. Again, this project demonstrates that pollution at our local beaches is not just the presence of plastic bottles and other trash, but the microorganisms living in the water have a source on land . The human effect on nature and our environment is not always visible, but it is still present. It is important for students to realize that just because we don’t see the harm doesn’t mean we aren’t doing damage. E. faecalis has the potential to harm human lives and students should be aware of the reciprocal damage of our actions to ourselves and our community.

Lesson Plan

What’s In Our Water?
5E lesson plans are ideal for science lessons, due to their comprehensive and exploratory nature. If completed correctly, the lesson should encourage students to take the lead. The Engage is the first of five, and acts as the attention-getter or focus of the lesson, introducing the topic and hopefully sparking interest. During the Exploration segment, students are engaged in hands-on, minds-on learning. Like the title suggests, students should be exploring the concept with guidance from the teacher, not direct instruction. The teacher should have some larger questions to focus the learning. After students have become familiar with the topic, the teacher will use the Explanation section to question students and encourage them to share what they thought, before sharing the basis of the lesson that curriculum has guided. This is also a good time to ask higher-level questions. In a traditional lesson plan, Elaboration is most similar to enrichment. The students are encouraged to go beyond the basis of the lesson and deepen understanding. During this segment, links to daily life can often be easily established. The fifth E stands for Evaluation, or the assessment phase of the lesson. This could be as simple as an Exit Ticket, or involve students creating and inventing. Just like any assessment, this is when the teacher is looking for demonstration that the students understood the lesson.

Subject area / course / grade level: Science/ELA 4th Grade
Materials:
“Waterway”
-Aquarium
-Rectangular Water Box
Watering Can and/or Spray Bottle
“Pollutants”
-Green Food Coloring (pesticides/fertilizer)
-Vegetable Oil (motor oil)
-Soil/Sand/Pebbles (erosion)
-Grass Clippings (or Shredded Paper) and Twigs
Gloves
Posterboard, markers, colored pencils

Lesson objective(s): The learned will be able to identify types of pollution that enter bodies of water through storm drains, as well as write an expository essay/create a poster which discusses preventative measures.

ENGAGEMENT
YouTube: Freddy the Fish Teaches About Stormwater or, for older students, Stormwater to Drinking Water
The students should be thinking about what is going on in our water, here in Corpus Christi. Although the video takes place in North Central Texas, ask students what body of water we should worry about. Ask them if/where they have seen storm drains. The students could complete a K-W-L chart on water pollution after watching the video. There could also be brainstorming on other ways water gets polluted.

EXPLORATION (2-3 days)
The students will use a model storm drain-water system (a plastic box with a hole cut in it, placed over a clear plastic box that has been filled with water) to brainstorm what goes into our water and how it might affect us and the environment. On the first day, the teacher will ask students to think of what might go into storm drains (they should have some ideas from the video) and model one of the pollutants (vegetable oil as motor oil) going into the system by placing the oil in the “storm drain” and using the watering can to wash it out into the “bay”. The students will be given time to brainstorm what else might go into the drains and how they could represent it in the model system.
The teacher will take some of the student ideas and provide their “pollutants” for the second day, making sure that they are materials that can be safely handled. These pollutants will also be rinsed out to the “bay”. The setup should be in a place where all students can see if/how the different materials change when they mix with the larger body of water. A record should be kept of which pollutants are still visible after being added to the larger tank, and at this stage the idea of harmful bacteria could be introduced. Students will draw and describe what the water looks like before and after.
Questions:
How would this end up in the water?
What have you seen in your neighborhood that could end up in a storm drain (and the bay)?
Is water the only thing in the bay?

EXPLANATION
Students will be encouraged to share their thoughts about what has happened in the “bay”, and how it relates to our own Corpus Christi Bay. Students should share the reasoning behind their thoughts and can be given time to brainstorm in groups or pairs if desired. They will also be asked to sort the pollutants into “natural” and “unnatural” and explain why they chose where to place the pollutants. The students can also decide whether a pollutant ending up in the bay is likely the result of an accident, or on purpose.
Questions:
Who could be harmed by what is in the water?
Do you think it would be easier to do something about pollution before or after the pollutants go in the water?

ELABORATION
Now that the water is polluted, students will need to brainstorm how they could take the pollutants out of the water again. Again, the teacher should provide examples of these pollutant removers as necessary. Once the water is polluted, however, the students must wear gloves to handle the water as they attempt to clean it. Students should be reminded of all the materials in the tank and asked to determine whether their location will make them easier or harder to clean up. Some of the materials will have completely dissolved into the water-make sure to question how the students could be sure they had cleaned those from the water as well.
Most vocabulary should be known, but pollutant and source may need to be refreshed, and some of the pollutants and cleaning methods brainstormed may need to be explained.
The link between the model tank and Corpus Christi Bay should be emphasized. In other locations, this could be linked to a similar body of water, like a river or lake.

EVALUATION
Students will write an expository essay on how they will prevent polluting our water and why it is important. They may also create an anti-pollution poster with a convincing tag line, which can be displayed.

Even though this activity specifically relates our model system to Corpus Christi Bay, storm drains are a common sight in communities and most should be able to find a waterway affected in a similar manner. Water pollution is everywhere, even in the places where you would least expect it.

TEKS:
§112.15. Science, Grade 4
(b) Knowledge and skills.
(1) Scientific investigation and reasoning. The student conducts classroom and outdoor investigations, following home and school safety procedures and environmentally appropriate and ethical practices. The student is expected to:

(A) demonstrate safe practices and the use of safety equipment as described in the Texas Safety Standards during classroom and outdoor investigations; and

(B) make informed choices in the use and conservation of natural resources and reusing and recycling of materials such as paper, aluminum, glass, cans

(2) Scientific investigation and reasoning. The student uses scientific inquiry methods during laboratory and outdoor investigations. The student is expected to:

(A) plan and implement descriptive investigations, including asking well-defined questions, making inferences, and selecting and using appropriate equipment or technology to answer his/her questions;and plastic.

(3) Scientific investigation and reasoning. The student uses critical thinking and scientific problem solving to make informed decisions. The student is expected to:

(C) represent the natural world using models and identify their limitations, including accuracy and size;

(5) Matter and energy. The student knows that matter has measurable physical properties and those properties determine how matter is classified, changed, and used. The student is expected to:

(C) compare and contrast a variety of mixtures and solutions such as rocks in sand, sand in water, or sugar in water.

(7) Earth and space. The students know that Earth consists of useful resources and its surface is constantly changing. The student is expected to:

(C) identify and classify Earth’s renewable resources, including plants, water, and animals; and the importance of conservation.

§110.15. English Language Arts and Reading, Grade 4

(18) Writing/Expository and Procedural Texts. Students write expository and procedural or workrelated texts to communicate ideas and information to specific audiences for specific purposes. Students are expected to:

(A) create brief compositions that:
(i) establish a central idea in a topic sentence;
(ii) include supporting sentences with simple facts, details, and explanations;
(iii) contain a concluding statement;