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 Bacterial Content of Water Bottles Researched by Marisol G.  2003-04

### PURPOSE

My purpose is to show that the bacteria content does increase in a water bottle when it is not properly washed between uses.  In the summer during swim team, I see my teammates fill the same water bottle every day for the whole season.  They do not wash the water bottles as evidenced by the lip-gloss colors that don’t go away.  The water bottles are stored in their swim bags with moldy dirty towels or they rattle under the seats of their parents’ cars.  Also I am doing this experiment in the winter when the temperatures are cooler and bacterial growth tends to be slower than during the hot summer months.  If the bacteria do grow in my experiment, the bacteria growth could be greater during the summer months.

My sister’s first grade class uses water bottles in the classroom.  I know that my sister does not always take a new water bottle as she is instructed.  In addition she just tosses the water bottle in her backpack with her snow boots and everything else, regardless of what we tell her.  I know her classmates are probably no different than her.

I would like to prove my hypothesis so I can teach others that their water bottle can make them sick if they don’t take precautions to protect themselves.

### HYPOTHESIS

I hypothesize that the bacteria content will increase in just a few days when you drink from a water bottle without washing it prior to filling the water bottle.

### EXPERIMENT DESIGN

The constant in this study were:

• The brand and the amount of water in each of the water bottles.
• The type of agar and agar plates.
• The incubator.
• That you use a new bottle each time you refill the test subject’s water bottles
• How the bacteria colonies were counted.
• The amount of days each subject drank from their individual water bottle
The manipulated variable was how the individual test subjects drank from and cared for the water bottles.  They did not change their normal behavior for this experiment.

The responding variable was the amount of growth of the individual samples.
To measure the responding variable I counted the number of colonies in the petri dishes after they had been incubated.

### MATERIALS

16 Kirkland brand water bottles, 16.9 fl.oz. (0.5 L)
1 Sharpie marker pen
6 Gallon size Ziploc bags
1 Comet cleanser
1 Liquid dish detergent
1 Roll paper towels
1 Lab coat
12 Vials BHI agar slants, 10 ml. each
1 Metal container to melt BHI agar in
1 Bunsen burner
6 Nalgene filtration units.  (115 ml capacity)
1 Pair of hot pad gloves
1 Trivet
1 Vacuum to filter the water
6 Petri dishes
1 Sterile scalpel
1 Pair of forceps
6 Disposable inoculating loops
1 Container with Stat III disinfectant
1 Incubator to incubate the bacteria
1 Stat lll liquid disinfectant
1 Hibiclens cleanser
1 Autoclave

### <>PROCEDURES

1. Experiment Day One

a. Mark five water bottles with a marking pen with the individual test subjects’ names.

b. Mark one water bottle “Control? And set aside, unopened.  Store in a one-gallon size Ziploc bag to prevent cross contamination from the environment.

c. Give each test subject his or her water bottle in the morning.  Instruct them to drink all the water from the bottle through out the day and to return it to you at the end of the day.

d. In the evening get the water bottles back from the test subjects.

2. Experiment Day Two

a. Clean sink and counter top area with Comet cleaner and paper towels.

b. Wash your hands with soap and water.

c. Get five new water bottles and place on paper towels on the counter top.

d. Place the test subjects’ individual water bottles on a paper towel on the counter top.

e. Place a third paper towel on the counter top that you will be actually working over.

f. Wash your hands again with soap and water.

g. Take the first subject’s water bottle.  Place it on the paper towel covered workspace.

h. Carefully remove the lid to the water bottle and place it topside down on the paper towel.

i. Open up a new, unused water bottle of the same manufacturing lot.

j. Over the paper towel covered work area, carefully pour the water from the new bottle into the used water bottle of the first test subject.   See figure 1.

k. Set the newly filled water bottle back on the work surface and put the used water bottle lid back on the used water bottle of the first test subject.  Do not use the new lid from the new water bottle.

l. Discard the new lid in the trash and the new water bottle in the recycling bin.

m. Set the re-filled water bottle of the first subject aside.

o. Place a new paper towel on your workspace on the counter top.  Repeat steps “g” to “o” until all five test subject’s water bottles is refilled.

p. Give each subject his or her individual water bottle.  Remind them that they are to drink all the water through out the day and to return them to you at the end of the day.

q. In the evening, get the water bottles back from all the test subjects.

3. Experiment Day 3

a.  Follow procedures for Experiment Day 2 steps “a” to “p.”

b. Draw a line with a marking pen on the first indentation from the bottom of the water bottle.  See Figure 2.

c. If a test subject’s water bottle has been indented reducing the volume of water the bottle can hold due to displacement, make a line on the second indentation from the bottom instead of the first to accommodate for the reduction of volume.

d. When you hand out the water bottles to the individual test subjects, instruct them to not drink below the line drawn on the water bottle.

e. Show the test subjects where the line on the bottle is to help them understand what you have instructed them to do.

f. Remind the test subjects that they are to drink water through out the day and to return the partially consumed bottle of water to you at the end of the day.

g. In the evening, collect the individual test bottles from all the test subjects.

h. Place each subject’s individual water bottle in its own Ziploc bag.

4. Test Day 1

a. Gather the five water bottles used by the individual test subjects; the water bottle marked “control” and the city water sample.  Take all samples to the lab for testing.

b. Upon entering the lab, put on a lab coat.

c. Obtain 12 test tubes of BHI agar slants, 10ml each.

d. Loosen the lid of each tube slightly and place in a metal container.  See Figure 3

e. Add tap water to the metal container to cover the bottom quarter of the test tubes.

f. Place metal container with the test tubes and water over a Bunsen burner.  Heat.

g.  Check the test tubes after 17 minutes to see if the agar has melted in all of the test tubes.  If they are not completely melted, check the test tubes again after 20 minutes has elapsed. See Figure 4.

h. While the BHI slants are melting, label the Nalgene filter systems and the petri dishes with the test subject’s name and “control.”

i.  Open the first test subject’s water bottle.  Pour the test water into the top of the corresponding test subject’s Nalgene filter system to the 90ml line.  See Figure 5.

j. Repeat step h until each test subject’s water sample and the control water bottle sample is in the corresponding labeled Nalgene filter system.

k. Turn off the Bunsen burner.  With hot pad gloves move the metal container with the BHI slants to a trivet in the work area.

l. The first subject’s water sample in the Nalgene filter system is attached to a vacuum.  The vacuum is turned on.

m. When all the water has moved to the bottom half of the filter system, turn off the vacuum and remove the connecting hose.

n. Open the two test tubes of BHI slant and pour the melted agar into the first test subject’s labeled petri dish.  See Figure 6.

o. While the BHI is allowed to cool slightly, cut the perimeter of the filter in the Nalgene filter system along its perimeter with a sterile scalpel.  See Figure 7.

p.  Remove the filter from the filter system with sterile forceps.  Place the filter into the center of its corresponding petri dish with the slightly warm BHI agar.  See Figure 8.

q. With a sterile inoculating loop, gently push the filter into the melted BHI agar so it is fully immersed in the BHI agar and centered in the petri dish.  See Figure 9.

r. Place the lid on the petri dish and set aside until al the samples are prepared.

s. Repeat steps “h,” “I,” and steps “k” to “r” until all individual test samples and the control water bottle sample are prepared.

t. Place all prepared petri dishes in a CO2 incubator for 24 hours.

u. Store water samples in the Nalgene filter systems for re-testing, if needed.

v. Clean the workspace with Stat III disinfectant.

w. Wash your hands with Hibiclens

x. Remove lab coat.

y. Wash your hands again with Hibiclens.

5. Test Day 2

a. Return to the lab 24 hours after the individual petri dishes were prepared with the individual test samples.

b. Upon entering the lab, put on a lab coat.

c. Obtain two test tubes of BHI slant, 10ml each.

d. Take out the prepared petri dishes you placed in the incubator on Test Day 1.

e. Count the number of bacteria colonies in the petri dishes and record the data.  Return to the CO2 incubator.

f. Clean the workspace with Stat III disinfectant.

g. Wash your hands with Hibiclens

h. Remove lab coat.

i. Wash your hands again with Hibiclens.

6. Test Day 3

a. Return to the lab 24 hours after preparing the petri dish with the Selah city water sample.

b. Upon entering the lab, put on a lab coat.

c. Count the number of bacteria colonies in all petri dishes.  Record the data.

d. Place all petri dishes in an autoclave for sterilization prior to their disposal by the lab.

e. Clean the work space with Stat III disinfectant

f. Wash your hands with Hiebiclens

g. Remove lab coat.

h. Wash your hands again with Hiebiclens.

7.  Survey

a. Arrange with a classroom teacher for permission to conduct a survey in her classroom

b. Find out when information is sent home with students for their parents.

c. Set a time frame with the teacher for you to deliver the surveys and when they need to be returned by.

d. Deliver survey to teacher of the class you are requesting information from in the manner agreed upon.

e. Pick up surveys.

f. Compile the information.

### RESULTS

The original purpose of this experiment was to see if the bacterial content in water bottles would increase if you re-filled it without washing it between uses. I wanted to prove to people that re-filling water bottles without washing them between uses could be making you sick.
The results of this experiment was that the bacterial content in water bottles does increase when you simply re-fill your water bottle with out washing it prior to re-filling it. Figure 10 shows that test subjects number and 4 and 5 had results that were significantly lower than the three other test subjects but there still was bacterial growth compared to the control sample.

Figure 11, below, visually shows the comparison of the five test subjects and the control regarding the number of bacteria colonies that grew after three days of simply re-filling the water bottles.

Figure 12 is a startling show of the percent of bacterial growth that occurred in the five test subjects’ water samples.  Test subjects 1, 2, and 3 all had a high amount of bacteria colonies present compared to the control.  The bacteria growth shows an increase of 400% to 200,000%.

In Figure 13 you can see the bacteria colonies present on the BHI agar in the petri dishes of two test samples.   The bacteria looks like a sheen of lotion in the photo, but it really looked like vomit splashed on gelatin.  The bacteria in the petri dishes smelled like vomit.

I have a theory why Test Subject #5 had such a low count of bacteria colonies.  Test subject is a 6-year-old girl who has been on continuous antibiotics since August 2003 for kidney infections leading to kidney failure.  The antibiotics she took in November and December 2003 were Cezfil and Cefllaxen.  Both are broad-spectrum antibiotics that kill the bacteria common in the mouth.  In addition on December 23, she had surgery and was on IV antibiotics for several days followed by more Cezfil upon discharge.  She had been off of antibiotics a few days before she participated in the study.  Most likely, the bacteria that would normally be present in her mouth had been killed by the long-term use of those specific antibiotics and the bacteria, both harmful and beneficial, had not had much of a chance to return. Considering that the test subject is a notorious backwasher, and not always has the best hygiene, I thought that her bacterial colony count would have been the highest of all five test subjects but I was wrong.  Test subject #5 still did have a 400% increase in the bacteria colonies present

Test subject #4 had a low count but still had 2,000% increase in the amount of bacteria colonies present.  Test subject is a 46-year-old man who drank his water sample immediately after swimming in a chlorinated pool.  He did not drink the water through out the day as intended.  He had not eaten for 10 hours prior to his consuming the water and he had brushed his teeth prior to his morning swim workout.  I would assume that since he most likely had no food particles in his mouth, recently brushed his teeth, and had chlorinated pool water in his mouth just prior to consumption, that is why his sample had a relatively low bacterial colony count.

The survey to the parents of one class of first grade students proved interesting.  All of the parents responded to the survey.  Of 17 students, only one did not send a water bottle to school with their student.  The majority of the students use a reusable water bottle (Figure 14).

Most of the parents do wash the bottle before re-filling it bit a few do both methods as shown in Figure 15 above.  One parent did not think that there would be any bacterial growth as shown in Figure 16, but of the 16 other parents, half thought some bacteria would grow and half thought a lot of bacteria would grow.  I believe that the parents do understand that bacteria are there even if you don’t see them.

### CONCLUSION

My hypothesis was that the bacteria content would increase in just a few days when you drink from a water bottle without washing it prior to filling the water bottle.

The results indicate that this hypothesis should be accepted.

Because of the results, I wonder if instead of not washing the water bottle wash it in the dishwasher and see if it reduces the bacteria in the water bottle.

My findings should be useful to everyone, because their risk of getting sick is much lower if they wash their water bottle before refilling it.

If I were to conduct this experiment again I would test different brands of water bottles instead of testing one brand of water. That would prove which brand of water would have the most bacteria in their water.

ACKNOWLEDGEMENTS

First, I would like to thank Karissa Craig at Memorial Hospital Microbiology Laboratory for guiding me in the lab.  Without her I would have never been able to begin my experiment, let alone complete it.   Thanks to Memorial Hospital who allows students like me to use their  facilities for special science experiments.

I would like to thank Mrs. Tullis at Lince Elementary School for letting me hand out surveys on their water bottle practices to the parents of first grade students.  The students did an awesome job at being responsible for taking the surveys home and returning them.  I would also like to thank the students’ parents for taking the time to help me out simply by completing the surveys.  I hope to see those first graders participating in the science competition when they are in sixth grade.

I would like to thank Mr. Newkirk for getting me ready to go before both boards and giving me all of the paperwork I needed and informing me on the “project log”.   I would like to thank Mrs. Helms for just making me feel at home when I had to stay late after school to go before the IRB and the SRC.  I would like to thank Mrs. Hostetler for signing the form that allowed me to participate in a study that uses human subjects and a pathogenic agent even when she disagreed about  my ability to handle such a project.

I would like to thank both the IRB and the SRC for approving my experiment because if they didn’t approve of my science experiment I would have never been able to so my sciences experiment.

I also need to thank my parents.  I would like to thank my Dad for showing me the necessity of backing up my work.  He provided for me so I could have the tools needed to participate in this project.  I would like to thank my Mom for taking me to the library and showing me where to look for my research.  She showed me what information to write down to put in my bibliography.  My Mom also drove me to Memorial Hospital to do my experiment.  Without Mom and her wheels I would have never been able to do my experiment or do the necessary research.  When things weren’t going as planned, she picked me up dusted me off and got me going again.

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