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.
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.
The constant in this study were:
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 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 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.
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 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. 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.
n. Wash your hands.
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.
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.
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.
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.
Bacteria can cause serious diseases. People don’t realize bacteria are everywhere because they are so small so we can’t see them. One source of bacteria that people forget about is the bacteria that may be present in their water bottle. Often people simply fill water bottles with out washing them between uses. They may contaminate the water bottle from unwashed hands, a dirty kitchen, sharing with others, or letting the water bottle roll around on the floor of their car. There are simple steps that we can do to reduce the chance of bacterial contamination. By reducing our risks we may reduce the chance of illness.
Bacteria grow in warm and moist places, and in places where other forms cannot survive. “Bacteria (in a water bottle) feed on food particles, saliva, and sweat.”1 Bacteria are one-celled organisms that are so small that you can only see the bacteria under a microscope. Bacteria surround us every day; whatever we eat drink or touch has bacteria on it or in it. “On average, bacteria are about 1 micrometer (0.000039 inch) long and 0.5 micrometer in diameter.”2 “You can’t see the bacterial cells without a microscope but if a large number are present you can see the bacterial clusters they form.”3 To produce a cluster large enough to see, you need to collect a small number of bacteria and provide them with food. In time the bacteria will grow into a cluster large enough to see.
Types of Bacteria
There are four different types of bacteria. The bacteria that are round and sometimes linked together are called Cocci, the Bacilli resemble the shape of rods. Vibrios look like bent rods, and the spiral shaped bacteria are called Spirochetes or Spirilla.
“Certain bacteria, called aerobes, require oxygen to live, but others, known as anaerobes, can survive without it. Some anaerobes can exist either with or without oxygen. Other anaerobes cannot live with even a trace of oxygen in their environment.”4 In your mouth there are several types of bacteria such as:
- Streptococcus viriclasns
- Staphloccous, both aerobic and anaerobic types
Structure of a Bacterium
“A bacterial cell may have up to three layers. These layers surround the cytoplasm, which contains the cells nuclei. Hair like flagella extends through the layers in many types of bacteria. The flagella help the bacterium to move by means of a whip like motion.”6
Almost all kinds of bacteria have a tough outer shell called a cell wall. The cell wall gives the bacterium its shape and allows it to live in a wide range of environments. The capsule helps protect the bacterium from destruction. “All bacteria have a cell membrane, an elastic baglike structure just inside the cell wall.”7
Almost all bacteria reproduce asexually which means that the cells simply divide into two parts; this is called binary fission. The DNA in the new cell is identical to the original cell. Some bacterium can exchange its DNA by a simple sexual process that is called conjugation. Conjugation is a direct transfer of DNA from one type of bacteria cell that is called a male to another type of bacteria cell called a female. “Some bacteria can double their number every 20 minutes while others reproduce slowly.”8 “If one of these cells were given enough food, over a billion bacteria would be reproduced in 10 hours.”9
There are certain kinds of bacteria that are helpful. The bacteria in your body for example some of them help you digest your food and destroy harmful organisms in your body. The bacteria in the water and soil are very important in recycling carbon, nitrogen, sulfur and other chemicals used by living things. Bacteria help decompose dead animals into chemical elements. Other bacteria’s help make medications, drugs, and antibiotics.
There are thousands of types of bacteria only a few of them are harmful to humans. The harmful bacteria can get into your body system by openings in your skin like your nose, mouth, cut in your skin, and by digesting food. If harmful bacteria enter your body, the bacteria can make you sick. Depending on the type of bacteria and the health of the individual, a person can even die.
If people become in contact with a harmful bacteria they may become sick depending on the individual’s immune system. People who are particularly vulnerable to infections are more at risk of becoming quite ill compared to the rest of the population. Those who are considered to be high risk include “babies and young children, the elderly, pregnant women (because of the risk to the fetus), and people already seriously ill or whose immune systems are weakened.”10
Illness can be as simple as an infected skinned knee to meningococcal bacteria that infects the brain membranes. Food poisoning is very is most often caused by bacteria but people just think they have the flu. “Food borne illnesses affect millions of Americans each year but most cases are attributed to the flu, not from something they ate.”11 “Drinking water with bacteria in it may cause upset stomachs, diarrhea or more serious illnesses. Just one drink of water with these germs can make you sick.”12
Cross contamination is when bacteria from one source are in contact with another source. One example is when a person sneezes into their hand and then shakes hands with another person. The second person would then have the bacteria that the first person sneezed out of his body and any thing else that hand might have been in contact with. When re-filling a water bottle, you can cross contaminate the water bottle with any bacteria that might have been in the kitchen. You could easily hold the water bottle on the faucet or the lid could sit on the counter that had some bacteria splash on it while you were cutting up a chicken earlier. If the water bottle has a lid that just pulls up and does not screw off, the bacteria from your hands get on the lid that you drink through. Even when you simply drink from a water bottle, bacteria from your mouth will return to your water bottle by saliva and any water returning to the bottle (backwash). Since food particles also return you end up feeding the bacteria too. If you share a water bottle, even with a loved one, you can get their bacteria in your water bottle.
According to the Center for Disease Control to prevent infectious diseases, including those caused by bacteria, a person should follow the following seven steps:
- Wash your hands often
- Routinely clean and disinfect Surfaces
- Handle and prepare food safely
- Use antibiotics appropriately
- Keep pets healthy
- Avoid contact with wild animals
- Get immunized13
Some of these steps will prevent cross contamination. If you don’t do these things you could end up in a hospital room with a serious disease.
Antibiotics are powerful drugs used to treat illnesses caused by bacteria. Antibiotics don’t work against viruses and using unnecessary antibiotics can be harmful since it increases your chance of developing a drug-resistant bacteria. Common illnesses caused by bacteria include most ear infections, some sinus infection, strep throat, and urinary tract infections. “Each antibiotic works against specific types of bacteria. Some destroy only one of two kinds of bacteria.”14
Antibiotics work by prevention of the bacteria’s cell wall formation, disruption of the cell membrane of the bacteria or the disruption of the bacteria’s chemical processes. There are dangers to using antibiotics. Some people are allergic to antibiotics resulting in a reaction as mild as a rash to as severe as death. Antibiotics can kill off the harmless bacteria because the antibiotic does not always distinguish between bad and good bacteria. The harmless bacteria that are killed are frequently in a person’s mouth or digestive organs. In rare cases, a person’s organs and tissues can be damaged. According to Michelle Sviel, P.A., it is possible for antibiotics to kill enough in the mouth enough to prevent bacteria from a person’s mouth to be backwashed into a water bottle. If a person uses antibiotics for a long time it can harm certain organs. Streptomycin has caused kidney damage and deafness. Physicians prescribe antibiotics with high risks only when no other drug is effective.
Resistance is when bacteria tolerate antibiotics that used to kill them off. If this happens stronger antibiotics are prescribed that might increase your risk for reacting to the stronger antibiotic and developing resistance to the stronger antibiotic.
Bacteria and Laboratory Safety
Experimental science can be dangerous because events can happen quickly while you are doing an experiment. Basic procedures can help prevent serious accidents and exposure to pathogens. Good judgment or common sense is critical. If there is any question about the safety of a material or procedure the person should stop and find out for sure it is safe before they continue the experiment. To avoid accidents, pay close attention to your work, take your time and follow basic safety procedures For working with microorganisms you should follow these safety procedures:
- Clear all surfaces before beginning work.
- Read through the whole experiment before you start.
- Identify hazardous procedures and anticipate dangers.
- Follow all directions step by step and do one procedure at a time.
- Make sure there is adequate ventilation.
- Do not play around.
- Wear protective gear such as goggles, gloves, apron, or lab coat as needed.
- Do not wear contact lenses, open shoes, or loose clothing. Pull your hair back.
- Keep floor and work space net, clean, and dry.
- Clean up spills immediately and in the correct manner.
- Do not eat, drink, or smoke in the laboratory or workspace.
- Set up apparatus far from the edge of the desk.
- Use knives and sharp or pointed instruments with care.
- Pull plugs and not cords when inserting and removing electrical plugs.
- Do not use your mouth to pipette use a suction bulb or other devise.
- Clean glassware before and after use.
- Check glassware for scratches, cracks, and sharp edges.
- Clean up broken glassware immediately.
- Be careful using stepstools, chairs, or ladders.
- Use goggles, apron or lab coat, and gloves when boiling liquids.
- Keep your face away from test tubes and beakers
- Never leave heating apparatus unattended.
- Use safety tongs and heat resistant mitts.
- Turn off hot plates, Bunsen burners, and gas when you are done.
- Keep flammable substances away from heat.
- Have a fire extinguisher on hand.
Working with Microorganisms
- Assume that all microorganisms are infectious and handle them with care.
- Sterilize all equipment being used to handle Microorganisms.
- Thoroughly clean your work area and equipment.
- Be careful not to return chemicals or contaminated items to the wrong containers.
- Don’t dispose of materials in the sink unless instructed to do so.
- Wash your hands thoroughly.
- Clean up all residues and properly dispose of them.
- Dispose of all chemicals according to local, state, and federal laws.15
No matter what, remember to be safety conscious at all times.
To prevent bacterial illness we must take precautions. We know the basics to prevent getting sick, like washing hands and covering our mouths when we cough. Water bottles can become contaminated with bacteria easily. Consumers need to take more precautions to protect themselves from illness from the water in their water bottle.
“An Ounce of Prevention: Keeps the Germs Away.” http://www.cdc.gov/ncidod/op/antibiotics.htm 1/19/04.
“Bacteria in the Mouth.” http://www.vampirovibrio.com/micro.bacteria 1/27/04
Britannica Macropedia Ready Reference. Volume 1, 15th Edition. Chicago, IL. 2002. Pages 779-780.
Gardener, Robert. More Ideas for Science Projects. Chicago, IL. F. Watts. 1989.
Hillers, Val. You Can Prevent Food Poisoning. Pullman, WA. W.S.U. Cooperative Extension. 1993.
International Bottled Water Association firstname.lastname@example.org 12/27/03
“Kids can leave the can open.” American Society for Microbiology General Meeting. Florida, May 2001. http://www.nature.com/nsu/010531/010531-html 12/10/03
KLTV, ABC News. “Refilling Your Water Bottle Turns it into a Bottle of Bacteria.” http://www.kltv.com/Global/story.asp?S=1285710 12/27/03.
Litin, Scott C. M.D. Mayo Clinic Family Health Book. New York, NY. 2003.
McDowell, Natasha. “Mineral Water Contamination Claim: Signs of Virus From Human Feces Found in Bottled Water.” April 8, 2002. Nature Science Update. http://www.nature.com/nsu/020402/020402-9.html 12/10/03.
NBC News. “Refilling Your Water Bottle Could Expose You To Harmful Germs.” Columbus, OH. http://www.nbc4columbus.com/target4/2191190/detail.html 12/27/03.
Science Experiments on File. Facts on File, Inc. New York, NY. 2000.
Sviel, Michelle, P.A., Personal Interview. January 16, 2004.
Tortora, Gerald J., Funke, Berdell R., and Case, Christine L. Microbiology, An Introduction. Benjamin Cummings. 2003.
WETN News, Albany, NY. “Bottles Bacteria: The Dirty Danger of Re-using Water Bottles.” http://www.wten.com/global/story.asp?s=1171862&ClientType=Printable 12/27/03
“What May be in Drinking Water That is Not Safe – Drinking Water Should You be Concerned.” http://pueblo.gsa.gov/cic_text/children/healthyhome/water-7.pdf 1/12/04.
World Book Encyclopedia. 2004 Edition. Volumes A (1) and B(2). Chicago, IL. 2004.
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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