Introduction: (Initial Observation)
Manufacturers of different brands of mouthwash often claim that their brand is better, kills more bacteria, and prevents bad breath for a longer time.
Different brands of mouthwash also have different price ranges. Are you getting a better and more effective product when you buy a more expensive brand?
Like any other consumer product, mouthwash can be tested for effectiveness in removing bacteria and killing bacteria.
In this project, you will determine which mouthwash is more effective in killing germs. First select which brands of mouthwash you wish to experiment with. At least three different brands of mouthwash should be selected for your experiment. For better results, we recommend four or five different brands of mouthwash.
You need to have some bacteria growth experience for this project.
Information Gathering:
For each brand of mouthwash see if there is a company website and find any information the manufacturer provides about that specific mouthwash. Also, search the web for the term “mouthwash ingredients” and other related terms to find useful information about mouthwashes.
Mouthwashes are among the most popular oral hygiene products, and you can often find new brands on drugstore shelves. They serve, or claim to serve, various purposes: to cure or at least mask bad breath, fight cavities, prevent the buildup of plaque, and whiten teeth. Most are classified as cosmetic agents, meaning that the FDA regulates them only minimally. Therapeutic rinses, on the other hand, contain active ingredients that may protect against periodontal disease. They kill bacteria and reduce plaque and gingivitis.
Dental gum disease is caused mostly from a build-up of plaque, which is formed from bacteria and food films. This build-up between the gums and the teeth causes consequential gum damage and disease.
These films, if not removed, provide nutrition to oral bacteria, which excretes acids and in turn leads to tooth decay and cavities. By removing the food source of the bacteria, the chance of tooth decay can be significantly reduced.
Manufacturers typically make different claims about their brand of mouthwash and what their brand of mouthwash is designed to do. Below is an example from one manufacturer of mouthwash:
“Our unique mouthwash is formulated with a unique patented ingredient (Stabilized Chlorine Dioxide) that helps eliminate odors by dissolving food films, thus eradicating the true source of bad breath. This powerful ingredient is registered with the EPA, as an excellent bactericide, fungicide, and antimicrobial agent. Peppermint flavoring is also added to Eliminator’s formula to give it a minty-fresh taste.”
How do I grow bacteria?
To grow bacteria you make some nutrient gel and inoculate it with some sample bacteria from your hands, your kitchen sink or any other polluted source. Gel can hold water and food that bacteria need to grow. Nutrient gel is very similar to the gels that you eat at home as desert; however there are some differences too. The gelling agent used to make gel for growing bacteria is Agar. Agar is a gelling substance extracted from seaweeds. Agar is not a food for bacteria. While making agar gel, you must add some nutrients to that. Nutrients are food for bacteria. A good nutritious food for bacteria is chicken broth (like chicken soup).
Bacteria can grow in nutrient broth or on nutrient gel (also known as nutrient agar).
What is nutrient broth?
Nutrient broth is a clear liquid containing food or nutrients. You may make nutrient broth by boiling fat free chicken or beef in water. For a better nutrient broth, you may boil some fat free chicken or beef with some potato, some mushroom, some carrots and some beans. Think that you are making a delicious and nutritious soup for yourself; the only difference is that you want it to be clear when you make it for bacteria growth. After a while when bacteria grow in the nutrient broth, it will not be clear any more. (The clear soup becomes cloudy by bacteria).
We grow bacteria in nutrient broth when we are trying to reproduce them for further experiments.
What is nutrient agar?
Nutrient agar is the gel form of the nutrient broth. Agar is a gelling agent. To make the nutrient agar, you add some agar to the hot nutrient broth. The ratio of agar to broth is about 1 to 2 percent. Agar must be added gradually while you stir the solution continuously. If the agar precipitates, it will burn. When the agar dissolves fully in the hot boiling broth, then you are almost done and you can turn off the heat. Let it cool off a little. While the nutrient agar is still warm, start filling up the petri-dishes. To each petri-dish add enough nutrient agar to cover the bottom of the plate. Put the lid back immediately. The nutrient agar will fully cool off and form gel inside the petri-dishes.
We grow bacteria on nutrient agar when we want them to grow at their own place and form visible bacteria colonies. The gel does not allow the bacteria to move around or float around. Aerobic bacteria will grow at the surface of nutrient agar gel. Anaerobic bacteria may also grow inside the gel.
The nutrient agar you make must be free from bacteria. (it must be sterile). A chicken broth made in a pressure cooker is usually sterile. Pressure cookers get much hotter than regular pots.
PA.
Question/ Purpose:
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.
The purpose of this project is to find out which mouthwash is more effective in killing oral bacteria.
Identify Variables:
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
The independent variable is the type or brand of mouthwash.
The dependent variable is the effectiveness of a particular mouthwash in killing bacteria.
Controlled variables are:
- Exposure time
- Concentration
- Procedures of testing each mouthwash
- Type of bacteria
Hypothesis:
You will state which mouthwashes have been selected and then the hypothesis will state which of the mouthwashes is believed to be more effective. Your hypothesis may be based on your own intuition, gathered information, or past experiences with mouthwashes.
Experiment Design:
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Experiment 1: Comparing mouthwashes for their effectiveness in killing bacteria
Materials Needed are:
- 4-6 petri dishes with nutrient agar
- sterile blank disks
- inoculating loop
- sterile swaps
- 2-3 tubes of sterile nutrient broth
- forceps
- sterile water (Boiled tap water works fine in most cases)
- metric rulers
- Bunsen burner (You may use an alcohol burner or any other smokeless flame)
- samples of 4-6 mouthwashes, cleaners or mouthwashes
- Also required: Incubator set at 35ºC. (A warm cabinet may work as well)
Where to get the material?
All the required material can be purchased from laboratory suppliers or scientific suppliers. Find them locally by looking in your phone directory. Petri-dishes are more widely available. You can also buy petri-dishes from MiniScience.com or other online suppliers. They are sold separately and they are also included in bacteria growth kits.
If you cannot find sterile blank disks, use a paper puncher and cut them yourself from filter paper (coffee filter paper may be used as well).
An inoculating loop can be built using a thin solid wire. Just form one end of wire to the shape of a ring.
Sterile water can be made by boiling water in a pressure cooker for 10 minutes. If you do not have a pressure cooker, boil water in a kettle for 30 minutes. This is not a perfect solution, but it works good for the purpose of this project.
Any burner that has a gas flame or burns alcohol can be used instead of a Bunsen burner.
Any warm space about 35ºC can be used instead of an incubator. A box or a metal can painted black and placed under a desk lamp may be used as an incubator. You will need to adjust the distance of the desk lamp until the temperature inside the can stays around 35ºC.
Procedure:
1. The first step is to grow a sample of your teeth’s bacteria. To do this, take a sterile cotton swab and carefully run it back and forth along your front teeth right at the gum line (where the gums meet the teeth). Restrict your sample collection area to the top four front teeth.
2. Next, open a plate of nutrient agar and implant your bacteria while isolating them in different concentrations using the following method:
- Rub the swab over the top third of the plate (see left diagram below). Dispose of the swab.
- Flame your inoculating loop. Let it cool off. Turn the plate one a quarter of a turn. Rub the loop over the plate starting from some point of previous swab. (see center diagram below)
- Flame your loop again. Turn plate another quarter of a turn. Rub the loop over the plate as shown in the right diagram below.
3. Incubate these plates for 24 hours at 35ºC.
4. The bacteria that should be growing at this time should look like small, clear pinpoint dots. This is the bacteria that you want to subculture into your broth tube. Be careful not to touch large white colonies. These are yeast and not the cause of gum disease. Have your plate checked by your teacher before proceeding any further!!
5. Once your plate has been checked, follow the procedure below for setting up your broth culture:
- Flame your inoculating loop and cool.
- Select 5-6 colonies and pick them up with your inoculating loop.
- Remove the top of the broth tube and flame gently.
- Insert the loop with bacteria into the broth tube and swirl gently. Remove the loop and re-flame.
- Gently flame the top of the broth tube and recap.
6. Incubate your broth culture for 24 hours at 35ºC.
7. You are finally ready to set up your test. Record keeping is most critical from this point on. Make sure to label your dishes distinctly with permanent marker!! Follow the procedures below:
- On the outer bottoms of the petri dishes, mark each with an identifying number or letter that corresponds to one of the mouthwashes that you are going to test.
- Take a new sterile swab. Using the same technique as before, uncap your broth tube and dip the swab in. Recap the broth after removing the swab and flaming the side of the tube.
- Swab the entire plate. Turn the plate one quarter of a turn and swab the entire plate again. (Refer to the figure below.)
- Dispose of the swab.
- Flame your forceps. Pick up a sterile disk and briefly soak it in one of the mouthwashes you are testing. Place it in the center of your plate.
- Repeat this step for each of your mouthwashes and one with sterile water to serve as the control for the experiment.
8. Incubate your plates for 24 hours at 35ºC.
9. You are looking for clear spaces around your disk where no bacterial growth has taken place. These will look like little circles around your disk. These are called zones of inhibition. Your next step is to measure the diameter of these zones. The diameter is directly proportional to the effectiveness of the mouthwash that your are testing. Record all results in the table of your data section.
10. Dispose of all bacterial cultures.
Your results table may look like this:
| Brand of Mouthwash | Diameter of inhibition zone |
Make a graph:
You can also use a bar graph to visually present your results. Make one vertical bar for each brand of mouthwash you test. The height of the bar will represent the diameter of the inhibition zone.
SAMPLE ABSTRACT: The purpose of this experiment was to find which mouthwash product is the most effective against bacteria. To carry out this experiment Scope, Listerine, and distilled water were tested against mouth bacteria samples taken from an individual’s mouth. To test the Scope, Listerine, and distilled water, four paper disks were soaked in each product and then put into three separate petri dishes. Each disk was put into the quadrant of the petri dishes, one petri dish with the four disks soaked in Scope, another with the four disks soaked in Listerine, and the third petri dish containing the 4 disks soaked in distilled water. Swabbed on the petri dishes were bacteria samples taken from one individual’s mouth. The petri dishes were then sealed and put into an incubator at 37 degrees Celsius for 48 hours. After 48 hours, the petri dishes were taken out of the incubator and examined. The zone of inhibition of each disk in each quadrant of the petri dishes were measured to determine which product produced the largest average zone of inhibition. The zone of inhibition was measured in millimeters.
After completing the experiment, it was found that Listerine produced the largest average zone of inhibition with an average of 16.5 mm. The product Scope was the second most effective with an average zone of inhibition of 11.5mm, while distilled water did not produce a zone of inhibition. The distilled water was the least effective in killing bacteria.
Experiment 2: Simple Experiments
Introduction: Bacteria can grow on food and spoil the food. Bad odor, separation and loss of viscosity are conditions that may be used as indications for bacteria growth. For example bacteria can grow on milk and spoil it. Spoiled milk will coagulate so that the protein of the milk separates from the water. Cooked chickpeas spoil and have a very bad odor. Fresh chicken broth forms a gel in the refrigerator. When bacteria grow on chicken broth, they produce enzymes that change the gel back to liquid. Any of these properties may be used to test an anti bacterial substance.
Please note: I have never done this experiment. I just think that it will work. Please let me know what are your results. Project Advisor
Procedure: Make some chicken broth. Split it in two equal cups. In one cup add one spoon of mouthwash. Label the cups “mouth wash”. In the other cup (call it control experiment) add one spoon drinking water or distilled water or tap water. Label this cup “water”.
Leave both cups uncovered in the room for about 10 minutes. That usually is enough for bacteria from the air to enter both cups. If you want to make sure, you can also add one drop of polluted water in each cup.
Cover both cups and place them in the refrigerator. Inspect them after a few hours. Both must have formed gel. (If they are not, this will not work). If the gel is formed, then revisit them after 3 days to see if they are still gels. If the mouthwash is able to kill bacteria, then then the cup with mouthwash must remain gel and the other cup must change to liquid after a few days (3 to 9 days).
Variations: If instead of chicken broth you are using milk, then the sample with mouth wash must remain homogenized for a longer time than the sample without mouth wash. If you use cooked chickpeas, then the odor is the best indication to show if mouthwash has been able to kill bacteria. Cooked chickpeas may be tested with or without their soup.
Results of Experiment (Observation):
Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
Summary of Results:
All measurements should be recorded in the project book and may be presented in the final project report either as numerical data or shown as a bar graph. For each brand of mouthwash tested, the length of the bar should show the effectiveness of that specific mouthwash.
Following are two sample displays.
Another Sample abstract: The purpose of this experiment was to learn which mouthwash killed the most bacteria. To perform this experiment, six agar dishes were divided into quarters and swabbed with a sample of oral bacteria. Three sterile discs where then soaked in either Plax, Scope, Listerine, Cepacol, Tom’s of Maine or the Generic Brand. All three discs that were soaked in a certain type of mouthwash were then placed in one of the quadrants in the agar dish. A clean sterile disc was then placed in the last quadrant to act as the control. All six agar dishes were then placed in an incubator for 72 hours at room temperature.
Plax was found to be the most effective mouthwash, having a zone of inhibition of 13.7 mm. Scope, Listerine, Cepacol, Tom’s of Maine, and the Generic Brand all had zones of inhibition under 4 mm. Through this experiment, it was learned
that Plax is the best mouthwash to use.
Conclusion:
The conclusion to the report will state the ranking of the various mouthwashes, and decide if the original hypothesis was proved or disproved. The conclusions might also state any relationships you have noted between the popularity of a given brand of mouthwash and its effectiveness or its price.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
Possible Errors:
If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.
If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.
Question: What is the scientific name of the bacteria that we have grown?
Answer: Most bacteria have no specific names. Just call them bacteria. Scientists often name bacteria based on their shape or properties or some other specifications (i.e streptococcus) , but they are not “unique names”. They are almost like naming an animal as “fat brown mammal”.
Some bacteria have a name (e.coli for example), but that also does not mean much. It is like an animal called “dog”. Not all dogs are the same!
