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The Effect of Mouthwash on Alpha Streptococcus

Researched by Sierra S. 


The purpose of this experiment was to determine which mouthwash was most effective in killing alpha streptococcus.

I became interested in this idea while brushing my teeth and wondered if the bacteria in my mouth were being killed.

The information gained from this experiment would help consumers and dentists find the best solution for killing and preventing bacteria in the mouth.

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My hypothesis was that Listerene Antiseptic Mouthwash would kill the most alpha streptococcus.

I based my hypothesis on what Marie Clark, Microbiologist at Memorial Hospital, said: "Listerene as an antiseptic should be the best at inhibiting alpha streptococcus."  I also based it on the fact that it was approved by the American Dental Association (ADA).

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The constants in this study were: 

  • The amount of mouthwash used
  • Number of mouthwash types per plate
  • Number of control groups per plate
  • The type of plates used
  • Same blood and broth mixture in plates
  • The type of bacteria used
  • The approximate number of organisms per plate
  • Same death zone measurement method and tool

The manipulated variable was the type of mouthwash used. 

The responding variable was the size of the death zone around the area where the mouthwash was placed.

To measure the responding variable a caliper was used to measure the diameter of the circle of death zone in millimeters.

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2                         cotton swabs 
1                         blood agar plate 
1                         blood agar slant 
1                         inoculating loop 
1.8 ml                 saline solution (.9% density) 
1                         colorimeter 
3                         blood Mueller Hinton plates 
1                         10 microliter pipette (eye-dropper) 
1                          50 microliter pipette (eye-dropper) 
1                          incubator  
1                          calipers 
1                          autoclave

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1. Gather materials. 
2. Isolate alpha streptococcus: 
a. Swab mouth. 
b. Inoculate plate. 
c. Take inoculating loop, and streak plate for isolation. 
d. Streak plate for growth. 
e. Incubate overnight at 35*F at 7% CO2. 
f. Take predominate colony. 
3. Make suspension of organism: 
a. Mix organism in saline solution in glass vial. 
b. Insert in colorimeter and measure the density. 
c. Continue adding bacteria to the vial until it has reached about 80% density. 
d. Swab over Mueller Hinton plate with blood. 
4. Innoculate plate with mouthwashes 
a. Treat plate with 10 microliters of the first type of mouthwash to the right of the quadrant. 
b. Treat plate with another dose of 10 microliters of the mouthwash to the left of the other 10 microliter dose. 
c. Treat plate with 50 microliters of the mouthwash below the two 10 microliter dosages.
d. Add saline solution to the fourth quadrant for control. 
e. Repeat steps 4a-4c with three mouthwashes and one control (saline) on 3 plate. 
f. Incubate overnight at 35*F at 7% CO2. 
4. Measure zones of death with a calipers where mouthwash was placed in millimeters. 
5. Estimate the number of colonies within the death zones. 
6. Record data. 
7. All materials are put in the autoclave (heated at 250*F) to sterilize. 


The original purpose of this experiment was to determine which mouthwash was most effective in killing alpha streptococcus.

The results of the experiment were that the least amount of bacteria colonies grew where Rembrandt or Scope had been. The most bacteria grew where Fluorigard, Biotene, and Long’s brand.

See the graphs.

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My hypothesis was that Listerene Antiseptic Mouthwash would kill the most alpha streptococcus.

The results indicate that this hypothesis should be rejected because Listerene did not kill an average of 70 bacteria colonies in Trial 1 and 2, while other mouthwashes killed all colonies.

Because of the results of this experiment, I wonder if the mouthwash would be effective in killing other types of mouth bacteria. I also wonder if toothpaste would work in killing bacteria.

If I were to conduct this experiment again, I would use a different type of bacteria. I would also use separate plates for each mouthwash, and use varied amounts of mouthwash (such as 5, 15, 25, 40, and 55 microliter dosages). I would also use more brands of mouthwash, and do more trials.

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Human health is necessary for survival and happiness. Many diseases are caused by bacteria, which can be prevented or killed by the use of antiseptics.


Bacteria are tiny, one-celled microbes. Because of their ability to adapt to any environment, they are found almost everywhere. Bacteria break down materials into simple materials such as carbon, nitrogen, and other materials needed for new life. Unfortunately, some also cause diseases.

A wall that holds the bacteria organism together, and controls what enters and exits surrounds the one cell. The wall also affects how the bacteria react to certain antibiotics, antiseptics, and protective secretions such as tears and saliva. The bacteria’s wall sometimes has a sticky coating called a capsule that provides extra protection, and allows the cell to stick to others. When the bacteria have a limited food supply, they dehydrate and produce, thick spore coats. As spores, the bacteria can survive for hundreds, even thousands of years. Bacteria move using flagella, which are tiny structures that beat in a circular motion. Spirilla move in a "corkscrew" motion.  Some bacteria do not move at all.

Bacteria come in distinct shapes, and grouping types, and are named based on these. These three shapes are bacilli, which are rods, cocci, which are round, and spirilla, which are spirals. Bacteria group together in different ways. Diplo is where two bacteria join, strepto form chains, and staphlyo form clusters. The level of hemolysis further classifies bacteria. Hemolysis is when hemoglobin, the iron-protein that gives blood its red color and transports important nutrients, leaks out of red blood cells. The levels of leakage caused by bacteria are beta, completely hemolytic, alpha, moderately hemolytic, and gamma, which is non-hemolytic.

Although bacteria have no brain or nervous system, they have the ability to remember stimuli up to 60 seconds. They can also sense food or toxins.

Bacteria reproduce through two methods. The first is binary fission, where the cell divides in half, creating a new bacteria cell. The new cells are half the size, but grow. The new bacteria cell is an exact replica of the original cell. Binary fission may occur from every 15 minutes to every16 hours. The second method of reproduction is called conjugation. This is where to bacteria cells join together and exchange DNA.

Alpha Streptococcus

Alpha Streptococcus is hemolytic bacteria that are round and form chains with other organisms. Alpha streptococcus can cause diseases as strep throat, and play a large role in tooth decay. It may also damage body tissues such as the heart, and kidney

Human Defense Against Bacteria

The human body has ways to naturally protect itself from bacteria without the use of medications. Within the skin are glands, which secrete sweat and oil, which contain acid that kills and prevents the growth of bacteria on the skin. Openings in the body are covered with a layer of mucus that traps bacteria. If the bacteria get past this mucus, acids in the stomach kill them. If the bacteria get past the outer defenses and enter the skin, it will injure the cells. The cells release histamine that causes blood vessels near the wound to swell and increase blood flow to this area. The chemicals in the blood clot and seal off the cut, which prevents more bacteria from entering. When the cells release histamine, they attract white cells called phagocytes. These cells engulf bacteria, and kill the harmful microorganisms. They then die and excrete puss. Any bacteria that get past the phagocytes then have to defeat the immune system. The two million lymphocytes, also called T-cells, in the blood and lymph systems detect invaders such as bacteria and viruses. When they discover the cells they began producing antibodies, which destroy a specific type of foreign body. The lymphocyte's memory cells remember previously fought "enemies" and instantly produce antibodies. So, a disease that has already been fought doesn't have a good chance against the lymphocytes in the future. If it is a new "enemy" it may take the lymphocytes five to seven days to produce the maximum amount of antibody. If not enough are produced, the foreign cells may defeat the lymphocytes, and eventually kill the person.


In ancient times leprosy, tuberculosis, cholera and other diseases killed many people. People believed that disease was a punishment from the gods or a curse of an enemy. A theory called spontaneous generation was accepted for over 2000 years. Spontaneous generation was the belief that life could spring from non-living materials. In the 1860's Louis Pasteur and Robert Koch discovered evidence that diseases were caused by microorganisms, and life could not come from dead materials. Pasteur discovered this by isolating two types of bacteria on dying silkworms in France. Pasteur also discovered that microorganisms cause food and drink, like milk, to spoil and decompose. He invented a method called Pasteurization that destroyed bacteria in the milk using heat. Robert Koch discovered that bacteria cultures grew best on agar, a gelatin substance that comes from seaweed combined with broth for the bacteria to feed on.

Joseph Lister, a professor of surgery, read about the discoveries of Pasteur and Koch. He was convinced that bacteria caused infections that killed patients. So, Lister cleaned the area that was to be operated on, his hands, operating tools, and the air of the room with acid. As a result, the patient had no infections, and Lister's method was called anti-septic surgery.


Antiseptics have bacteria-fighting abilities. The chemicals within the antiseptic may cause an allergic reaction, or damage skin if not used correctly.


Wine and vinegar have been used as antiseptics for over  2,500 years. In addition to these, brandy, mercury, pitch, tar, and turpentine have been used as antiseptics throughout history.

Hundreds of year ago, a surgeon noticed that untreated wounds smelled unpleasant, and began treating them with various substances. Some materials prevented infection, but the patient later died from the harm of the materials used. In the mid-1800’s Ignaz Semmelweis, successfully used a mild solution of lime chloride. Later, in the mid-1860’s, Joseph Lister used a carbolic acid to prevent infection in a patient he operated on. Together, Lister and Semmelweis lead to the use of mild yet powerful antiseptics that are common today.


Dentistry is the job of diagnosing, treating and preventing diseases of the teeth, and tissue of the mouth. The branches are general dentistry, which covers most areas, orthodontics, the correction of the teeth, oral surgery, surgical correction of the teeth, periodontics, the treatment of the bones and tissue that support the teeth, prosthodontics, the replacement of missing or damaged teeth, oral pathology, the diagnosis and treatment of mouth diseases, pediatric dentistry, which specializes in the treatment of children’s mouth and teeth, and enthodontics, the prevention, treatment and diagnosis of diseases of dental tissues.

Structure of the Tooth

"Each tooth is composed of a crown which projects above the gums, and a root, which is hidden beneath them.  The tooth narrows at the neck, where the root and crown meet.  The visible crown varies in shape, depending on the function of the tooth.  Much of a tooth is formed by its root or roots.  A canine tooth has a single root whereas a molar has two or three small ones fused together.  Third molars may have as many as four or five roots."  (pp. 17-18, Dental Health by Dorothy Siegel). 

The tooth is in a jaw socket, held in by the strong connective fibers of the periodontal ligament. It attaches the tooth to the jawbone. The ligament contains blood vessels, nerves and bone-building materials. As long as it remains healthy, it will continue to nourish the tooth, keeping the tooth alive.

Purpose and Care of Teeth

The mouth is the first step in digestion,  and the teeth play an important role. They chop up the food into a smaller size, and saliva further processes it. It is then swallowed, and the food continues through the digestive system.

The remains of food and bacteria harden on the tooth. This thin, sticky substance is called plaque. Plaque eats sugar and food on the tooth, and produce acids that eat away the tooth. Decay causes a hole in the tooth called a cavity, which is drilled out and filled with a substance such as gold, porcelain or plastic.

 The decay of the tooth can be prevented if plaque is properly removed. The first method of removal is tooth brushing. The bristles of the tooth scrape away the plaque. Toothpaste, fluoride, which helps strengthen the tooth enamel, and mouthwash help prevent decay. Mouthwash is a liquid that contains ingredients that help kill bacteria, loosen plaque, get to places where the toothbrush and dental floss miss, and add fluoride to the enamel. 

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"Dentistry." World Book Encyclopedia of Science: The Human Body 1997. pp. 108

Facklan, Howard and Margery. Bacteria New  York: Twenty-first Century Books, 1994. pp. 7-10, 12, 15, 17-20, 22-24

"Hemoglobin." Microsoft Encarta Encyclopedia Deluxe. 2001.

Martin, Chris. "Dentistry." Microsoft Encarta Encyclopedia Deluxe. 2001.

"Mouth." Illustrated Science Encyclopedia. 1997. vol. 14. pp. 1273

Nucci, Mary and Abuchowski, Abraham. "The Search for Blood Substitutes." Microsoft Encarta Encyclopedia Deluxe. 2001.

Rowdowskas, Chris A. Jr., "Antiseptic." World Book Encyclopedia. 1998. vol. 1. pp.

Siegel, Dorothy. Dental Health New York: Chelsea House Publishers, 1994. pp. 14-15, 19-24, 105-106

"Streptococcus." [online] Available: 

"Streptococci." [online] Available

Wortel, John P. "Dentistry." World Book Encyclopedia. 1995. vol. 5. pp. 143.

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This science project could not have been completed without the assistance of several people. I would like to thank all of them for their help and support.

-My mother for driving me to and from the hospital, and other places. 
-My father for also driving me places. 
-Marie Clark, Microbiologist at the Memorial Hospital, for helping me with my  experiment. 
-Karissa Kreig at the Memorial Hospital, for assisting me in obtaining bacteria on the days Marie was gone. 
-Mr. Newkirk for helping me narrow my topic, and edit my journal and research report. 

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