Summer Science Academy Experiment:

The Antibacterial Action of Mouthwash


GRADES: 6-12


TIME NEEDED: 15 minutes (day 1); 30 minutes (day 2); 1 hour (day 3)


Mouthwashes are widely advertised as being helpful in controlling bad breath (halitosis), cleansing the mouth, and promoting oral hygiene. Many mouthwashes claim to "kill the germs that cause bad breath". With this experiment, you will demonstrate the effectiveness of mouthwashes in inhibiting the growth of bacteria obtained from your mouth and saliva.


What is in a Mouthwash?


Most commercial mouthwashes contain more than one active ingredient and are advertised as providing several benefits to the user. The effectiveness of any mouthwash is influenced by dilution with saliva, time of exposure, and contact with organic matter in the mouth, which can alter the action of the mouthwash.


Each mouthwash is a unique combination of compounds designed to promote oral hygiene. Some of the active ingredients commonly found in mouthwashes and their functions are:


  1. Antibacterial and antifungal agents, which reduce the number of microorganisms in the mouth. Examples are hexylresorcinol, thymol, benzethonium chloride, cetylpyridium chloride, boric and benzoic acids, hexitidine, and chlorine-liberating compounds such as hypochlorous acid.
  2. Oxygenating agents, which are active against anaerobic bacteria of the mouth. Their effervescence helps to remove unhealthy tissue. Examples are hydrogen peroxide and perborate.
  3. Astringents, which cause local blood vessels to contract and thereby reduce swollen tissues. Examples are alcohol, zinc chloride, zinc acetate, alum, and organic acids such as tannic, acetic, or citric acid.
  4. Anodynes, which alleviate pain and soreness. Examples are phenol derivatives, oil of wintergreen and eucalyptus oil.
  5. Buffers, which reduce the acidity in the mouth resulting from fermentation of food debris by microorganisms. Examples are sodium perborate and sodium bicarbonate.
  6. Deodorizing agents, which neutralize odor from decomposing oral debris. An example is chlorophyll.
  7. Detergents, which reduce surface tensions, thereby allowing ingredients to become more soluble, and which also degrade bacterial cell walls, causing the bacteria to lyse. In addition, the foaming action of detergents helps to wash microorganisms out of the mouth. An example is sodium laurel sulfate.


Inactive ingredients included in mouthwashes are:


  1. Water, which makes up the largest percentage by volume.
  2. Sweeteners, such as glycerol, sorbitol, caramel, and saccharin.
  3. Coloring agents.
  4. Flavorings, such as oil of wintergreen, spearmint oil, peppermint oil, eucalyptus oil, and orange oil. In this experiment, you will isolate and characterize microorganisms growing in your mouth and determine the effect of mouthwash on these microorganisms.


Materials Needed


tongue depressor

sterile cotton swabs

blood agar plates

sterile inoculating loops


gram stain chemicals



compound microscope


Experimental Procedure


Day 1:


  1. Use a sterile cotton swab to wipe the inside of your mouth. After obtaining the specimen, roll the swab back and forth several times on one section of the blood agar plate. Use a sterile inoculating loop to streak out an area of the culture. This technique will hopefully assure that the culture will show isolated colonies, instead of just a big smear of many colonies. Incubate the plate overnight at 37o C.
  2. Choose a mouthwash and use at least a 10 ml sample to swish around your mouth for about 1 minute. Repeat step 1 now that you have cleaned your mouth with the mouthwash.


Day 2:


  1. Examine your cultures. Is there any difference between the 2 plates, before and after using the mouthwash? Be sure to look through the plates to note any change in the sheep red blood cells of the medium. Some organisms will cause a total clearing of the red blood cells; this is referred to as beta hemolysis. Some organisms will cause an incomplete change or greening; this is referred to as alpha hemolysis. The normal flora streptococci usually produce alpha hemolysis.
  2. Note and draw the different types of colonies.
  3. Use a sterile inoculating loop to touch a colony and streak it onto a sector of a blood agar plate. Repeat this with each different type of colony present. USE A NEW LOOP FOR EACH COLONY. This will allow you to isolate each colony as a pure culture and to determine which types of organisms are killed by the mouthwash and which are sill growing. Incubate the plates at 37 C overnight.


Day 3:


  1. Perform a Gram stain on each of your pure cultures, using your plate from yesterday. Record the results of the Gram stain for each of the different colonies tested. What types of microorganisms are still growing after the mouthwash treatment?