Re: How can I tell what kinds of bacteria are in your mouth after brushing?

Hi Christine -

I'd recommend "rinsing" your tooth brush in sterile saline (salt water solution with about as much "salt" as is found in human blood), then spread some of the saline solution on agar in Petrie plates to see what grows. You should be able to find most of the things you'd need to do this at a local drug store or perhaps through your school science lab:

1. New unused toothbrushes - use a new one each time you begin an experiment so you don't worry about any bugs that were on the toothbrush previously.
2. Sterile saline (try the drug store).
3. Latex or PVC gloves - as a safety precaution when handling your specimens. Again, try the drug store.
4. Sterile testubes - try your school lab or a scientific supply house. The tubes should be wide enough for you to place your toothbrush into the tube.
5. Sterile pipettes - You can use glass or disposable 1 and 5 milliliter plastic pipettes to measure your sterile saline and transfer equal volumes into the testubes. You'll also need a "bulb" that fits on the end of the pipette which is used to "suck" fluid into it. You may be able to obtain some through your school, or again, through a scientific supply house.
6. Agar plates - these can be purcahsed commercially. I'd recommend a form of "nutrient agar" such as LB agar (Luria-Bretani agar), or BHI agar (brain heart infusion agar). You could also try sheeps blood agar which will let you see if bacteria from the mouth are capable of hemolyzing, or "bursting open" red blood cells.
7. A new box of toothpicks. You will use these to streak your plates with saline that has been used to rinse a toothbrush.
8. An incubator if your shcool has access to one - you'd like to incubate the plates at 37 degrees Celcius. If you don't have an incubator, take an old piece of tupperware, store your plates in this container, and incubate them at room termperature ~ 25 degrees Celcius.

Then, to carry out your experiment:

1. Wear gloves and protective clothing during your experiments. Some bacteria that live in your mouth are classified as pathogens (disease causing), and you don't want to expose your skin or other body parts to the colonies you grow. Be certain to wash your hands when you're done with experiments or after every time you handle the plates.
2. Use a sterile pipette to place 1-5 milliliters in 3 testubes before each experiment. The volume of saline should be enough to cover the bristes of the toothbrush.
   • Label one "negative control" - you won't put anything in this tube, but will streak a small amount to the agar to make certain your saline is sterile.
   • Label the 2nd "positive control" - spit into this one, so you know what was growing in your mouth before you brushed.
   • Label the 3rd "experimental" - you'll spit into this one after brushing.
3. Spit into the 2nd tube.
4. Brush your teeth with a new toothbrush, then spit into the 3rd tube.
5. Mix the solution in the tubes.
6. With sterile pipettes (one for each tube!) transfer 0.1 milliliters to a fresh agar plate.
7. From your toothpick box, take a toothpick and use it to move the fluid all over the plate. Start in the fluid and make straking motions back and forth across the plate until it is evenly spread. It may take a bit of practice to do this without gouging the agar - though if you gouge it, it won't wreck your experiment. If you want to practice the technique beforehand, try making the streaking motions on some jello :).
8. Incubate your plates for 24 hours at 37 degrees C, or for 48 hours at room temperature.
9. Check the number of colonies on each plate, and record characteristics such as their color morphology and size. For "morphology" you'll want to note whether the colony is smooth, wrinkled or "glistening" for instance. Do you note any differences in the number of colonies as well as the types that grow when you compare plates before and after you brushed?
10. Repeat the above experiment at least 3 times so you have multiple data points on which to base your assuptions.

You should also ask your science teacher if you have reagents for making stained slides of bacteria, such as a Gram stain. With the use of a microscope, you can then look at the morphology of the bacterial cells, and make some assuptions about what types of bacteria they may be. However, this may be far beyond what you need to do for the science fair, and may also be beyond the capabilities of most middle schools.

Hope this helps..

-L. Bry, MadSci Admin