|MadSci Network: Biochemistry|
Most bacteria have very few friends, but that isn't a reason to kill all of them. The ones we most want to kill in our mouths are the ones that cause cavities in our teeth and gum disease. After that, we would like to get rid of the ones that make our breath smell bad, and bacteria in our mouths that might make someone else sick ("infectious bacteria"). Unfortunately, you probably won't have the means to tell the different kinds of bacteria apart, so killing as many as possible becomes the objective. If you could kill all of the bacteria, your mouth would be "sterile." This is not likely to be accomplished, even if you gargled with bleach (don't' try it!!!), and it might not be desirable in any case. Bacteria are a normal part of our lives — you shouldn't leave home without them.
At some point, you are going to need some grown-up help. It's not that working with bacteria is always dangerous — most bacteria are pussycats. The main problem is something called "aseptic technique." You need someone to teach you how to handle samples so that only the bacteria of interest are present — not bacteria from your fingers, your breath, the air in the room, or the containers you put your samples in. Maybe your science teacher has had a microbiology course and can help you learn the tricks. If there is a college or university fairly nearby, sometimes faculty members (or even students) there are willing to help a young person get a start. If you can't find an adult to work with, you'll have to get a book and read about this. There may be some on-line resources for this, too; I haven't looked. There will also be some expense, because you will probably have to buy some supplies and sterilize them, or buy supplies that are already sterile.
OK, so your project is about mouthwash. You don't want to kill skin bacteria, or soil bacteria, or water bacteria. The first question is: Do I want to kill bacteria IN my mouth or FROM my mouth? When planning a research project, you need to think of what you want to measure and how you are going to measure it. You could rinse your mouth with "Product A," and then take some water and swoosh it around in there and then find out how many bacteria came out in the water. Or, you could rinse your mouth with "Product A," and then take a sterile cotton swab and wipe whatever you could get off of a few teeth, trying to keep the method standard from trial to trial, and then try to measure how many bacteria were on the swab. Some people would say you could sample the top of you tongue with a swab, or scrape it with a toothpick — I've never tried that. Anyway, whatever you do with Product A, you do the same thing with Product B and Product C, till you run out of products or ambition. The trouble with this is that you can't do these trials one right after another. Hopefully, Product A will have killed or removed many of your mouth bacteria; if you follow right away with Product B, there won't be as many bacteria there to kill, and there may still be some of Product A hanging around to confuse the experiment. If you wait till next meal or the next day, to get some fresh bacterial build-up, you surely won't have identical conditions twice in a row. Different foods, and even different times of the day, can influence what bacteria are in your mouth.
One alternative is to get bacteria from your mouth, but try to kill them out where you can control the experimental conditions. You could take some tap water (it might be better to boil it first, to kill any bacteria it had; if you do this, let the water cool completely before you put it into your mouth) and swoosh it around in your mouth for a minute or more, then spit it into a bowl. Now you have a mouth-bacteria "soup." It needs to be mixed carefully, to distribute the bacteria uniformly without adding any that didn't come from your mouth. Then, you could dispense it into small, sterile containers (maybe test tubes) with a sterile pipette (something like an eyedropper). If you don't have pipettes with measuring marks on them, just count the drops so that everything comes out even. You need as many containers as there are products to test, plus one or two. Then you need that number of pipettes, to add the mouthwash or plain water to each. The plain water (in at least one, maybe two of the containers) is called a "control"; you use this or these containers to see whether the mouthwash is any more effective than water. The reason for two controls, rather than one, is to see how uniform your method is; if you get very different numbers of bacteria from the two water treatments, it's going to be very difficult to take your mouthwash results seriously. Anyway, you need to standardize how you put the mouthwash into the tubes, and you need to plan a standard dilution that will maybe stop the action of the mouthwash after a standard period of time. When you use a mouthwash, you probably only swoosh it around for half a minute or so before you spit it out; but some of it stays in your mouth for a few minutes, until it gets diluted by your saliva till it has no more effect, or until you put something else in your mouth that counteracts it. I think giving the mouthwash in the test tubes 5 minutes to kill all the bacteria it can is OK, but you may want to make the time shorter or longer. Then, you need to stop the action of the mouthwash, either by diluting it with at least 9 volumes of sterile water, or maybe by adding some bacteriological broth that ties up the active ingredients in the mouthwash (like when you put some more food in your mouth and chew it). Keep the time standard, for each of the mouthwashes, and also for the controls; treat each tube the same.
Finally, you have to find out how many bacteria are left. A common way to do this is to spread the samples on the surface of an agar medium. This is a gelled material, usually in a plastic "petri plate" with a lid. It has nutrients to make the bacteria grow, and the agar holds the nutrients so that the bacteria grow and form "colonies," if there aren't too many bacteria. Various kinds of spreaders can be used to distribute a few drops of the sample on the agar surface. It is best to store the plates upside down at body temperature (98.6 degrees Fahrenheit = 37 degrees Celsius) for 24 hours. If you can't find an incubator that will hold the plates at this temperature, just put them in a comfortably warm place (where temperatures are fairly steady) for 24 hours. Be sure to get a good thermometer and record the temperature you used.
You don't want the whole agar surface covered with a "lawn" of bacteria. Plate count agar (it has various other names) is usually used in petri plates about 4 inches in diameter; for accurate counting, the number of colonies that form on the plate should be between 30 and 300. If there are many more, you'll have to learn to dilute the samples accurately, so the numbers aren't too large. Ideally, your controls will let you figure how many bacteria were in the original sample (in "colony-forming units," or "CFU"). You consider the average number of colonies you saw on your control plates (should be more than one), times the dilution. Then, compare these with what came from the tubes with the mouthwash. If you had to use different dilutions of some of them, so as to get between 30 and 300 colonies per plate, you'll have to adjust for the different dilutions. An effective mouthwash should have killed some of the bacteria — if you had to count the control plates that had a one-to-one thousand dilution, you may have to count the mouthwash plates at one-to-one hundred or less. Any experiment of this kind should be repeated several times, to ensure that the results come out the same. Because your measuring devices are not super-accurate, you can expect some variation from trial to trial. In order to get consistent, meaningful results, you have to learn to do dilutions and counts accurately, and do everything the same way every time.
There are some faster, high-tech ways to estimate the relative numbers of bacteria in your samples, if you can find someone who has the equipment. One measures the energy component (ATP) of the bacteria by how much light it generates in a standard test with luciferin — the material that glows in fireflies' tails. This system is used to measure how clean the work surfaces are in some food processing plants; if you have any contacts in the food industry, they may be able to help you. All you need at this point is something that will tell you whether there are less bacteria in the samples treated with mouthwash than in those with water, and whether there is any difference among the mouthwashes. Plate count agar lets most kinds of bacteria grow, if they like the temperature. There are some mouth bacteria that only grow in the absence of oxygen ("anaerobes") that won't grow on your plates, but this can't be helped. The main things is to learn how to carry out the experiments so that you don't get a lot of bacteria that don't have anything to do with your mouth. Of course, if you have a college with a school of dentistry nearby, they will have even better ways of running experiments like this, but schools of dentistry are usually not that nearby. Your own dentist might put you in touch with a laboratory that does his microbiology work, and they might be willing to help.
You have "bitten off" quite a project. Good luck with it!
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