Methods and Materials Section

Note: I think it lost all the italics, etc. when I copied/pasted it here, but oh well.

It's a little more than a page in Word, but if we still need to make our paper longer, I can definitely do images.

Go ahead and send me edits/comments/suggestions via email whenever...

--------------------------

Methods and Materials
Our attempts to test the efficacy of mouthwash on mouth bacteria and E. coli started off with a series of unsuccessful experiments involving the use of two types of filter paper soaked in mouthwash and filling holes bored in the agar with mouthwash. These experiments produced no noticeable zones of inhibitions for most of the mouthwashes, and bacteria seemed to grow freely, forming a lawn that covered the entire plate.
Finally we decided to try an entirely new method of conducting this experiment: one that had been used by others who had also wanted to test the efficacy of mouthwash on bacteria. The paper, titled “Antiseptic effect of a novel alcohol-free mouthwash: A convenient prophylactic alternative for high-risk patients,” was authored by Paul Bahna, Hend A. Hanna, Tanya Dvorak, Ara Vaporciyan, Mark Chambers, and Issam Raad and published online on June 22, 2006. We modified the procedure by using pure mouthwash from the bottle instead of a modified solution. We also used different amounts of mouthwash and bacteria solutions and a different type of agar (triptic soy broth with bacteriological agar).
Using sterile technique throughout the experiment, we started off by creating multiple capped test tubes filled with nutrient triptic soy broth (100 mL of deionized water to 3 g of nutrient broth). In the end, we used three of these tubes: two filled with about 7 mL of broth each and one filled with about 15 mL of broth.
To set up our bacteria cultures, we labeled one of the 7 mL test tubes “E. Coli” and the other “Mouth.” We dipped a sterile cotton swab into the non-triclosan-resistant E. Coli bacteria culture tube from our previous experiment and inserted into the first tube, replacing the cap immediately afterwards. We then took another sterile cotton swab and swabbed out the inside of Beatrice’s mouth, inserting the swab into the second tube and replacing the cap immediately afterwards. The two tubes were allowed to incubate, and the third one was put into the refrigerator. (Our incubation time is not exact, as we didn’t monitor it very closely and ended up having to sub-culture our bacteria into two more nutrient broth-filled test tubes, simply because the bacteria had been incubating for about a week, and the concentration was too high to be useful. After sub-culturing the bacteria, we allowed it to incubate for approximately 24 hours.)
Finally, when we were ready to conduct our experiment, we created 8 plates of agar using bacteriological agar and triptic soy broth in the ratio of 100 mL of deionized water to 3 g of triptic soy broth to 1.5 g of bacteriological agar. In the meantime, we prepared our solutions that we would use for spreading on the agar plates.
Using micropipettes, we set up four beakers with 990 μL of nutrient broth in each. We added 10 μL of the mouth bacteria solution to one and 10 μL of the E. coli bacteria solution to the other to perform a 1:100 dilution. We swished the beakers around to allow each solution to mix before taking 10 μL of the new 1:100 mouth bacteria solution and adding it to a fresh beaker of nutrient broth. We did the same with the E. coli bacteria solution. Thus, in the final two beakers, we had a 1:10,000 dilution of mouth bacteria solution and E. coli bacteria solution.
We pipetted 200 μL of each of the solutions into four 1.5 mL eppendorf tubes, ending up with eight eppendorf tubes with 200 μL of solution in each. We then took 200 μL of each type of mouthwash solution and added each solution to two tubes: one of mouthwash and one of E. coli. We used a total of four types of mouthwash solution: Scope Original Mint (15% alcohol), Listerine Antiseptic (26.9%), Tom’s of Maine Spearmint (alcohol-free, containing aloe vera and witch hazel), and plain deionized water as a control. We then incubated the tubes for ten minutes at 37 degrees Celsius.
After ten minutes, we removed the tubes from the incubation chamber and took 100 μL of the liquid from each tube and put it onto a plate. Using a sterile inoculating loop, we plated the liquid. The plates were labeled and turned upside-down to incubate for 24 hours. (At this point in our experiment, however, we broke the E. coli: Scope plate, so we had only the other seven plates to incubate. We plated the liquid the next day on a new plate, but this difference in methods could explain the results we obtained, which are discussed in the next section.)
The next day, we counted bacteria colonies simply based on how many small dots there were. Possible errors in counting will also be discussed further on in the paper.