with all the pictures, the file is taking FOREVER to attach to the e-mail and i am giving up. you can just take my word for it that the pictures are there and the data table is formatted. sarah - should i give this to you in bio class tomorrow via a flash drive? will we have time to do some editing?
Counting Methods:
Individual colonies on the plates were counted as “one,” regardless of size. There were visibly more colonies on the Mouth: Tom’s plate, but the colonies on the Mouth: Water plate each had a larger area (see Figures 1 and 7). Thus, the net area covered by the colonies was about the same, though numerically Mouth Tom’s had more colonies.
When the bacteria did not form colonies, but instead covered dense areas of the plate, the diameter of the area covered was measured in centimeters. This method was used for the E. Coli: Tom’s plate (see Figure 2) and the E. Coli: Water plate (see Figure 4). It is important to note that this method does not take into account a dense film of colonies outside the area measured.
The E. Coli: Scope plate did not grow anything. The method used for creating this plate was slightly different from those used creating the other plates. The Scope was left in with the incubating E. Coli bacteria for approximately 24 hours. The other plates had their respective mouthwashes left in with the incubating bacteria for only ten minutes. Thus, the lack of bacteria on the E. Coli Scope plate could either be because the Scope killed all the bacteria or because the experiment failed. Either way, the results cannot accurately be compared with the other trials.
Mouth E. Coli
Water 330 colonies 4.65 cm diameter
Tom's 721 colonies 3.3 cm diameter
Listerine 2 colonies 4 colonies
Scope 45 colonies 0 colonies
Table 1. Results observed on plates
Figure 1. Mouth: Water plate
Figure 2. E. Coli: Water plate. Approximate circle whose diameter was measured for our results shown in red circle.
Figure 3. Mouth: Scope plate
Figure 4. E. Coli: Tom’s plate
Approximate circle whose diameter was
measured for our results shown in red circle.
Figure 5. Mouth: Listerine plate
Figure 6. E. Coli: Listerine plate
Figure 7. Mouth: Tom’s plate
Figure 8. E. Coli: Scope plate
Sunday, June 3, 2007
Discussion Section
Hey guys, the discussion is 2 and 1/3 pages long 1.5 spaced and 3 pages when double spaced. I'm still working on the future research part but it shouldn't take long. Also Lisa's is the first part and mine begins with "While our hypothesis tested to be correct..."
Our hypothesis of the efficacy of three mouthwashes on both oral and E. coli bacteria was supported by our results. As shown in Table 1 (don't know if it'll actually be table 1), Listerine Antiseptic mouthwash was the most effective at killing both the oral flora colonies and the E. coli colonies. Listerine showed only 4 colonies on the oral bacteria plate and 2 colonies on the E. coli plate, both results much lower than Scope, Tom's and water. The next most effective mouthwash was Scope. Scope showed 45 colonies on the oral plate. Tom's appeared to have only a minor influence on the growth of either bacteria and had results similar to those of water for both bacterial colonies.
As stated in the introduction, Listerine contains thymol, a chemical with strong antiseptic properties, as its active ingredient. It also contains ethanol, sodium benzoate, and benzoic acid, all of which have antibacterial properties. Listerine's superior efficacy is most likely a result of its high percentage of ethanol (26.9%). Like Listerine, Scope contains both sodium benzoate and benzoic acid as well as the additional antibacterial cetylpyridinium chloride. It does not contain thymol (the active ingredient in Listerine) and its ethanol content is much lower—only 15%. Because of this, it is less effective at killing bacteria. Despite being less effective at killing bacteria, Scope may in fact be more effective than Listerine at eliminating bad breath. The inclusion of cetylpyridinium chloride (CPC) in Scope specifically targets the sulfur compounds in one's mouth that produce especially malodorous breath[1].
Tom's contains no proven antiseptics or antibacterials. Its active ingredients, witch hazel and ascorbic acid, both have astringent properties which could make it harder for bacteria to survive. This explains why the E. coli colony for Tom's is slightly smaller than that of water—3.3cm in Tom's to 4.65cm in water. Tom's is also alcohol-free, which is good in terms of oral comfort, but ineffective in the sense that bacteria aren't actually being eliminated.
The alcohol in both Scope and Listerine may also be promoting halitosis. Alcohol is a desiccant—it dries out the mouth, eliminating the natural antibacterials that humans produce in their saliva. Without this natural aid in eliminating bad breath, the compounds that are not eliminated by the antibacterials in the mouthwash are left to grow unmasked by the mouth's saliva, leading to bad breath[2]. (this is not an incredibly reliable source, but i thought the point they make is interesting)
While our hypothesis tested to be correct, our lab was not without experimental errors. As outlined in the methods and materials section, we used sterile technique as well as sterile equipment such as cotton swabs and inoculating loops throughout the experiment; however, the use of the word “sterile” is only nominal. For example [put example here]. Another error in experiment to identify is the fact that the liquids were not spread evenly along each plate or equally in comparison to other plates. This existing inconsistency may have resulted in more or less coverage of bacteria killed in some plates. We also plated the liquids one after another and did not keep track of the amount of time that each soaked for. Having this initial imbalance of different soaking times could have caused some plates to have had more exposure to their respective liquids, therefore producing better results. Not only did the soaking times differ, but the incubating times as well. As stated earlier in the paper, before we began plating, two tubes were incubated longer than the third. Another part to note is that because we broke the E.coli: Scope plate, we had to re-plate the E.coli: Scope and incubate it independently from the other plates. The incubation times differed again at this point in the experiment and created more discrepancies.
Once results were produced, we collected the data by counting the colonies on the bacteria plates. We did this by marking the plates above the colonies with whiteboard markers. We split the task amongst the group instead of having a consistent counter. This may be classified as another error because one group member could have had a different idea as to how big each colony was, thus allowing room for deviation. Also, the counting of the colonies was not done in the most accurate fashion; another experimental error to add to the list. In addition to all the errors above, our group did not have enough time to do more trials. We were not able to produce realistic results (for example at one point our water had a zone of inhibition) until our last test, which we only had enough time to create one generation for. Therefore our conclusions are heavily dependent on our one successful experiment.
Having had the experience of executing a number of unsuccessful tests and trials, our group would do a few things differently if we were to run the experiment again. Recurrent errors that were identified relate to the lack of consistency such as the uneven plating of the liquids, the inaccuracy of soaking and incubation times, and the potential various methods used to count the colonies. It is imperative that every aspect of the experiment is done in the same vein. We would need to create a system to make sure that each plate received even amounts of their respective liquids and were spread evenly, record exact times for soaking and incubation to have homogenous numbers and resulting data, and lastly form a technique allowing for uniformity in the counting of colonies (or perhaps have one person perform the task).
[1] http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-adv.htm&r=1&f=G&l=50&d=PTXT&S1=%226723305%22&OS=
[2] http://www.therabreath.com.au/mouthwash.html
Our hypothesis of the efficacy of three mouthwashes on both oral and E. coli bacteria was supported by our results. As shown in Table 1 (don't know if it'll actually be table 1), Listerine Antiseptic mouthwash was the most effective at killing both the oral flora colonies and the E. coli colonies. Listerine showed only 4 colonies on the oral bacteria plate and 2 colonies on the E. coli plate, both results much lower than Scope, Tom's and water. The next most effective mouthwash was Scope. Scope showed 45 colonies on the oral plate. Tom's appeared to have only a minor influence on the growth of either bacteria and had results similar to those of water for both bacterial colonies.
As stated in the introduction, Listerine contains thymol, a chemical with strong antiseptic properties, as its active ingredient. It also contains ethanol, sodium benzoate, and benzoic acid, all of which have antibacterial properties. Listerine's superior efficacy is most likely a result of its high percentage of ethanol (26.9%). Like Listerine, Scope contains both sodium benzoate and benzoic acid as well as the additional antibacterial cetylpyridinium chloride. It does not contain thymol (the active ingredient in Listerine) and its ethanol content is much lower—only 15%. Because of this, it is less effective at killing bacteria. Despite being less effective at killing bacteria, Scope may in fact be more effective than Listerine at eliminating bad breath. The inclusion of cetylpyridinium chloride (CPC) in Scope specifically targets the sulfur compounds in one's mouth that produce especially malodorous breath[1].
Tom's contains no proven antiseptics or antibacterials. Its active ingredients, witch hazel and ascorbic acid, both have astringent properties which could make it harder for bacteria to survive. This explains why the E. coli colony for Tom's is slightly smaller than that of water—3.3cm in Tom's to 4.65cm in water. Tom's is also alcohol-free, which is good in terms of oral comfort, but ineffective in the sense that bacteria aren't actually being eliminated.
The alcohol in both Scope and Listerine may also be promoting halitosis. Alcohol is a desiccant—it dries out the mouth, eliminating the natural antibacterials that humans produce in their saliva. Without this natural aid in eliminating bad breath, the compounds that are not eliminated by the antibacterials in the mouthwash are left to grow unmasked by the mouth's saliva, leading to bad breath[2]. (this is not an incredibly reliable source, but i thought the point they make is interesting)
While our hypothesis tested to be correct, our lab was not without experimental errors. As outlined in the methods and materials section, we used sterile technique as well as sterile equipment such as cotton swabs and inoculating loops throughout the experiment; however, the use of the word “sterile” is only nominal. For example [put example here]. Another error in experiment to identify is the fact that the liquids were not spread evenly along each plate or equally in comparison to other plates. This existing inconsistency may have resulted in more or less coverage of bacteria killed in some plates. We also plated the liquids one after another and did not keep track of the amount of time that each soaked for. Having this initial imbalance of different soaking times could have caused some plates to have had more exposure to their respective liquids, therefore producing better results. Not only did the soaking times differ, but the incubating times as well. As stated earlier in the paper, before we began plating, two tubes were incubated longer than the third. Another part to note is that because we broke the E.coli: Scope plate, we had to re-plate the E.coli: Scope and incubate it independently from the other plates. The incubation times differed again at this point in the experiment and created more discrepancies.
Once results were produced, we collected the data by counting the colonies on the bacteria plates. We did this by marking the plates above the colonies with whiteboard markers. We split the task amongst the group instead of having a consistent counter. This may be classified as another error because one group member could have had a different idea as to how big each colony was, thus allowing room for deviation. Also, the counting of the colonies was not done in the most accurate fashion; another experimental error to add to the list. In addition to all the errors above, our group did not have enough time to do more trials. We were not able to produce realistic results (for example at one point our water had a zone of inhibition) until our last test, which we only had enough time to create one generation for. Therefore our conclusions are heavily dependent on our one successful experiment.
Having had the experience of executing a number of unsuccessful tests and trials, our group would do a few things differently if we were to run the experiment again. Recurrent errors that were identified relate to the lack of consistency such as the uneven plating of the liquids, the inaccuracy of soaking and incubation times, and the potential various methods used to count the colonies. It is imperative that every aspect of the experiment is done in the same vein. We would need to create a system to make sure that each plate received even amounts of their respective liquids and were spread evenly, record exact times for soaking and incubation to have homogenous numbers and resulting data, and lastly form a technique allowing for uniformity in the counting of colonies (or perhaps have one person perform the task).
[1] http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-adv.htm&r=1&f=G&l=50&d=PTXT&S1=%226723305%22&OS=
[2] http://www.therabreath.com.au/mouthwash.html
Saturday, June 2, 2007
Introduction
Hi guys,
Here's the introduction. It is two and a quarter pages including the heading and footnotes. I lost some formatting when I pasted it in here.
I tried to mention our previous triclosan lab, since that is one of the requirements. Also, I referenced two scientific papers (three are required).
Here is a description of what an introduction should include:
"The purpose of this section is to state the objective of your study. You will present relevant background information in order to familiarize the reader with your research topic. For example, background information on how enzymes catalyze reactions, and why they are important in organisms, would be appropriate in your enzyme report. Your hypothesis should be clearly stated here, along with a well supported, scientifically valid rationale for the hypothesis. Your hypothesis should be no more than one or two sentences. Your hypothesis can be followed by a set of predictions based on your hypothesis. Finally, discuss the variables you will measure in order to test your hypothesis."
Yay! please edit!
Sarah
Sarah, Beatrice, Deborah, Lisa, Victoria
Advanced Biology Period 1
May 19, 2007
Effectiveness of Mouthwashes in the Inhibition of the Growth of E. coli and Oral Flora
After inducing resistance to triclosan in E. coli, our group was interested in testing the efficacy of a product with similar anti-bacterial properties. We seized on mouthwash as a common household item whose effectiveness as an anti-bacterial agent is very important to consumers.
The primary purpose of most mouthwashes is to kill oral bacteria in order to prevent bad breath and/or fight plaque and gingivitis[1]. The active ingredients in mouthwashes vary, from thymol[2] (a plant-derived antiseptic[3]) and sodium benzoate[4] to witch hazel and vitamin C[5]. In order to conduct the most informative assay of the effectiveness of mouthwash, we chose to compare several brands which vary in both their advertising claims and their active ingredients. The products chosen were Listerine Antiseptic Original, Scope Original Mint, and Tom’s of Maine Natural Cleansing Mouthwash in Spearmint. The active ingredients of Listerine Original include thymol, methyl salicylate, menthol, and eucalyptol. Their stated purpose is fighting plaque and gingivitis. Thymol is an antiseptic while menthol[6], eucalyptol[7], and methyl salicylate[8] (oil of wintergreen) are flavoring agents. Listerine also contains denatured alcohol (26.9%) “as a solubilizer for flavors and active ingredients”[9], benzoic acid, poloxamer 407, sodium benzoate, and caramel[10]. The company claims that Listerine Original “kills germs that cause plaque, gingivitis and bad breath” and “provides 24-hour protection against plaque and gingivitis germs when used morning and night”10.
Tom’s of Maine contains witch hazel and ascorbic acid (Vitamin C) for an “astringent feel”, aloe vera for a “soothing feel”; and glycerin, spearmint oil, and menthol for flavor5. Poloxamer 335 is used for dispersion of flavorings, and water is included to improve the consistency of the product. Advertising for this mouthwash emphasizes its lack of alcohol5.
The ingredients of Scope Original Mint include water, alcohol (15 WT%), glycerin, polysorbate 80, flavor, sodium saccharin, sodium benzoate, cetylpyridinium chloride, benzoic acid, blue 1, and yellow 54. Scope is classified by its parent company as “a cosmetic mouthwash” designed to kill the bacteria that cause bad breath[11].
We hypothesized that Listerine would be most effective in preventing the growth of bacteria, that Scope would have some preventative effect, but not as great as that of Listerine, and that Tom’s of Maine would not prevent bacterial growth. Our rationale for this hypothesis was that if Listerine kills 99.9% of bacteria on contact, as the company claims, it will prevent the growth of bacteria on a plate. Also, independent clinical studies have shown that Listerine does significantly reduce plaque and gingivitis after six months of use[12]. We concluded that Listerine would work better than Scope in preventing bacterial growth because Scope’s advertising does not claim to kill 99.9% of bacteria. In addition, Listerine has the American Dental Association (ADA) seal of approval for preventing plaque and gingivitis[13]. Scope does not have this accreditation[14]. However, because Scope does contain anti-bacterial agents, we decided that it should have some ability to prevent the growth of bacteria. Conversely, the lack of an antimicrobial ingredient in Tom’s of Maine’s led us to hypothesize that it would not inhibit bacterial growth.
A variable used to test this hypothesis included the concentration of bacteria used on the plates to which the mouthwashes were applied (1:10, 1:100, 1:1000, and 1:10,000). A second variable was the testing of the mouthwashes’ effectiveness against both E. coli and an oral culture. We hypothesized that the mouthwashes would be more effective against oral bacteria than E.coli because oral bacteria are the organisms they were designed to kill and there should be little if any E. coli in the mouth.
[1] “Oral Health Care Products: Mouthrinses”
[2] http://www.pfizerch.com/product.aspx?id=428
[3] Shapiro and Guggenheim
[4] http://www.pg.com/content/pdf/01_about_pg/msds/health_care/oral_care/Scope_Liquids.pdf
[5] http://www.tomsofmaine.com/toms/product.asp?dept%5Fid=350&pf%5Fid=MW
[6] http://en.wikipedia.org/wiki/Menthol
[7] http://en.wikipedia.org/wiki/Eucalyptol
[8] http://www.chemistrystore.com/methyl_salicylate.htm
[9] http://www.pfizerch.com/faq.aspx?brand=298#FAQ935
[10] Listerine.com
[11]http://pg.custhelp.com/cgi-bin/pg.cfg/php/enduser/std_adp.php?p_faqid=5592&p_created=1114095268&p_sid=dV3e5gCi&p_accessibility=0&p_lva=&p_sp=cF9zcmNoPTEmcF9zb3J0X2J5PSZwX2dyaWRzb3J0PSZwX3Jvd19jbnQ9NTgmcF9wcm9kcz00JnBfY2F0cz0wJnBfcHY9MS40JnBfY3Y9JnBfcGFnZT01&p_li=&p_topview=1
[12]http://jada.ada.org/cgi/content/full/132/5/670?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=1&andorexacttitle=and&andorexacttitleabs=and&fulltext=listerine&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT
[13] http://www.ada.org/ada/seal/pro-com1.asp?company=Johnson%20%5e%20Johnson%20Consumer%20%5e%20Personal%20Products%20Worldwide
[14] http://www.ada.org/ada/seal/sealsrch.asp
Here's the introduction. It is two and a quarter pages including the heading and footnotes. I lost some formatting when I pasted it in here.
I tried to mention our previous triclosan lab, since that is one of the requirements. Also, I referenced two scientific papers (three are required).
Here is a description of what an introduction should include:
"The purpose of this section is to state the objective of your study. You will present relevant background information in order to familiarize the reader with your research topic. For example, background information on how enzymes catalyze reactions, and why they are important in organisms, would be appropriate in your enzyme report. Your hypothesis should be clearly stated here, along with a well supported, scientifically valid rationale for the hypothesis. Your hypothesis should be no more than one or two sentences. Your hypothesis can be followed by a set of predictions based on your hypothesis. Finally, discuss the variables you will measure in order to test your hypothesis."
Yay! please edit!
Sarah
Sarah, Beatrice, Deborah, Lisa, Victoria
Advanced Biology Period 1
May 19, 2007
Effectiveness of Mouthwashes in the Inhibition of the Growth of E. coli and Oral Flora
After inducing resistance to triclosan in E. coli, our group was interested in testing the efficacy of a product with similar anti-bacterial properties. We seized on mouthwash as a common household item whose effectiveness as an anti-bacterial agent is very important to consumers.
The primary purpose of most mouthwashes is to kill oral bacteria in order to prevent bad breath and/or fight plaque and gingivitis[1]. The active ingredients in mouthwashes vary, from thymol[2] (a plant-derived antiseptic[3]) and sodium benzoate[4] to witch hazel and vitamin C[5]. In order to conduct the most informative assay of the effectiveness of mouthwash, we chose to compare several brands which vary in both their advertising claims and their active ingredients. The products chosen were Listerine Antiseptic Original, Scope Original Mint, and Tom’s of Maine Natural Cleansing Mouthwash in Spearmint. The active ingredients of Listerine Original include thymol, methyl salicylate, menthol, and eucalyptol. Their stated purpose is fighting plaque and gingivitis. Thymol is an antiseptic while menthol[6], eucalyptol[7], and methyl salicylate[8] (oil of wintergreen) are flavoring agents. Listerine also contains denatured alcohol (26.9%) “as a solubilizer for flavors and active ingredients”[9], benzoic acid, poloxamer 407, sodium benzoate, and caramel[10]. The company claims that Listerine Original “kills germs that cause plaque, gingivitis and bad breath” and “provides 24-hour protection against plaque and gingivitis germs when used morning and night”10.
Tom’s of Maine contains witch hazel and ascorbic acid (Vitamin C) for an “astringent feel”, aloe vera for a “soothing feel”; and glycerin, spearmint oil, and menthol for flavor5. Poloxamer 335 is used for dispersion of flavorings, and water is included to improve the consistency of the product. Advertising for this mouthwash emphasizes its lack of alcohol5.
The ingredients of Scope Original Mint include water, alcohol (15 WT%), glycerin, polysorbate 80, flavor, sodium saccharin, sodium benzoate, cetylpyridinium chloride, benzoic acid, blue 1, and yellow 54. Scope is classified by its parent company as “a cosmetic mouthwash” designed to kill the bacteria that cause bad breath[11].
We hypothesized that Listerine would be most effective in preventing the growth of bacteria, that Scope would have some preventative effect, but not as great as that of Listerine, and that Tom’s of Maine would not prevent bacterial growth. Our rationale for this hypothesis was that if Listerine kills 99.9% of bacteria on contact, as the company claims, it will prevent the growth of bacteria on a plate. Also, independent clinical studies have shown that Listerine does significantly reduce plaque and gingivitis after six months of use[12]. We concluded that Listerine would work better than Scope in preventing bacterial growth because Scope’s advertising does not claim to kill 99.9% of bacteria. In addition, Listerine has the American Dental Association (ADA) seal of approval for preventing plaque and gingivitis[13]. Scope does not have this accreditation[14]. However, because Scope does contain anti-bacterial agents, we decided that it should have some ability to prevent the growth of bacteria. Conversely, the lack of an antimicrobial ingredient in Tom’s of Maine’s led us to hypothesize that it would not inhibit bacterial growth.
A variable used to test this hypothesis included the concentration of bacteria used on the plates to which the mouthwashes were applied (1:10, 1:100, 1:1000, and 1:10,000). A second variable was the testing of the mouthwashes’ effectiveness against both E. coli and an oral culture. We hypothesized that the mouthwashes would be more effective against oral bacteria than E.coli because oral bacteria are the organisms they were designed to kill and there should be little if any E. coli in the mouth.
[1] “Oral Health Care Products: Mouthrinses”
[2] http://www.pfizerch.com/product.aspx?id=428
[3] Shapiro and Guggenheim
[4] http://www.pg.com/content/pdf/01_about_pg/msds/health_care/oral_care/Scope_Liquids.pdf
[5] http://www.tomsofmaine.com/toms/product.asp?dept%5Fid=350&pf%5Fid=MW
[6] http://en.wikipedia.org/wiki/Menthol
[7] http://en.wikipedia.org/wiki/Eucalyptol
[8] http://www.chemistrystore.com/methyl_salicylate.htm
[9] http://www.pfizerch.com/faq.aspx?brand=298#FAQ935
[10] Listerine.com
[11]http://pg.custhelp.com/cgi-bin/pg.cfg/php/enduser/std_adp.php?p_faqid=5592&p_created=1114095268&p_sid=dV3e5gCi&p_accessibility=0&p_lva=&p_sp=cF9zcmNoPTEmcF9zb3J0X2J5PSZwX2dyaWRzb3J0PSZwX3Jvd19jbnQ9NTgmcF9wcm9kcz00JnBfY2F0cz0wJnBfcHY9MS40JnBfY3Y9JnBfcGFnZT01&p_li=&p_topview=1
[12]http://jada.ada.org/cgi/content/full/132/5/670?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=1&andorexacttitle=and&andorexacttitleabs=and&fulltext=listerine&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT
[13] http://www.ada.org/ada/seal/pro-com1.asp?company=Johnson%20%5e%20Johnson%20Consumer%20%5e%20Personal%20Products%20Worldwide
[14] http://www.ada.org/ada/seal/sealsrch.asp
Friday, June 1, 2007
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.
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.
From Beatrice's e-mail
2) Due dates/times are as follows:
- Sunday noon: post or email your section of the lab report
- Sunday 4 pm: Send edits of the section back to the person who wrote it
- Sunday 6 pm: Final version of your section due - send to Sarah
- Sunday 8 pm: Send powerpoint presentation slides to Beatrice
If you're not home on Sunday afternoon or whatever....send us an email or something and try to send out your section before hand...
(this is mostly for my benefit, so I can go to one website instead of fishing through my messy inbox)
- Sunday noon: post or email your section of the lab report
- Sunday 4 pm: Send edits of the section back to the person who wrote it
- Sunday 6 pm: Final version of your section due - send to Sarah
- Sunday 8 pm: Send powerpoint presentation slides to Beatrice
If you're not home on Sunday afternoon or whatever....send us an email or something and try to send out your section before hand...
(this is mostly for my benefit, so I can go to one website instead of fishing through my messy inbox)
Wednesday, May 30, 2007
Possible Experimental Errors
Not sterile
Not soaking for the same amounts of time
Not incubating for the same amounts of time
Not spreading evenly
Not having enough time to do more trials
Counting - different people counted
Not THE MOST ACCURATE COUNTING EVER
Not soaking for the same amounts of time
Not incubating for the same amounts of time
Not spreading evenly
Not having enough time to do more trials
Counting - different people counted
Not THE MOST ACCURATE COUNTING EVER
Title of Our Lab Report
Hi Guys!
What do you think of this title?:
"Effectiveness of Mouthwashes in the Inhibition of the Growth of E. coli and Oral Flora"
- Sarah
What do you think of this title?:
"Effectiveness of Mouthwashes in the Inhibition of the Growth of E. coli and Oral Flora"
- Sarah
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