Laporte Introduction Hundreds of years ago thieves would steal from grave sites and would take jewels

and valuables from the bodies of the deceased rich. Although the bodies were covered in diseases some thieves would never get sick because they used oil that had cinnamon and other spices inside and they would use this to fight the bacteria that they got on them. (Secret of thieves) Escherichia coli or E. coli for short, is normally a helpful bacteria that lives in many animals including humans digestive track and there they help break down food particles, but certain strains of this bacterium can get into the blood stream and cause many problems, this illness is usually a kind of food poisoning because uncooked cattle or poultry can be infested with these E. coli strains. Kanamycin is a powerful antibiotic that kills many harmful kinds of bacteria such as E. coli and salmonella. It was picked for the experiment because of its availability and ease of use.

Kanamycin kills bacteria by causing the bacterias ribosomes to mistranslate genes correctly and will not be able to reproduce thus stopping the growth and eventually killing the bacteria. The kanamycin was used to see how effective the cinnamon and change in temperature would be by creating a zone of inhibition. Cinnamon is a common used spice that has been used for many things for hundreds of years. Although its uses range from a cooking ingredient to a mosquito repellant (Cinnamon coughs), recently it is being recognized and tested on its antibiotic properties (New York Times); In fact, cinnamon was found extremely effective in a study done at Kansas State University where about one teaspoon of cinnamon was placed in an E. coli filled jug of apple juice and after three days killed almost all of the Escherichia coli (Science daily).

Laporte Temperature, a common variation when moving meats from where they are cut to a kitchen also affects E. coli and the way it grows, E. coli normally grows best at about 37 C

because that is the temperature of the human digestive system (BioWeb). During the experiment the temperature of its environment was changed to see a difference in how much it grows. E. coli can cause a very dangerous illness and is normally treated with a powerful antibiotic. In this experiment the antibiotic kanamycin was used in the form of small discs and placed in petri dishes inoculated with E. coli and agar mixed with cinnamon. The data would be expressed in the diameter of the zone of inhibition measured in centimeters. It was expected that a decrease in either cinnamon or temperature would cause a decrease in the antibiotics zone of inhibition. The experiment was designed to show how the cinnamon would affect the antibiotic properties of the kanamycin discs and see if cinnamon could in the future be used as a substitute for food preservatives.

Experimental Design Materials (3) Test Tube Virginia Brand Nutrient Agar (15) Petri Dishes McCormack Cinnamon Marker E. coli inoculated Petri Dish 35 C Fisher Isotemp 500 Series Incubator Stirring Magnet Water Dropper 1 ml Transfer Loop 36 C Baxter Tempcon Incubator 37 C Baxter Tempcon Incubator Distilled Water Corning Brand Stirrer/Hotplate Kanamycin Antibiotic Sleeve Humdolt Bunsen Burner Ruler 1000 ml Flask

Laporte 3 Procedure: 1. Create five Petri dishes, one of each kind of treatment; (+,+) (+,-) (-,+) (-,-) and standard. Making a Petri dish: 2. Set temperature of Corning Brand Stirrer/Hotplate to ten. 3. Pour 300 ml of tap water into 1000 ml flask. 4. Put stirring magnet into flask and set stirring speed to four. 5. Place flask onto the hotplate. 6. Measure 6 grams of Virginia Brand Nutrient Agar Figure 1.

7. Slowly pour the agar into the flask try to get as little as possible on the edges as seen in figure 1. 8. Wait about 10 minutes for agar to be mixed well. The solution will begin to look like apple juice. 9. During this time measure the cinnamon that will be used in the experiment, measure out three differ .1 grams of cinnamon and place them in separate piles. 10. After the agar has been heated it will remain hot for about 15 to 20 minutes. At this time label the petri dishes; name, treatment, date. This should look like figure 2.

Labeling and treatment:

Laporte 4 Each value of either cinnamon or the temperature it would be placed in. these are labeled according to the amounts in each. In the Petri dish of (-,+) the cinnamon is the first symbol and the temperature is the second. There will 5 different kinds of kinds of Petri dish a (+,+) (+,-) (-,+) (-,-) and standard.

There will be 15 petri dishes at the end of the experiment three of each kind.

Figure 2. Petri dish of Experiment

11. To make a petri dish with the low value of cinnamon with the agar still hot, pour .1 grams of cinnamon into the flask, replace the flask onto the hotplate now turned off. Put the stirring magnet on three to stir the cinnamon. At this point be careful, if the flask is stirred too fast the cinnamon will bubble this is not wanted as bubbles shouldnt be put in the Petri dishes. 12. After a minute of stirring remove the flask from the hotplate 13. Pour just enough of the agar into the Petri dish to fill the bottom. This Petri dish should be labeled either (-,+) or (-,-) leave the dish still for about ten minutes and until cool and solidified. 14. This will need to be done to both Petri dishes that are the low values. 15. One Petri dish of the standard value of cinnamon needs to be made, to do this add another .1 grams of cinnamon to the flask of agar. 16. Put the flask back onto the hot plate with the stirring magnet on three. 17. Wait about one minute for the cinnamon to be mixed with the agar then remove.

Laporte 5 18. Pour just enough agar to fill the bottom of the Petri dish. This dish will be labeled (S) for the standard and should be left still for ten minutes to cool. If at any point of this process there are bubbles in the flask try to keep them out of the Petri dishes as much as possible. 19. The third value of cinnamon is the (+) or .3 grams of cinnamon. Carefully add another .1 gram of cinnamon to flask. 20. Return the flask to the hotplate and return the stirring magnet to three. 21. Wait about one minute then remove flask. 22. Pour the agar into the high cinnamon value petri dishes; just enough to fill the bottom. Let cool for ten minutes. E. coli inoculation: 23. With the Petri dish inoculated with E. coli, a test tube, distilled water, and the agar Petri dishes nearby, use the water dropper to put in five ml of distilled water into a test tube. 24. Sterilizes your transfer loop by placing it under the Bunsen burner. 25. Wipe the transfer loop across the E. coli Petri dish, then place the loop into the test tube swish it around to get the E. coli into it. 26. Use the water dropper to place the water inoculated with E. coli into each Petri dish, one ml per dish. 27. Swish each dish around then open it up to pour out the excess water, try to keep the water off your hands. Take the antibiotic out of its plastic holder and push them onto opposite sides of the petri dish.

Laporte 6 28. Place the Petri dishes into their designated incubators. A (+) means put it into the Baxter Tempcon Incubator set at 37 C, a (S) means place the Petri dishes into the Baxter Tempcon Incubator set at 36 C, and a (-) means to place it into the Fisher Isotemp 500 Series at 35 C. 29. Wait 24 hours until removing Petri dishes. Gathering Data: 30. Use the ruler to measure the zone of inhibition created by the antibiotic. Measure one way for a first diameter then measure perpendicular to that direction to get 2 diameters, average the diameter for each zone of inhibiton. 31. Repeat steps 1-30 two more times for three different DOEs.

Data and Observations Data: Table 1 Data from Petri Dishes

DOE 1 nd 2 rd 3 th 4 th 5 th 6 Average
st

Diameter of Zone of Inhibition (cm) (Cinnamon, Temperature) (+,+) (+,-) (-,-) (-,+) 2.10 2.10 2.35 2.15 2.20 2.40 2.45 2.50 2.25 2.30 2.25 2.40 2.40 2.20 2.50 2.00 2.30 2.35 2.30 2.60 2.40 2.35 2.65 2.35 2.275 2.283333 2.416667 2.333333

s 2.40 2.05 2.40 2.45 2.05 2.30 2.73

As shown in Table 1, six Designs of experiments were completed, each Petri dish had two sets of data that could be collected and fifteen Petri dishes were made. The DOEs were done five at a time with one set from each group. After three days all data was collected. Table 2 Design of Experiment Values

Laporte 7 Cinnamon (gram) Standard .2 Temperature (C) Standard 36

.1

+ .3

35

+ 37

The values in Table 2, were found for specific reason; the cinnamon values were picked based on just how little cinnamon was required to have a change in the growth of E. coli .1, .2, and .3 were found because the experiment was made to find out how a little change in a persons life, such as ingesting a minute quantity of cinnamon, could increase their health. (foods) The DOEs temperature values were found at 37 C because 37 C is the temperature that E. coli grows best in.(Estapa) and was lowered from 37 C -35 C to see the effect of growth it has. Also it is important that when the cinnamon is mixed with the agar it is stirred well, but too fast will cause it to bubble. Observations: Table 3 Observations During the Experiment
Date 3/23/2012 Observation Agar for the first two DOEs was made, when the agar was stirred too much the agar got bubbles in it, and some bubbles did get in the Petri dishes when poured. Then the Petri dishes were placed in the refrigerator for the night. Petri dishes from March 23 were inoculated with the E. coli bacterium then placed in their designated incubators. During this time a flask of Agar was set on the hot plate and heated. This flask had double the quantity of agar and water. Today two sets of Petri dishes, DOEs 3-6, were made. These dishes were then place in the refrigerator for overnight storage. The March 23rd Petri dishes were removed from their incubators and the data th collected. The March 24 set, with twice the originally plan number of Petri dishes were inoculated with E. coli, and placed in their designated incubators. This was due to a lack of sufficient time to have the extra DOE done on another day.

3/24/2012

3/25/2012

Table 3 shows the process of how the experiment was run as well as any changes in the procedures, each day the daily activities were recorded the collection of the trial data took three days.

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(+,+)

Figure 3. Petri Dishes of DOE one and two

Figure 3 shows the Petri dishes of DOE 1 and 2, the zone of inhibition can be seen here around each white disc of the antibiotic Kanamycin the (+,-) dish contains the highest amounts of cinnamon, (.3 g) and was exposed to the lowest temperature, (35 C). The dish in the center, S, was the Standard group and had the middle quantity of cinnamon, (.2 g) and was expose to the middle temperature, (36 C). As seen in table 2 each dishes symbols corresponded a temperature and quantity of cinnamon. Each had a unique exposure to either temperature or cinnamon levels. Using a marker a Petri dishes zone of inhibition, (one of two) was circled and the distance across one of the circles was measure with a ruler in centimeters in perpendicular directions and averaged. This gave twice the amount of data per dish because of two zones. After one dish was marked labeled and the data placed in the table it was placed aside to be discarded later. Each dish was marked in this way. After that days dishes data was collected the Petri dishes were placed in the garbage can be thrown away.

Laporte 9 Data Analysis and Interpretation Data Analysis: Table 4 Values Used in Experiment Cinnamon (gram) Standard .1 .2

+ .3

35

Temperature (C) Standard 36

+ 37

Table 4 describes the quantities and temperature used in the experiment. The symbols were used to mark the Petri dishes and show the values in the experiment. Table 5 Data Collection
DOE 1st (+,+) 2.10 2.20 2.25 2.30 2.40 2.40 (+,-) 2.10 2.40 2.30 2.20 2.35 2.35 (-,-) 2.35 2.45 2.25 2.50 2.30 2.65 (-,+) 2.15 2.50 2.40 2.00 2.60 2.35

2nd
3rd

Table 5 shows the data collect for all three designs of experiment.

Standards
3.8

Centemeters

3.4 3 2.6 2.2 1.8 1.4 1 0 1 2 3 4 5 6 7 Numbers of Trials

Figure 4. Standards Throughout Experiment As seen in figure 4 the standards indicate that there is a change in length of about .4 cm.

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Figure 5. Effect of Temperature Table 6 Effect of Cinnamon data Cinnamon (-) .1 gram (+) .3 grams 2.4166667 2.275 2.3333333 2.2833333 Avg =2.375 Avg =2.27916665 As shown in Figure 5 the effect of Cinnamon is -.09583335 cm. On average, as cinnamon increases, the diameter of the zone of inhibition decreases by -.09583335 cm.

Figure 6. Effect of Temperature Table 7 Effect of Temperature Temperature (-) 35C (+) 37C 2.4166667 2.275 2.2833333 2.3333333 Avg =2.35 Avg =2.304167 As show in Figure 6 the effect of Temperature is -.045833 cm. On average, as temperature increases, the diameter of the zone of inhibition decreases by -.045833 cm.

The Grand average was found at 2.327083333

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Table 8 Interaction effect data

2.45 2.4
2.35 2.3333

Temperature
2.416666

DOE Solid Segment Dotted Segment

(-) 35C (+) .3g (-) .1g 2.28333 333 2.41666 6

(+)37C 2.275 2.3333

2.3
2.25

2.28333333 2.275

2.2 (-) 35C Temperature (+)37C

Figure 7. Graph of Interaction Effect Using Figure 7 and Table 8 the slope of the solid line segment was subtracted with the slope of the dotted segment and the interaction effect of the cinnamon was .412502. -.00833 -.08333333= .412502 2 2

A test of significance can be done on the effect of each variable, cinnamon and temperature. -.0958333= .4 -.045833 .4 .038 .4

0.239583375 2 false. 0.11458252 false. 0.0952 false. None of the values were found significant.

Cinnamon

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Interpretation: In Figure 2 the graph describes the effect of the cinnamon on the diameter of the zone of inhibition. As can be see the effect, -.09583335, was negative. This means that the cinnamon, instead of being helpful, and increasing the size of the zone of inhibition it decreased its side and increased the growth of the E. coli bacterium. In Figure 3 the graph describes the effect of temperature on the diameter of the zone of inhibition. Just as the first effect, its effect, -.045833, was negative. This means that the temperature like cinnamon when increased will decrease the diameter of the zone of inhibition. Figure 4 shows the slopes of the cinnamon and Temperatures effect. With the slopes subtracted the interaction effect was found at .038 cm. This means that by an amount of .038 cm the diameter of the zone of inhibition was increased. This is due to the combined effect of the cinnamon and temperature treatment. In Figure 1 the standards had a range of .4 cm. This means that all the standard experiments were very close to each other in length. The range of standards helped with the test of significant effects and after being completed it was found that the experiment and all of the values meant to affect the growth around the zone of inhibition were ineffective.

Conclusion The data collected from the experiment drove the researcher to reject the hypothesis that an increase in the temperature or quantity of cinnamon would cause an increase in size of the zone of inhibition. The experiment was to test how cinnamon and

Laporte 13

temperature would help with the antibiotic effects of the antibiotic kanamycin. The experiment was a two factor DOE where the researcher placed 15 Petri dishes of E. coli with three different levels of cinnamon into three different incubators for one day to find the diameters of the zone of inhibition on each Petri dish. The results showed that the increase of cinnamon had a negative effect of the zone of inhibition and caused the zone to decrease in size. The increase in the temperature also had a negative effect causing the zone of inhibition to decrease. The effect of the cinnamon is because of the cinnamon being insoluble in liquid, and could have not been dispersed properly to be effective. The temperature could have been ineffective because E. coli could grow better at a temperature slightly higher than 37 C rather than slightly lower. There were some design flaws in how the experiment was made. If the experiment was to be redone it would be better for the researcher that the E. coli was suspended in a free liquid such as water and the cinnamon placed in as cinnamon sticks. This would solve the issue that was keeping the cinnamon from dispersing throughout the liquid. During the experiment due to lack of time two tests were run on the same day. This should be prevented by giving the experiment plenty of time for the tests to be run, the tests should take three days but give the test more time in case of errors. Further research should be done on the effect of cinnamon on E. coli, this research can help further the knowledge of the antibiotic properties of spices in how they work and how they kill or inhibited some parts of the bacteria to be effective, and further studies can lead to not needing artificial preservatives added to our foods. It would be extremely beneficial to use natural preservatives that do not require man-made chemicals

Laporte 14

and can be add to foods without the chance of any harmful effects. Although modern food preservatives are heavily studied and strictly regulated natural preservatives that are not made in a lab could provide many advantages. They would be less expensive to add to foods as they do not have to be manufactured chemically, and they would be more easily accepted because they would be recognizable spices and herbs that have been used for centuries. Throughout the experiment the researcher learned how E. coli grows and reproduces as well as how it is killed by antibiotics and preservatives, and how temperature and cinnamon interact with the growth of this bacterium.

Works Cited "ANTIMICROBIAL EFFECTS OF SPICES AND HERBS." ANTIMICROBIAL EFFECTS OF SPICES AND HERBS. Web. <http://www.hi-tm.com/Documents/Spices.html>. "Cinnamon, Ground." The Worlds Healthiest Foods. George Mateljan Foundation. Web. <http://www.whfoods.com/genpage.php?tname=foodspice>. "Cinnamon Is Lethal Weapon Against E. Coli O157:H7." ScienceDaily. ScienceDaily, 06 Aug. 1999. Web. <http://www.sciencedaily.com/releases/1999/08/990806074926.htm>. "Escherichia Coli." Escherichia Coli. Web. <http://bioweb.uwlax.edu/bio203/s2008/moder_just/reproduction.htm>. "KidsHealth." E. Coli. Web. <http://kidshealth.org/kid/stay_healthy/food/ecoli.html>. O'connor, Anahad. "REALLY?; THE CLAIM: Cinnamon Oil Kills Bacteria." The New York Times. The New York Times, 08 Sept. 2009. Web. <http://www.nytimes.com/2009/09/08/health/08real.html>.

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Pilewski, Victor Pilewski. "Why We Use Spices." Why We Use Spices. 12 Dec. 2001. Web. <http://webpub.allegheny.edu/employee/r/rmumme/FS101/ResearchPapers/VictorPilews ki.html>. "Thieves Oil Blend - Arm Yourself with the Power of Thieves." Home of THIEVES ESSENTIAL OIL and Products Based on Thieves Oil. Web. <http://www.secretofthieves.com/>. Thorp, Susanna. "Preserving the Living and the Dead." Spices:. Web. <http://www.newag.info/02-1/focuson/focuson8.html>.