1. Do More Expensive Hand Sanitizers Inhibit Bacterial Proliferation Better Than Less Expensive Ones? By: Henry Microbe Grade 7

2. Question / Problem To determine which hand sanitizer will inhibit the proliferation of Escherichia coli (E. coli ) for the lowest cost.

3. Background Research A. Bacteria 1. Kingdom a. Eubacteria 2. Prokaryotic Cells 3. Methods of Classifying Bacteria a. Shape 1. Coccus - Round 2. Bacillus - Rod 3. Spirillum - Spiral b. Gram Staining 1. Gram + = purple 2. Gram - = red c. Clustering / Grouping 1. Diplo= two bacteria 2. Strepto= chain of bacteria 3. Staphylo= cluster of bacteria 4.E. coli A. Bacillus bacteria B. Gram - C. Normally found in the intestines of animals D. Diseases Caused by E. coli 1. Urinary Tract Infection 2. Diarrhea 3. Nausea 4. Possible Death

4. Hypothesis As the price of the hand sanitizer increases its ability to inhibit bacterial proliferation will increase.

5. Variables Responding / Dependent Constants 1- Pure slant of E. coli 21- Sterile Tryptic Soy Agar (TSA) plates 84 - Sterile blank discs Incubated at 37° C Incubator used Method of determining the average zone of inhibition Nuisance Minor fluctuations in temperature and humidity in the incubator Age & viability of the E. coli Irregular zones of inhibition The hand sanitizers being tested Swan, Germ-x Aloe, Purell Adv., Germ-x Foaming, Germ-x The ability of the hand sanitizers to inhibit bacterial proliferation Manipulated / Independent

6. Materials Used Pen Paper Incubator 21 Sterile Tryptic Soy Agar (TSA) Petri Dishes 84 Sterile Blank Discs Sterile Saline Water (H 2 0) Hydrogen Peroxide (H 2 0 2 ) Sterile Latex Gloves Sterile Tweezers Sterile Beakers Black Sharpie Marker.22µm Millex Millipore Filter Goggles Sterile Nutrient Broth Hand Sanitizers Tested: Swan Brand $0.37/100mL Germ-x Aloe $0.55/100mL Purell Advance $1.13/100mL Germ-x Foam $1.19/100mL Germ-x $1.33/100mL

7. Experimental Procedure Step 1 Obtain needed materials Step 3 Sterilize work area with Hydrogen Peroxide Step 4 Inoculate 50mL of sterile nutrient broth solution with E. coli & incubate for 72 hours at 37 º C Step 5 Inoculate 21 Tryptic Soy agar plates from Step 2 with 1 mL of the nutrient broth/E. coli solution prepared in step 4 Step 6 Dip a sterile disc into a sterile saline water solution (- Control) Step 7 Place the sterile disc from Step 6 in the center of one of the quadrants on one of the TSA plates Step 2 Divide 21 Tryptic Soy agar (TSA) plates into 4 equal size quadrants Step 8 Repeat steps 6-7 using 3 more test discs Step 9 Repeat steps 6-8 using 2 more TSA plates prepared in Step 2 Step 10 Repeat Steps 6 – 9 for peroxide (+ Control) and each of the hand sanitizers being tested Step 11 Incubate all Petri dishes for 72 hrs. at 37 º C Step 12 Determine and record the average zone of inhibition for each of the sterile discs dipped in the solutions being tested

8. Determining the Average Zone of Inhibition for Hydrogen Peroxide (+ Control) Sterile Disc Dipped in H ₂ O ₂ Photo: A Optional Slide

9. Which Hand Sanitizer Inhibits Bacteria the Best for the Lowest Cost? Average Zone of Inhibition (mm) Disc Tested Hydrogen Peroxide (+ Control) Sterile Saline (- Control) Swan Brand ($0.37 /100 mL) Germ X Aloe ($0.55 /100mL) Purell Adv. ($1.13 /100mL) Germ X Foaming ($1.19 /100mL) Germ X ($1.33/ 100mL) 1 7.50.05.04.06.04.06.0 2 10.50.08.04.51.03.56.0 3 10.00.03.55.013.53.55.5 4 11.00.06.54.511.03.54.5 5 10.50.08.55.07.03.54.5 6 6.00.010.05.07.54.0 7 9.00.01.0 7.03.55.0 8 10.00.08.05.04.54.05.0 9 10.50.01.04.55.53.54.5 10 10.50.04.04.56.04.05.0 11 10.00.05.55.04.53.55.0 12 9.50.06.05.59.01.06.0 Average9.580.005.584.466.883.465.50

11. Two Sample Independent T-Test Sterile Saline H 2 O (- Control ) Significance Level = 0.05 Claim: µ 1 = µ 2 Confidence Level = 95% - Control vs.P-valueResult Swan Brand ($0.37 / 100 mL) 0.0023 Hypothesis Accepted Germ X Aloe ($0.55 / 100 mL) 0.0450 Hypothesis Accepted Purell Adv. ($1.13 / 100 mL) 0.0031 Hypothesis Accepted Germ Foaming ($1.19 / 100 mL) 0.0420 Hypothesis Accepted Germ X ($1.33 / 100 mL) 0.038 Hypothesis Accepted Optional Slide

12. Two Sample Independent T-Test Hydrogen Peroxide (+ Control) Significance Level = 0.05 Claim: µ 1 = µ 2 Confidence Level = 95% + Control vs.P-valueResult Swan Brand ($0.37 / 100 mL) 0.0510 Hypothesis Rejected Germ X Aloe ($0.55 / 100 mL) 0.0400 Hypothesis Accepted Purell Adv. ($1.13 / 100 mL) 0.0148 Hypothesis Accepted Germ Foaming ($1.19 / 100 mL) 0.0550 Hypothesis Rejected Germ X ($1.33 / 100 mL) 0.0410 Hypothesis Accepted Optional Slide

13. Based on the data I collected from my experiment I concluded that the data partially supported my hypothesis. As the price of the hand sanitizer increases its ability to inhibit bacterial proliferation will increase. Hand Sanitizer Cost/100 mL Average Zone of Tested Inhibition (mm) Swan Brand $0.37 5.58 Germ-x Aloe $0.554.46 Purell Advance $1.136.88 Germ-x Foam $1.193.46 Germ-x $1.335.50 Conclusion Actual Results Hypothesis H 2 O 2 (+ Control) $0.35 9.58 Saline (- Control) $0.22 0.00

14. Conclusion Based on the Two Sample Independent T-Test that I performed on the data that I collected from my experiment I concluded that the data partially supported my hypothesis. Hypothesis As the price of the hand sanitizer increases its ability to inhibit bacterial proliferation will increase. Results Hand Sanitizer Cost/ (P Value) (Hypothesis) (P Value) (Hypothesis) Tested 100mL Swan Brand $0.37 0.0023 Accepted 0.0510 Rejected Germ-x Aloe $0.55 0.0450 Accepted 0.0400 Accepted Purell Advance $1.13 0.0031 Accepted 0.0148 Accepted Germ-x Foam $1.19 0.0420 Accepted 0.0550 Rejected Germ-x $1.33 0.0380 Accepted 0.0410 Accepted H 2 O 2 (+Control) $0.35 Saline (-Control) $0.22 Confidence Level = 95% Saline Vs Sanitizer Significance Level = 0.05 H 2 O 2 Vs Samitizer Conclusion Based on Statistical Analysis Optional Slide

15. Future Improvements / Changes Test more discs for each of the hand sanitizers being tested. (≥ 30 discs) Perform more trials (3 – 10 trials) Use a different protocol for determining the ability of hand sanitizers to inhibit bacterial proliferation. Purchase a greater number of hand sanitizers

16. Future Experiments / Extensions Design an experiment in order to determine the effect of hand sanitizers on a variety of different types of bacteria. Perform an experiment in order to determine the ability of natural herbs and spices to inhibit bacterial proliferation. Design and perform an experiment in order to determine whether bacteria will develop a resistance to various antibacterial chemicals. Perform an experiment in order to test the ability of different types of anti bacterial hand soaps to inhibit bacterial proliferation. Design and perform a similar experiment with hand soap

17. Real Life Applications The results obtained from my experiment could be used by cafeterias, restaurants, and hospitals to purchase the least expensive but most effective hand sanitizer. The results obtained from my experiment could assist my family to purchase the least expensive and most effective hand sanitizer for home use. This information could assist retailers in determining the most effective hand sanitizer to purchase for the least cost.

18. Bibliography of Sources "Clinical Infectious Diseases." Consumer Antibacterial Soaps: Effective or Just Risky? N.p., n.d. Web. 05 Dec. 2013. "On the Structure of Eukaryotic, Prokaryotic, and Viral Chromatin." On the Structure of Eukaryotic, Prokaryotic, and Viral Chromatin. N.p., n.d. Web. 05 Dec. 2013. "Zone of Inhibition." - Definition from Biology-Online.org. N.p., n.d. Web. 04 Feb. 2014. "10-2 The Antibiotic Disk Sensitivity Test." Microbiology Labs. N.p., n.d. Web. 05 Feb. 2014. "Escherichia Coli." Wikipedia. Wikimedia Foundation, 27 Jan. 2014. Web. 05 Feb. 2014. "Wash Your Hands." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 11 Dec. 2013. Web. 03 Feb. 2014.

19. Gram Staining Procedure Procedure: A. Slant Cultures 1. Prepare and heat-fix smears. 2. Prepare the smears of S. epidermidis and N. sicca on a second slide. Heat-fix. 3. Stain the slides as follows: a. Flood the crystal violet for one minute. b. Pour off excess dye and wash gently in tap water and drain the slide against a paper towel. c. Expose the smears to Gram's iodine for one minute by washing with iodine, then adding more iodine and leaving it on the smear until the minute is over. d. Wash with tap water and drain carefully. (Do not blot.) e. Wash with 95% alcohol for 30 seconds. f. Wash with tap water at the end of the 30 seconds to stop the decolorization. Drain. g. Counterstain with 0.25% safranin for 30 seconds. h. Wash, drain, blot, and examine under oil. i. Draw the cells showing morphology, grouping, and relative sizes. Color a few of the cells of each bacterial species to show the Gram reaction. j. Save these slides and the ones from parts B & C of this exercise to use at the next lab period. Optional Slide

20. Active Ingredients Hand Sanitizer Active Ingredient Percentage Swan Brand($0.37/100mL)Ethyl Alcohol 65% Germ-x Aloe($0.55/100mL)Ethyl Alcohol 62% Purell Advance($1.13/100mL) Ethyl Alcohol 70% Germ-x Foaming($1.19/100mL)Benzalkonium Chloride 0.13% Germ-x($1.33/100mL) Ethyl Alcohol 63% Optional Slide

21. Sterile Saline Water (- Control) Optional Slide Photo: B