Artificial Sweetener Effects on Microbial Flora Thomas Peilert Central Catholic High School
Problem Can the artificial sweetener Equal Original affect survivorship of Escherichia coli?
Microbial Flora Present in the respiratory, urinary, and digestive tracts as well as on skin Consists of mostly bacteria, few protists and fungi Most are harmless, but some can cause diseases Influences human anatomy, physiology, and overall health 10^14 bacteria are harbored within and on humans
Escherichia coli Cells are rod shaped and usually about 2 micrometers in length. Part of the human flora; found in the human colon and digestive tract. One of the most studied and widely understood types of bacteria. Reproduces rapidly Gram negative bacteria
Equal Original Sweetener Artificial Sweetener Zero Calorie Common in U. S.
Equal Original Sweetener (Ingredients) Dextrose Maltodextrin Aspartame Acesulfame Potassium
Purpose To investigate the effect of Equal Sweetener on E. coli survivorship
Null Hypothesis Equal supplementation will not significantly affect the survivorship of E. coli Alternative Hypothesis Equal supplementation will have a significant, positive affect on E. coli survivorship.
Materials LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) DH5- alpha E. coli Spreader bar Turn table Ethanol Sterile test tubes Micropipettes and tips Incubator SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl Equal Original Sweetener) Bunsen burner Hot plate Stirrer Bar Vortex
Procedure for Experiment 1. E. coli was grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/ml. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/ml. 5. A 10% Equal solution was sterile filtered. 6. The experimental variable was mixed with the appropriate amounts of SDF to create concentrations of 0%,.1%, 1%, and 10%.
Procedure for Experiment Solution ml of sterile water ml 10% Equal Solution 0% (control) 9.9 0.0 0.1 0.1% 9.89 0.01 0.1 1% 9.8 0.1 0.1 10% 8.9 1 0.1 ml of E. coli 7. 100 µl of cell culture was then added to Equal Sweetener solutions, yielding a final volume of 10 ml and a cell density of approximately 10^3 cells/ml. 8. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
Procedure for Experiment (Continued) 9. After vortexing to evenly suspend the cells, 100 µl aliquots were removed from the tubes and spread on LB-agar plates. 10. The plates were incubated at 37 C for 24 hours. 11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Effect on Survivorship 260 257 Average CFUs 195 130 65 150 178 197 P-value 0.000862 0 Control.1% 1% 10% Concentrations of Equal
Dunnett's Test E. coli T Crit.= 2.31 Concentration of Equal T-value interpretation 0.1% 1.564 Not Significant 1% 2.765 Significant 10% 5.977 Significant
Conclusions The alternative hypothesis was supported in all solutions except.1% as the 10% and 1% solutions significantly enhanced survivorship of E. coli
Limitations Limitations Plating was not perfectly synchronized Only four concentrations of Equal Only one type of exposure Only one exposure time Only survivorship was analyzed
Extensions Extensions Growth rate experiment Vary exposure times Vary type of exposure (Infusion) Wider range of concentrations Test different types of microbial flora
Works Cited Gallus, Scotti S. "Aspartame." Aspartame. American Cancer Society, n.d. Web. 24 Dec. 2015. "Ingredients." Ingredients. Merisant Company, n.d. Web. 25 Dec. 2015. Wilder, Jonah. "E. Coli." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 21 Dec. 2015. Web. 25 Dec. 2015. Mayo Clinic Staff. "Nutrition and Healthy Eating." Artificial Sweeteners and Other Sugar Substitutes. Mayo Clinic, 2 Feb. 2015. Web. 24 Dec. 2015. Pfeffer, M., S. Ziesenitz, and G. Siebert. "Acesulfame K, Cyclamate and Saccharin Inhibit the Anaerobic Fermentati." On of Glucose by Intestinal Bacteria. Springer International Publishing AG, 19 May 2015. Web. 25 Dec. 2015. Shell, Ellen Rupple. "Artificial Sweeteners May Change Our Gut Bacteria in Dangerous Ways." Scientific American. Scientific American Magazine, 14 July 2015. Web. 25 Dec. 2015. Holstein, Albert F. "Johns Hopkins: Diabetes Compares Non-caloric Sweeteners: Special Reports." Health After 50. Remedy Health Media, 2 Mar. 2014. Web. 24 Dec. 2015.
Data Control.1% 1% 10% 210 162 191 288 212 183 186 119 262 178 250 272 225 175 251 280 211 180 260 298 224 165 261 244 215 182 250 301 220 173 181 260