Effects of Splenda on E.coli Survivorship. Noah Stickel Central Catholic High School Grade 9 First Year of PJAS

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1 Effects of Splenda on E.coli Survivorship Noah Stickel Central Catholic High School Grade 9 First Year of PJAS

2 Microbial Flora Normal populations of eukaryotic fungi, protists, and bacteria, varies between individuals Not much is known about the association between humans and their flora Effects can be mutualistic, parasitic, pathogenic, and commensal Normal Flora provide nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes Supplements/Foods consumed by humans might have unintended effects on these important functions of the flora

3 Splenda Composed of Sucralose, Maltodextrin, and dextrose Primary Ingredient is Maltodextrin Maltodextrin is a polysaccharide, created from starch in partial hydrolysis, and is easily digested by the Human Body Most Popular Artificial Sweetener, sales exceeded $212 million in 2010

4 Table Sugar Also known as Sucrose Disaccharide, composed of hydrogen, oxygen, and carbon Sugar cane extract was sugar used

5 Escherichia coli More commonly known as E. coli Gram - bacteria Common laboratory model Widely Accessible Found in most water bodies Common mammalian endosymbiont Can cause meningitis and urinary tract infections

6 Purpose Will a common artificial sweetener have an effect on bacterial survivorship? If E. coli survivorship is altered, this could cause harm to humans, due to microbial flora imbalances.

7 Hypothesis Null Hypothesis- E. coli Survivorship WILL NOT be effected by Splenda Alternative Hypothesis: Splenda WILL significantly REDUCE survivorship of E. coli

8 Materials Ethanol (sterilization of instruments) Latex gloves E. coli DH5 alpha Micropipette Test Tubes SDF (Sterile Dilution Fluid), (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Bunsen burner Spreader bars Matches Sterile pipette tips Incubator Vortex sidearm flasks Splenda Sucrose

9 Procedure (Liquid Pulse) 1. E. coli was grown overnight in sterile LB media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was 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 culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml % stock solutions of Sucrose and Splenda were sterile filtered through a 0.22 micron syringe filter. The stocks were mixed with the appropriate amounts of sterile water to create concentrations of 1%, 0.1%, and 0.01% (and 0%).

10 Table of Concentrations 0% Carbo 0.01% Carbo 0.1% Carbo 1% Carbo Microbe 0.1 ml 0.1 ml 0.1 ml 0.1 ml SDF 9.9 ml ml 9.89 ml 9.8 ml Carbo 0 ml ml 0.01 ml 0.1 ml Total 10 ml 10 ml 10 ml 10 ml

11 Procedure µl aliquots of cell culture was then added to the solutions, yielding a final volume of 10 ml and a cell density of approximately 10 3 cells/ml. 7. The solutions were vortexed and allowed to sit at room temperature for a 15 minute incubation period. 8. After vortexing to evenly suspend the cells, 100 µl aliquots were removed from the tubes and spread on LB plates. 9. The plates were incubated at 37 C for 24 hours. 10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

12 100 ul 100 ul 10 8 cells/ml (E. coli) 10 5 cells/ml (E. coli) 10 3 cells/ml with carbo 10 2 cells

13 Agar Infusion Procedure 1. Sterilized Carbo was infused into the LB agar media by spread plating 2 different volumes: 200uL (0.1%) and 20uL (0.01%) 2. The plates were incubated for 60 minutes to promote infusion uL aliquots from the CONTROL TUBE was than added to the infused plates 4. After vortexing to evenly suspend the cells, 100 µl aliquots were removed from the solution and spread on the pre-prepared LB plates. 5. The plates were incubated at 37 C for 24 hours. 6. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

14 Effects of Sucrose on E. coli Survivorships (Liquid Pulse) Colonies % 0.01% 0.10% 1% P Value: 9.02 E [Sucrose]

15 T Values of Sucrose Sucrose T Value Interpretation 0.01% 4.6 SIGNIFICANT 0.1% 17.6 SIGNIFICANT 1% 5.6 SIGNIFICANT

16 Effects of Splenda on E. coli Survivorships (Liquid Pulse) P Value: 9.43 E-24 Colonies % 0.01% 0.10% 1% 20 0 [Splenda]

17 Splenda T Values Splenda T Value Interpretation 0.01% 17.7 SIGNIFICANT 0.1% 23.7 SIGNIFICANT 1% 33 SIGNIFICANT

18 180 Effects of Splenda on E. coli Survivorships (Agar Infusion) Colonies [Splenda] P Value: 2.19 E- 15 0% Lo (1%) Hi (10%)

19 Infusion T Values

20 Conclusion In both experiments, Splenda appeared to cause E. coli survivorship to INCREASE. Also in the Liquid Pulse experiment, the Sucrose appeared to aid E. coli survivorships, except in the 1% concentration. The NULL HYPOTHESIS is REJECTED, Splenda did effect E. coli survivorship. The ALTERNATE HYPOTHESIS is also REJECTED, Splenda appeared to aid E. coli survivorship.

21 Conclusion Splenda appeared to increase E. coli survivorship, this could be potentially harmful to humans. If a human digested large quantities of Splenda, this could cause the human to reach a microbial imbalance, which cause the human to become more prone to disease.

22 Limitations Plating was not perfectly synchronized More types of artificial sweeteners should be tested, to allow for more variance in the data. Artificial Sweetener could be tested on additional models, such as human cells LB plates do not properly represent conditions inside the human digestive system Only survivorship was tested

23 Extensions Test higher Concentrations Use Multiple Types of Artificial Sweeteners Instead of LB, a human cell culture should be used to more accurately form conclusions about effects of Splenda on the human body Shocking finding, sucrose reduced survivorship more than splenda, and at 1%, appeared to reduce survivorship REPEAT EXPERIMENT Growth curve experiment

24 Works Cited/ Acknowledgements Mr. Mark Krotec, for supplies, such as pipettes, LB plates, and E. coli CMU Biology Department for use of facilities to make plates and additional lab equipment Central Catholic High School, for use of facilities to conduct experiments

25 Acknowledgements Mr. Mark Krotec, for supplies, such as pipettes, LB plates, and E. coli CMU Biology Department for use of facilities to make plates and additional lab equipment Central Catholic High School, for use of facilities to conduct experiments

26 Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column ANOVA rce of Varia SS df MS F P-value F crit Between G E Within Gro Total Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column Column Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA ce of Varia SS df MS F P-value F crit ANOVA rce of Varia SS df MS F P-value F crit Between G E Between G E Within Gro Within Gro Total Total