By Tony Patsy, Pittsburgh Central Catholic, Grade 9 ARTIFICIAL SWEETENER EFFECTS ON MICROBIAL FLORA

By Tony Patsy, Pittsburgh Central Catholic, Grade 9 ARTIFICIAL SWEETENER EFFECTS ON MICROBIAL FLORA

PROBLEM Saccharin is consumed by many humans on a day to day basis. Overconsumption of saccharin may cause different types of cancer within the human body. However, what it does on microbial flora is unknown.

BACKGROUND ON SACCHARIN Artificial sweetener since its discovery in 1879. Manufactured by combining antralnalic acid with nitrous acid, sulfur dioxide, and ammonia. No nutritional value. Popular because it contains no calories, and does not add unwanted weight. 300 times sweeter than sugar. Derivative of coal tar. Despite this, saccharin was taken off of the carcinogenic list in 2010 by the FDA, and its safety is still debated.

BACKGROUND ON E. COLI Escherichia coli is a large and diverse group of gram (-) bacteria. Common symbiont in the digestive system of mammals. Causes several diseases if too much of it exists inside the intestines, or if it exist in the bloodstream. Standard prokaryotic model. Lab strain DH5 Alpha used as model bacterium.

PURPOSE To determine whether saccharin affects the survivorship of microbial flora, such as E. Coli.

HYPOTHESIS Null- Saccharin will have no significant effect on the survivorship of E. Coli. Alternate- Saccharin will significantly reduce survivorship on E. Coli.

MATERIALS Micropipettes Sterile pipette tips Saccharin, C7H5NO3S, (stock concentration 10%) Escherichia coli (DH5-Alpha) 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) Spreader Labeling Tape Marker Vortex Test tube rack Sidearm flask Klett Spectrophotometer Ethanol Bunsen Burners Matches Latex gloves

PROCEDURE 1. E. coli was grown overnight in sterile LB media. 2. A sample of this culture was added to LB media in a sidearm flask. 3. The culture was incubated until a density of 50 Klett spectrophotometer units was reached. This represents a density of 10^8 cells/ml. 4. The culture was then diluted in a sterile dilution fluid to a density of 10^5 cells/ml. 5. Stock of Saccharin was pipetted out of the bottle (assumed to be sterile because of an non-broken seal) and was diluted to concentrations of 0%, 0.1%, 1%, and 10% in sterile dilution fluid.

CONCENTRATION CHART Concentration 10% 1% 0.1% 0% Sterile Dilution Fluid 8.9mL 9.8mL 9.89mL 9.9mL E. Coli.1mL.1mL.1mL.1mL Saccharin Solution 1mL.1mL.01mL 0mL Total Volume 10mL 10mL 10mL 10mL

PROCEDURE (CONTINUED) 6. 0.1mL of cell culture was then added to the test tubes, yielding a final volume of 10mL and had a cell density of approximately 10^3 cells/ml/ 7. The solution was mixed by vortexing. 8. After vortexing to evenly suspend cells, 0.1 aliquots were removed from the tubes and spread on LB agar plates. 9. The plates were incubated at 37 degrees Celsius for 24 hours. 10. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

DATA CHART Survivorship Saccharin 10% Saccharin 1% Saccharin.1% Saccharin 0% Trial One 600 775 1016 1228 Trial Two 590 842 1062 1236 Trial Three 610 986 950 1196 Trial Four 604 826 945 1120 Trial Five 602 848 970 1280 Trial Six 594 841 1009 1208 Average 600 853 991.83 1211.33

Colonies EFFECTS OF SACCHARIN ON E. COLI 1400 1200 P Value: 4e-14 1000 800 600 400 Colonies 200 20 0 Saccharin 0% Saccharin Saccharin.1% 1% Percent Saccharin Saccharin 10%

Colonies EFFECTS OF SACCHARIN ON E. COLI 1400 P Value: 4e-14 1200 1000 800 600 Average 400 200 0 Saccharin 0% Saccharin.1% Saccharin 1% Saccharin 10% Percent Saccharin

DUNNETT S TEST RESULTS Results T-Value T-Critical Variation 10% Saccharin 21.2926 3.29 Significant 1% Saccharin 12.4806 3.29 Significant 0.1% Saccharin 7.6451 3.29 Significant

CONCLUSION Null Hypothesis Rejected: Saccharin did decrease survivorship of E. Coli at all concentrations.

LIMITATIONS Synchronization when spreading plates was not exact. Only one species of bacteria was tested. Some cells could have had longer or shorter exposure times. Only survivorship was tested.

EXTENSIONS More trials to create a better basis for evaluating the results. Using sucralose or aspartame instead of saccharin. Testing a gram (+) species of bacteria, such as Staphylococcus epidermidis. Prolonged exposure test using saccharin infused into the agar. Using an antibiotic resistant strain of bacteria for the test model. A growth curve could be performed.

REFERENCES 1. http://www.naturalhealthsherpa.com/sacchar in-danger-side-effects/52849 2. http://www.infoedulcorants.org/pdf/saccharin_en.pdf 3. http://www.webmd.com/a-to-z-guides/e-coliinfection-topic-overview

ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 6 3600 600 51.2 Column 2 6 5118 853 4958.4 Column 3 6 5952 992 2044.4 Column 4 6 7268 1211.333 2837.867 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1180849 3 393616.2 159.1676 4E-14 3.098391 Within Groups 49459.33 20 2472.967 Total 1230308 23

DUNNETT S TESTS Column 1 Average of Variable Average of Control Mean Squred Within Groups Replicates Resulting T-value 600 1211.333333 2472.967 6 21.29264157 Column 2 Average of Variable Average of Control Mean Squared Within Groups Replicates Resulting T-value 853 1211.333333 2472.967 6 12.48069231 Column 3 Average of Variable Average of Control Mean Squred Within Groups Replicates Resulting T-value 991.8333333 1211.333333 2472.967 6 7.645149659