The Antibacterial effect of Caffeine on E.coli Thomas Hartnett Central Catholic High School Grade 9

Red Bull A common energy drink consumed worldwide most popular in the world Active ingredients Glucose, Sucrose, Caffeine (80mg, of Caffeine) Amino acid Taurine 28g carbs, 27g combined of glucose and sucrose Average levels of Vitamin B12 (80% of daily serving) Also a large mount of B6 (250% of daily serving)

Caffeine Molecular Formula: C8H10N4O2 Average mass: 194. 191 Da Monoisotopic mass : 194.0803383 Symmetric name: 1, 3, 7-trimethyl-3, 7-dihydro-1H-purine-2, 6-Dione

Human Interaction with Caffeine In North America, 90% of Adults consume caffeine on a daily basis It is the most addictive psychoactive drug Coffee arabica, or coffee plant is the most common source of caffeine consumption for humans Improves mental awareness Also used in order to treat; headaches, asthma, type 2 diabetes, etc. However with this extensive use, caffeine dependence is as real concern

Escherichia coli (E.coli) Model Bacteria Highly common bacteria, found in a wide variety of species as well as in nature Constantly used in scientific research A rod-shaped microbial flora, ~2 micrometers in diameter Hundreds of different species and strains Certain strains derived from E.coli can be extremely dangerous to humans among other animals. Reproduction rate: 20 min Gram negative bacteria

Purpose To determine the effects of Caffeine (Red Bull) on E.coli survivorship

Alternative Hypothesis Null Hypothesis Caffeine (Red bull) will not have a significant effect on the survivorship of E.coli Alternative Hypothesis Caffeine(Red Bull) will have a significant effect in altering the Survivorship of E.coli

Materials Red Bull Escherichia coli (DH5-alpha) Micropipettes + sterile tips Macro pipettes Spreader Bars LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO 4, 10 mM MgSO4, 1mM NaCl) Vortex Incubator (37 degrees C) Ethanol Sterile capped test tubes with sterile distilled water 0.22 Micron sterile filter

Procedure (Liquid Pulse) Bacteria(E.coli) were grown overnight in sterile LB media Samples of the overnight culture were added to fresh media in a sterile sidearm flask The cultures were then placed in incubators until a density of 50 Klett spectrophotometer units were reached Th cultures were diluted in sterile dilution fluid to a concentrations approximately 10E5 cells/ml A number of concentrations of Red Bull (0%, 0.1%, 1%, 10%)were created in test tubes contains sterile dilution fluid resulting in 9.9ml per tube 100 μL of E.coli was then added to the test tubes, resulting ion a final concentration of 10 ml. The solutions were then mixed by way of vortexing to allowed to incubate at room temperature for exactly 5 minutes 100 μL of each tube were added to the 8 LB plates as replicates The plates were incubated for 24 hours The colonies were then counted.

Concentration Chart Concentration 0% 0.1% 1% 10% E.Coli 0.1mL Variable Sterile dilution fluid 9.9mL 9.89mL 9.8mL 8.9mL Total volume 10mL

Procedures (Agar Infusion) Plates were infused with Red Bull According to the chart below Plates were allowed to incubate for 1 hours, to promote infusion 100 μL from control tube to the plates then spread plated The resulting colonies were counted visually, each colony being assumed to have arisen from one cell. High Low Sterile Dilution Fluid 0 μL 180 μL Variable 200 μL 20 μL

Statistical tests ANOVA Statistical Dunnett's Test Analysis of Variance- Multiple mean comparison, allows you to compare different groups of statistical data P-value is smaller than the Alpha Value (0.05)= variation Dunnett's Test This is a follow up stats test, which examines the individual variation between each of the concentrations compared back to the control

0% 0.1% 1% 10% P= 3.3E-10

Dunnett's Test (Liquid Pulse) Concentration T value Significance 0.1 1.89 Not Significant 1 8.20 Significant 10 8.82 T-crit= 2.3

Control Low High P- value<10E-6

Dunnett's test (Agar Infusion) Concentration T-Value Significance Low 2.37 Not significant High 9.84 Significant T-crit= 3.47

Statistical Analysis Liquid Pulse The P- value was lower than 0.05, the null is rejected Dunnett's test revealed significant variation in every group except for the 0.1% Agar Infusion The P- value was less than 0.05, the null was rejected Dunnett's test revealed that only the high concentration had a significant effect

Conclusions The null hypothesis was rejected in both the Agar infusion and in the Liquid pulse Since Caffeine is in fact antibacterial in nature, these results most likely strain form one of the numerous other active ingredients found in the Red Bull.

Possible Limitations Spread plating was not 100% synchronized, causing unnecessary stress/ contaminants to the exposed tubes of solution Survivorship was the focus growth rate not examined Only one species Only one variable tested There were only 5 different concentrations of variable tested

Extensions Steps will be taken in order to insure the synchronicity for spread platting Both Survivorship and Growth rate will be tested More of a variety of species will be tested Caffeine will be tested solely A higher number of concentrations will be tested

Resources "Escherichia Coli."MicrobeWIki.Kenyon.edu, n.d. Web. 20 Dec. 2016 "E. Coli (Eschericia coli)." CDC.gov. US Center for Disease Control and Prevention, 29 December 2013. Web. 12 December 2016 Cherchi, C., and A.Z. Gu. Effect of Bacterial Growth stage resistance to Caffeeine. Diss. Northwestern University, 2011. Web 15 December 2016

Data ( Liquid Infusion) 0% 0.1% 1% 10% 70 81 119 97 68 66 103 115 74 72 107 110 63 75 111 99 78 79 106 83 89 105 67 86 96 95 88 102

Data (Agar Infusion) 0% Low High 70 92 98 68 79 120 74 72 128 63 89 113 78 85 131 88 125 67 64 117 75 100

ANOVA Stats Liquid Exposure Agar Infusion