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

2. 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)

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

4. 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

5. 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

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

7. 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

8. 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

9. 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.

10. 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

11. 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

12. 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

13. 0% % % %
P= 3.3E-10

14. 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

15. Control Low High
P- value<10E-6

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

17. 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

18. 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.

19. 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

20. 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

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

22. 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

23. 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

24. ANOVA Stats
Liquid Exposure
Agar Infusion