Effects of E-vapor Juice on Microbial Flora Joe Ebbert Pittsburgh Central Catholic HS Grade 10 4th Year in PJAS

Rationale "Vaping" has recently become a extremely popular habit for young people 10% of Americans and 14% of teenagers use electronic cigarettes According to "pro-vape" people and organizations, 0mg nicotine e- cigarettes have very little detrimental effects

E-Cigarette Juice Cuttwood Unicorn Milk Inhaled via an e-cigarette, or “vape pen” Not FDA approved Contains propylene glycol and vegetable glycerine, both individually have minor effects on humans if inhaled 0 mg nicotine juice used—nicotine is not the variable in focus One of the most popular brands of vape juice

Escherichia coli Diverse and large group of bacteria Found in the intestines of most mammals Strains are mostly not pathogenic Gram (-) Widely used model of microbial flora on humans

Staphylococcus epidermidis Bacteria found on healthy human skin Gram (+) Forms 1/2-2 mm colonies after incubation in agar Widely used microbial flora in or on humans

Purpose To test the effects of 0 mg nicotine Cuttwood Unicorn Milk vapor juice on E-coli and Staph bacteria populations.

Hypotheses Null hypothesis: Cuttwood Unicorn Milk will not have a significant effect on E-coli and Staph survivorship Alternate hypothesis: Cuttwood Unicorn Milk will have a significant effect on E-coli and Staph survivorship

Materials Escherichia coli (E-coli) Staphylococcus epidermidis (Staph) Cuttwood Unicorn Milk (0mg nicotine) Micro pipettes Sterile pipette tips Spreader bars Ethanol Bunsen burner Vortex Incubator Test tubes Sterile Dilution Fluid (100mM KH2PO4, 10mM MgSO4, 1mM NaCl LB plates + media (1% tryptone, 0.5% yeast extract, 1% NaCl

Liquid Pulse Procedure Bacteria (E.coli and Staph) were grown overnight in sterile LB media. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 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 10⁷ cells/mL. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. A 10% stock solution of vapor juice and sterile water was made. The following components were added to sterile test tubes.

Table of Concentrations Liquid Pulse 0% 0.01% 0.1% 1% Juice Solution (10%) 0mL 0.01mL 0.1mL 1mL SDF 9mL 8.99mL 8.9mL 8mL E. Coli/Staph Total Volume 10mL

Liquid Pulse Procedure cont’d These solutions were then vortexed and allowed to sit at room temperature for 15 minutes. After vortexing to allow even suspension, 100 μL aliquots were removed from the tubes and spread on LB-agar plates. The plates were incubated at 37°C for 24 hours. The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

Infusion Procedure 0.2mL of e-vapor juice concentrations (0%[Zero], 1%[Low], 10%[High]) were spread on LB agar plates and incubated for 1 hour, allowing the bacteria to absorb the chemicals. 100μL aliquots of bacterial suspensions from the original control tube were spread onto the infused plates. The plates were incubated at 37°C for 24 hours. The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

E. Coli p-value=1.26x10⁻¹³ Staph p-value=1.05x10⁻¹³

Dunnett’s Tests: Liquid Pulse E. coli t-value t-critical Significance 0% to 0.01% 8.699124935 3.18 SIGNIFICANT 0% to 0.1% 12.93941885 0% to 1% 26.0100334 Staph t-value t-critical Significance 0% to 0.01% 8.268115788 3.18 SIGNIFICANT 0% to 0.1% 16.74199533 0% to 1% 18.3881278

E. Coli p-value=6.48x10⁻⁸ Staph p-value=1.96x10⁻⁸

Dunnett’s Tests: Infusion E. coli t-value t-critical Significance 0% to 1% 11.11934932 4.30 SIGNIFICANT 0% to 10% 18.50737336 Staph t-value t-critical Significance 0% to 1% 10.67291662 4.30 SIGNIFICANT 0% to 10% 21.34583325

Conclusions The null hypothesis was REJECTED for all concentrations (liquid pulse and infusion). Experiment shows that Cuttwood Unicorn Milk (0mg nicotine) electronic vapor juice does have a significant and harmful effect on Staph and E. coli.

Limitations Only survivorship tested Vitality of bacteria may have varied One exposure period Limited replicates

Extensions More assessments Different brands of electronic vapor juice More exposure times for the liquid pulse experiment More levels of nicotine

Resources http://www.wisegeek.com/what-is-vegetable-glycerin.htm http://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=240 http://www.cdc.gov/ecoli/ http://www.horizonpress.com/staph

Anova: Single Factor E-coli Liquid Pulse SUMMARY Groups Count Sum Average Variance 0% 6 3620 603.3333 212.2667 0.01% 3222 537 168.4 0.10% 3028 504.6667 202.6667 1% 2700 450 114.4 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 73873.83 3 24624.61 141.1692 1.26E-13 3.098391 Within Groups 3488.667 20 174.4333 Total 77362.5 23  

Anova: Single Factor Staph Liquid Pulse SUMMARY Groups Count Sum Average Variance 0% 6 2736 456 304.4 0.01% 2294 382.3333 175.8667 0.10% 1841 306.8333 349.3667 1% 1753 292.1667 122.9667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 102846.3 3 34282.11 143.9518 1.05E-13 3.098391 Within Groups 4763 20 238.15 Total 107609.3 23  

Anova: Single Factor E-coli Infusion SUMMARY Groups Count Sum Average Variance zero 4 4291 1072.75 2292.25 low 3397 849.25 68.91667 high 2803 700.75 62.91667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 280518 2 140259 173.5819 6.48E-08 4.256495 Within Groups 7272.25 9 808.0278 Total 287790.3 11  

Anova: Single Factor Staph Infusion SUMMARY Groups Count Sum Average Variance zero 4 3690 922.5 150.3333 low 3172 793 36.66667 high 2654 663.5 696.3333 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 134162 2 67081 227.8223 1.96E-08 4.256495 Within Groups 2650 9 294.4444 Total 136812 11