The Effects of Cigarette and Cigar Smoke on Microflora

1. The Effects of Cigarette and Cigar Smoke on Microflora Garett Mann 9th Grade Pittsburgh Central Catholic High School

2. The purpose of this experiment is to examine the effects of cigarette and cigar smoke on human microflora, specifically E. coli (Escherichia coli).

3. Smoking Impacts • Over 1 billion people in the world smoke • Approximately 5 million people die from a tobacco-related illness every year, equating to one person every six seconds • Smoke exposure is linked to and may cause: • Lung Cancer • Mouth Cancer • Larynx Cancer • Pharynx Cancer • Esophageal Cancer • Kidney Cancer • Cervix Cancer • Liver Cancer • Bladder Cancer • Pancreatic Cancer • Stomach Cancer • Colon Cancer • Myeloid Leukemia • Microflora effects?

4. Microbial Flora • Not much is known about the association between humans and their flora. • Mutualistic, parasitic, pathogenic, and commensal • Normal Flora provide nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes • Can smoke products alter the health of the human micro biome

5. Escherichia coli (E. coli) • Large and diverse group of gram (-) bacteria • Free living, symbiont, or pathogen • Live in the intestinal tract of many mammals • Most strains are not pathogenic • Serve as a common microflora model

6. Alternative Hypothesis Cigarette and cigar smoke will significantly reduce the survivorship of E. coli. Null Hypothesis Both cigarette and cigar smoke will not significantly reduce the survivorship of E. coli.

7. Materials • One pack Cigarettes (Newport) • Two packs Cigars (Black Magic) • Lighter • Spreader bars • YEPD Agar Plates (1% Yeast Extract, 2% Peptone, 2% Glucose) • Escherichia coli C600 • SDF (Sterile Dilution Fluid)- 100mm, KH2Po4, 10mm,MgSO4, 1mm NaCl • Sterile Test Tubes • Ethanol • Burner • Sidearm Flask • Turn-table • Vortex • Incubator • Cone/Beaker • Filter Paper • Velcro • Aluminum Foil • Timer

8. Procedure 1. Bacteria (E.coli) were grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represented a cell density of approximately 108 cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 103 cells/mL. 5. 100 Microliters were spread on plates which were then labeled with appropriate trial information and exposed to smoke as follows. 6. Velcro was placed on the bottom of each agar plate. 7. Aluminum foil was placed on the bottom of smoke box and the smoke box and lid were sanitized with ethanol solution. 8. Six agar plates, that were labeled one minute cigarette, were attached to the smoke box lid, with covers on them. 9. Two cigarettes were lit, using the lighter for 45 seconds or until all of the edges were lit and orange.

9. Procedure (continued) 10. The two lit cigarettes were placed on the foil inside the smoke box. 11. The lids on the agar plates were quickly removed and the smoke box lid was then placed on to smoke box and snapped closed. 12. The timer was started for 1 minute. 13. When timer went off, the smoke box lid was removed, the agar plates were covered with their lids, and removed. 14. Once all smoke dissipated, the smoke box was sanitized, and six new agar plates were attach to smoke box lid. 15. Steps 7-13 were repeated, but the timer was set 5 minutes, then repeated again for 10 minutes. 16. The experiment was repeated with cigars being substituted for the cigarettes and steps 7-13 were repeated for 1 minute, 5 minute, and 10 minute intervals. 17. Plates were incubated at 37°C overnight and the colonies were counted.

10. Variables • The independent variable is the different types of smoke the microflora was exposed to. • The dependent variable is how many colonies of E. coli grow. • The constants: • Volume of smoke • Amount of E. coli placed on agar plates • Time intervals of smoke exposure • Incubation time • Temperature of incubator

11. T-crit=3.10 T=7.80 Rejects Null

13. Conclusion • The data supported the alternative hypothesis, cigarette and cigar smoke will have a significant effect on the survivorship of microflora. • The null hypothesis was rejected for both cigarette and cigar smoke. • Overall cigars at ten minutes had the least amount of colony growth with an average growth of 695 colonies.

14. Limitations • Small counting errors • Inconsistent burning process • Small inconsistency in synchronizing exposures in plating • Concentration of smoke unknown • Only one organism used • Limited exposure times

15. Extensions • Test and quantify additional concentrations of smoke • Test different brands of cigarettes and cigars to see if the amount of nicotine impacted the results • Test multiple types of microflora • Test other cellular heath effects such as growth rate • Wider range of exposure times • Synergistic effects?

16. Bibliography • 2015 Data Release. 3 Nov. 2017, https://www.cdc.gov/nchs/nhis/nhis_2015_data_release.htm. • American Cancer Society | Information and Resources about for Cancer: Breast, Colon, Lung, Prostate, Skin. https://www.cancer.org. Accessed 30 Dec. 2018. • Benowitz, Neal L. “Nicotine Addiction.” The New England Journal of Medicine, vol. 362, no. 24, June 2010, pp. 2295–303. PubMed Central, doi:10.1056/NEJMra0809890. • Brook, I. “The Impact of Smoking on Oral and Nasopharyngeal Bacterial Flora.” Journal of Dental Research, vol. 90, no. 6, June 2011, pp. 704–10. PubMed, doi:10.1177/0022034510391794. • Cigar Smoking Among Teenagers -- United States, Massachusetts, and New York, 1996. https://www.cdc.gov/mmwr/preview/mmwrhtml/00047667.htm. Accessed 30 Dec. 2018. • Edwards, Phil. “What Everyone Gets Wrong about the History of Cigarettes.” Vox, 18 Mar. 2015, https://www.vox.com/2015/3/18/8243707/cigarette-rolling-machines. • FastStats. 30 Aug. 2018, https://www.cdc.gov/nchs/fastats/smoking.htm. • Health Risks of Smoking Tobacco. https://www.cancer.org/cancer/cancer-causes/tobacco-and-cancer/health-risks-of-smoking-tobacco.html. Accessed 30 Dec. 2018.

17. Bibliography • Jelic, Sanja, and MD. “6 Facts About Cigar Smoking and Your Health That Might Surprise You.” Verywell Mind, https://www.verywellmind.com/facts-about-cigar-smoking-2824739. Accessed 30 Dec. 2018. • Media, Parscale. The History of Cigars - Finck Cigar Company. http://www.finckcigarcompany.com/cigar-advisor/the-history-of-cigars/. Accessed 30 Dec. 2018. • “Nicotine Dependence: Symptoms, Causes, and Treatment.” Medical News Today, https://www.medicalnewstoday.com/articles/181299.php. Accessed 26 Dec. 2018. • “Nicotine: Facts, Effects, and Addiction.” Medical News Today, https://www.medicalnewstoday.com/articles/240820.php. Accessed 26 Dec. 2018. • Medical News Today, https://www.medicalnewstoday.com/articles/240820.php. Accessed 27 Dec. 2018. • Ogba, Ofonime, et al. Effect of Tobacco Smoking on Oral Microbial Flora and the Relationship with Oral Health in Calabar, Nigeria. Vol. 6, May 2017. • Terry, Luther L., “Smoking and Tobacco Use; 50th Anniversary Surgeon General’s Report.” Smoking and Tobacco Use, 13 Aug. 2018, http://www.cdc.gov/tobacco/data_statistics/sgr/2012/.

18. Final Results

19. Pictures

20. Anova Cigar Data

21. Anova Cigarette Data

22. Dunnett’s Test