Combustion Products Effects on Yeast Population Growth

1. Combustion Products Effects on Yeast Population Growth Jack Leech Pittsburgh Central Catholic High School Grade 9

2. Car exhaust • Car exhaust was used because it is a very common source of gaseous pollution, and contains elements that are known to cause health problems. • The internal combustion engine gives off carbon monoxide, carbon dioxide, oxides of nitrogen, hydrocarbons, sulfur dioxide, and micro-particles.

3. Cigarette Smoke • Cigarette smoke also has harmful effects through all of the thousands of poisons it releases when burned. • The main pollutants are acetone, carbon monoxide, arsenic, benzene, butane, cyanide, nicotine, lead, formaldehyde, and many others.

4. Charcoal Smoke • Charcoal smoke was used because it is also a common fuel source. • Burned charcoal creates hydrocarbons and soot particles. In addition, the charcoal used in the experiment was soaked in kerosene, which when burned produces carbon monoxide.

5. Background Information • The smoke sources were chosen because they are known to have an effect on the environment, and now the effect on living cells would be tested.

6. Background Information (cont.) • The cells that would be tested on was a species of yeast known as Saccharomyces cerevisiae. This cell was used because it is very similar in its structure to the human cell, and so the results that would be drawn could be associated with human cells.

7. Purpose: • To find out if common combustion products have an effect on the growth of yeast colonies. • If smoke does have an effect on yeast, which specific smoke source has the greatest effect on yeast growth; car exhaust, cigarette smoke, or charcoal smoke.

8. Hypothesis • Null- the smoke sources will have no significant effect on yeast survivorship. • Alternative- based on the excessive amounts of toxins, smoke will have a significant negative effect in the destruction of yeast populations.

9. 48 YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose (dextrose)) Sterile capped test tube with Sterile Dilution Fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Cigarettes Charcoal Access to car for exhaust (car used was a 2003 Honda CR-V) Butane lighter Shoebox with a hole cut in the side (with plates in box, attach to exhaust pipe) Micropipette Permanent marker Aluminum foil Plate spreader Ethanol (used to sterilize plate spreader) Materials

10. Procedure (Indirect exposure) • S.c. Yeast was grown overnight in sterile YEPD Media. • A sample of the overnight culture was added to fresh media in a sterile sidearm flask. • The culture was placed in a shaking water bath (30 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/ml. • An aluminum foil chimney was created and placed over the smoking sources for intervals of 1 minute and 2 minutes. • After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates • The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/ml. • The plates were incubated at 30 C for 48 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

11. Procedure (Direct exposure) • S.c. Yeast was grown overnight in sterile YEPD Media. • A sample of the overnight culture was added to fresh YEPD media in a sterile sidearm flask. • The culture was placed in a shaking water bath (30 C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107cells/ml. • The cell culture was diluted in sterile dilution fluid to a concentration of approximately 103cells/ml. • After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on 24 plates • An aluminum foil chimney was created and placed over smoking sources for 1 minute and 2 minutes. The plates were incubated at 30 C for 48 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

12. Indirect Exposure P=0.2 P=0.02 .06 .04 .24 .003 .09 .2 1 Minute Exposure 2 Minute Exposure Time Exposed to Smoke Sources

13. Direct Exposures P=0.0003 P=2.6*10-5 .006 .0006 .002 .0005 .003 .0001 1 Minute Exposure 2 Minute Exposure Time Exposed to Smoke Sources

14. Results Smoke source Car exhaust P-value (.24) > than .05 (Insufficient data to have effect on control) Indirect 1 Min. exposure Charcoal Smoke P-value (.04) < than .05 (Significant effect on control) Cigarette Smoke P-value (.003) < than .05 (Significant effect on control) P-value (.09) > than .05 (Insufficient data to have effect on control) Car exhaust Indirect 2 min. exposure Charcoal Smoke P-value (.06) > than .05 (Insufficient data to have effect on control) Cigarette Smoke P-value (.2) > than .05 (Insufficient data to have effect on control)

15. Results Smoke source Car exhaust P-value (.002) < than .05 (Significant effect on control) Direct 1 Min. exposure Charcoal Smoke P-value (.0006) < than .05 (Significant effect on control) Cigarette Smoke P-value (.003) < than .05 (Significant effect on control) P-value (.0005) < than .05 (Significant effect on control) Car exhaust Direct 2 min. exposure Charcoal Smoke P-value (.006) < than .05 (Significant effect on control) Cigarette Smoke P-value (.0001) < than .05 (Significant effect on control)

16. Interpretation • After reviewing results, a trend was discovered. • The direct exposure had a much more destructing effect on the yeast. • In addition, the time exposed had little to do with the yeast destruction, but instead the exposure was what determined the survivorship.

17. Conclusions • After reviewing results, the null hypothesis was partially rejected. There was sufficient evidence to see that the direct exposure smoke did have a negative effect on the yeast cells, but the indirect was not as effective. • Based on the p-values of the smokes compared to the control, the conclusions were that cigarette smoke is the most effective smoke out of the others in destroying yeast cells. • In addition, although the indirect exposure did have an effect it was not enough to prove that the smoke was effecting the yeast cells • The most effective form of cell destruction resulted from the direct exposure.

18. Limitations • Such error could have been not exposing the yeast completely to the smoke source if smoke escaped through the chimney or if wind was a factor. • Because the plates were open, possible contamination could have been a factor. • Other reasons cold have been not vortexing the yeast before each trial, therefore pipetting a wrong concentration.

19. Extensions • Further extensions on the experiment would be to use different kinds of smoke to expose to the cells. • Also, the individual ingredients of products could be used to see which is the most effective. For example, use the separate poisons of a cigarette instead of the entire cigarette.

20. Works Cited • “Modern Day Pollutants” Robert Ingald • www.bbc.co.uk • www.epa.gov • www.sciencedirect.com • www.tobaccoscam.uscf.edu