EXPERIMENT TO DETERMINE IF CHILI POWDER AND CLOVES AFFECTS THE GROWTH OF Saccharomyces cerevisiae .

1. EXPERIMENT TO DETERMINE IF CHILI POWDER AND CLOVES AFFECTS THE GROWTH OF Saccharomyces cerevisiae. Mike Bohner Pandelee Mikroudis Jason Hinkle

2. Team of Investigators Jason Hinkle Mike Bohner Pandelee Mikroudis

3. Basic Information • Saccharomyces cerevisiaeis commonly known as the baking/brewing yeast. • S. cerevisiae ferments the sugars found in flour. This gives off carbon dioxide and alcohol. As the carbon dioxide gets trapped inside the dough, it forces the dough to rise. Figure 1: Fermentation of S. cerevisiae

4. Purpose: The purpose of this experiment was to determine if cloves and chili powder inhibit the growth of Saccharomyces cerevisiae. Through background research, we have learned that evidence directly supports that the yeast, S. cerevisiae, grown in culture is inhibited by cloves (25mg/mL and 100mg/mL) and chili (25mg/mL and 100mg/mL) when incorporated into nutrient agar (De et al., 1999). Figure 2: Scanning electron micrograph of S. cerevisiae.

5. H We hypothesize that the growth of S. cerevisiae will be inhibited by the spices chili powder and clove. Herein, we replicated the work of De et al., (1999) in order to partially repeat their experiment and to analyze the antifungal affects of the spices chili powder and cloves specifically. y o h s s p t e i

6. Methods There were five Petri dishes used: • Two possessing only nutrient agar for control purposes • One possessing nutrient agar inoculated with S. cerevisiae • One possessing nutrient agar heavily concentrated with cloves and inoculated with S. cerevisiae • One possessing nutrient agar heavily concentrated with chili powder and inoculated with S. cerevisiae

7. Methods The “Streak Plate Method” Figure 3: The Streak Plate Method 1. The “Streak Plate Method” was used for inoculation of appropriate experimental plates. 2. All plates, experimental and controls, were incubated for 5 days at 37O C.

8. Controls • Two Petri dishes possessing only nutrient agar were designated as controls. • The agar control remained unopened throughout the experiment to rule out contaminated nutrient agar. • The air control was exposed to the air in order to disregard bacterial and fungal species that may have traveled as spores and contaminated the experimental plates.

9. Methods Following the incubation period, a piece of transparent graph paper was placed over the appropriate Petri dishes in order to analyze and quantitate yeast growth. Observations were made using a stereoscopic microscope. The number of fungal colonies were counted in a one square cm. area of the appropriate Petri dishes. Then the average number of colonies in one square cm. area was multiplied by the total area of the Petri dish to get the average number of colonies found in the entire Petri dish. (A= π x r2) Figure 4: Photograph showing the use of transparent graph paper to quantitate yeast growth.

10. Results

11. Results: Nutrient Agar Control Figure 5: Nutrient agar control showing no microbial growth.

12. Results: Species 2 Open Air Control Species 1 Figure 6: Open air control showing microbial growth

13. Results: S. Cerevisiae grown on Nutrient Agar Figure 7: S. cerevisiae grown on nutrient agar

14. Results: Bacteria colony S. Cerevisiae grown on Chili Powder Figure 8: S. cerevisiae grown on nutrient agar embedded with chili powder Note: The bacteria found growing in the chili powder was negated by the open air control

15. Results: S. Cerevisiae grown on Cloves Figure 9: S. cerevisiae grown on butrient agar embedded with cloves Note: No S. cerevisiae was found growing on cloves

16. Results: S. cerevisiae S. cerevisiae S. cerevisiae Nutrient Open air On nutirent on chili on cloves agar control Agar powder control Figure 10: Histogram plotting the numbers of S. cerevisiae colonies verses designated Petri dish environment.

17. Conclusion • Our hypothesis was not fully supported by the data. • While cloves was a potent inhibitor of S. cerevisiae, chili powder was not. • Our data suggests that chili powder actually enhanced the growth of S. cerevisiae.

18. Further Analysis • According to previous research by De et al., 1999 , chili powder should have inhibited the growth of S. cerevisiae at 25mg/mL and 100mg/mL. We believe that when the Petri dish of chili powder was created, most of the powder settled at the bottom of the Petri dish leaving the top with a small concentration of chili powder (<25mg/mL). If we were to repeat this experiment, we would prepare our own Petri dishes to ensure an accurate concentration of chili powder.

19. Further Analysis • The transparent graph paper method was not entirely accurate. In order to accurately calculate the number of colonies, a more precise method would is needed to count the number of colonies inside an area of a cm2. • A more precise method needs to be used to count the total area that the fungus covered, which could significantly affect our results.

20. Further Analysis • In future experimentation we will more closely examine the chemical constituents in cloves in order to understand the cellular mechanisms behind its inhibition of S. cerevisiae growth. • To do this we will employ electron microscopy and florescence microscopy techniques.

21. References • Morgan, I. G, and Brown Carter, M.E., Investigating Biology. Benjamin/Cummings Publishing Co., Inc. 2002. • Minakshi De, Amit Krishna De, and A. B. Banerjee. (1999). Antimicrobial Screening of Some Indian Spices. Phytotherapy Research, 13 (7), 616-618.

22. Producers: Jason Hinkle, Pandelee Mikroudis, Mike Bohner SPECIAL THANKS TO: Dr. McLaughlin Mazin Albert Samer Moussa