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

2. 2 Team of Investigators Pandelee Mikroudis Mike Bohner Jason Hinkle

3. 3 Basic Information Saccharomyces cerevisiae is 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. 4 Purpose: 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 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). (De et al., 1999). Saccharomyces cerevisiae. The purpose of this experiment was to determine if cloves and chili powder inhibit the growth of Saccharomyces cerevisiae. Figure 2: Scanning electron micrograph of S. cerevisiae.

5. 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 p o t h e s i s

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

7. 7 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 37 O C. Methods Figure 3: The Streak Plate Method

8. 8 Controls Two Petri dishes possessing only nutrient agar were designated as 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 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. 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. 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. 10

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

12. 12 Results: Open Air Control Species NumberShapeMarginSurfaceColorSize Species OneRoundSmooth Light blue1/2cm Species TwoIrregularLobateSmoothOrange1cm Species 1 Species 2 Figure 6: Open air control showing microbial growth

13. 13 Results: S. Cerevisiae grown on Nutrient Agar Total area of Petri dish Total area covered by S. cerevisiae Average number of colonies found in one cm 2 Estimated number of colonies found in Petri dish  x r 2  x 3.8cm 2 45cm 2 10%55 colonies/cm 2 55 colonies x 45cm 2 = 2,475 colonies 2,475 colonies x.10 = 248 colonies Figure 7: S. cerevisiae grown on nutrient agar

14. 14 Results: S. Cerevisiae grown on Chili Powder Total area of Petri dish Total area covered by S. cerevisiae Average number of colonies found in one cm 2 Estimated number of colonies found in Petri dish  x r 2  x 3.8cm 2 45cm 2 25%87 colonies/cm 2 87 colonies x 45cm 2 = 3,915 colonies 3,951 colonies x.25 = 978 colonies Bacteria colony Note: The bacteria found growing in the chili powder was negated by the open air control Figure 8: S. cerevisiae grown on nutrient agar embedded with chili powder

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

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

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

18. 18 Further Analysis According to previous research by De et al., 1999, chili powder should have inhibited the growth of According to previous research by De et al., 1999, chili powder should have inhibited the growth of 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. 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. 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 cm 2. 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 cm 2. A more precise method needs to be used to count the total area that the fungus covered, which could significantly affect our results. A more precise method needs to be used to count the total area that the fungus covered, which could significantly affect our results.

20. 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 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. 21 References Morgan, I. G, and Brown Carter, M.E., Investigating Biology. Benjamin/Cummings Publishing Co., Inc. 2002. 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. Minakshi De, Amit Krishna De, and A. B. Banerjee. (1999). Antimicrobial Screening of Some Indian Spices. Phytotherapy Research, 13 (7), 616-618.

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