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EXPERIMENT TO EXPERIMENT TO DETERMINE IF CHILI DETERMINE IF CHILI

POWDER AND CLOVES POWDER AND CLOVES AFFECTS THE GROWTH AFFECTS THE GROWTH

OF OF Saccharomyces cerevisiaeSaccharomyces cerevisiae..

Mike Bohner Pandelee Mikroudis Jason Hinkle

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Team of Investigators

Pandelee Mikroudis

Mike Bohner Jason Hinkle

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Basic InformationBasic 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

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Purpose:Purpose:

Through background research, we have Through background research, we have learned that evidence directly learned that evidence directly supports that the yeast, supports that the yeast, S. cerevisiaeS. cerevisiae, , grown in culture is inhibited by cloves grown in culture is inhibited by cloves (25mg/mL and 100mg/mL) and chili (25mg/mL and 100mg/mL) and chili (25mg/mL and 100mg/mL) when (25mg/mL and 100mg/mL) when incorporated into nutrient agar incorporated into nutrient agar

(De et al., 1999). (De et al., 1999).

The purpose of this experiment was to determine if cloves and chili powder inhibit the growth of Saccharomyces cerevisiae.Saccharomyces cerevisiae.

Figure 2: Scanning electron micrograph of S. cerevisiae.

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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. yy

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Methods Methods There were five Petri dishes used: There were five Petri dishes used: Two possessing only nutrient agar for control Two possessing only nutrient agar for control

purposespurposes One possessing nutrient agar inoculated with One possessing nutrient agar inoculated with S. cerevisiae One possessing nutrient agar heavily concentrated One possessing nutrient agar heavily concentrated

with cloves and inoculated with with cloves and inoculated with S. cerevisiae One possessing nutrient agar heavily concentrated One possessing nutrient agar heavily concentrated

with chili powder and inoculated with with chili powder and inoculated with S. cerevisiae

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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.

MethodsMethods

Figure 3: The Streak Plate Method

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ControlsControls

Two Petri dishes possessing only nutrient agar were Two Petri dishes possessing only nutrient agar were designated as controls. designated as controls.

The agar control remained unopened throughout the The agar control remained unopened throughout the experiment to rule out contaminated nutrient agar. experiment to rule out contaminated nutrient agar.

The air control was exposed to the air in order to disregard The air control was exposed to the air in order to disregard bacterial and fungal species that may have traveled as spores bacterial and fungal species that may have traveled as spores and contaminated the experimental plates.and contaminated the experimental plates.

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MethodsMethodsFollowing the incubation period, a piece Following the incubation period, a piece of transparent graph paper was placed of transparent graph paper was placed over the appropriate Petri dishes in order over the appropriate Petri dishes in order to analyze and quantitate yeast growth. to analyze and quantitate yeast growth. Observations were made using a Observations were made using a stereoscopic microscope. stereoscopic microscope.

The number of fungal colonies were The number of fungal colonies were counted in a one square cm. area of the counted in a one square cm. area of the appropriate Petri dishes. Then the average appropriate Petri dishes. Then the average number of colonies in one square cm. area number of colonies in one square cm. area was multiplied by the total area of the was multiplied by the total area of the Petri dish to get the average number of Petri dish to get the average number of colonies found in the entire Petri dish. colonies found in the entire Petri dish. (A= π x r2)(A= π x r2)

Figure 4: Photograph showing the use of transparent graph paper to quantitate yeast growth.

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Figure 5: Nutrient agar control showing no microbial growth.

Results:Results:

Nutrient Agar Control

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Results:Results:

Open Air Control

Species Number Shape Margin Surface Color Size

Species One Round Smooth Smooth Light blue 1/2cm

Species Two Irregular Lobate Smooth Orange 1cm

Species 1

Species 2

Figure 6: Open air control showing microbial growth

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Results: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 cm2

Estimated number of colonies found in Petri dish

x r2

x 3.8cm2

45cm2

10% 55 colonies/cm2 55 colonies x 45cm2 = 2,475 colonies2,475 colonies x .10 = 248 colonies

Figure 7: S. cerevisiae grown on nutrient agar

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Results: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 cm2

Estimated number of colonies found in Petri dish

x r2

x 3.8cm2

45cm2

25% 87 colonies/cm2 87 colonies x 45cm2 = 3,915 colonies3,951 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

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Results: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

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Results:

Figure 10: Histogram plotting the numbers of S. cerevisiae colonies verses designated Petri dish environment.

histogram plotting the number of S. cerevisiae colonies

0

200

400

600

800

1000

1200

S. cerevisiaeon nutrient

agar

S. cerevisiaeon chilipowder

S. cerevisiaeon cloves

nutrient agarcontrol

open aircontrol

Environment

Nu

mb

er o

f co

lon

ies

histogram plotting the number of S. cerevisiae colonies

S. cerevisiae S. cerevisiae S. cerevisiae Nutrient Open air

On nutirent on chili on cloves agar control

Agar powder control

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ConclusionConclusion

Our hypothesis was not fully supported by Our hypothesis was not fully supported by the data. the data.

While cloves was a potent inhibitor of While cloves was a potent inhibitor of

S. cerevisiaeS. cerevisiae, chili powder was not. , chili powder was not. Our data suggests that chili powder actually Our data suggests that chili powder actually

enhanced the growth of enhanced the growth of S. cerevisiae.S. cerevisiae.

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Further AnalysisFurther Analysis

According to previous research by De et al., 1999 , According to previous research by De et al., 1999 , chili powder should have inhibited the growth of chili powder should have inhibited the growth of

S. cerevisiae at 25mg/mL and 100mg/mL. We believe at 25mg/mL and 100mg/mL. We believe that when the Petri dish of chili powder was created, that when the Petri dish of chili powder was created, most of the powder settled at the bottom of the Petri most of the powder settled at the bottom of the Petri dish leaving the top with a small concentration of dish leaving the top with a small concentration of chili powder (<25mg/mL). If we were to repeat this chili powder (<25mg/mL). If we were to repeat this experiment, we would prepare our own Petri dishes to experiment, we would prepare our own Petri dishes to ensure an accurate concentration of chili powder. ensure an accurate concentration of chili powder.

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Further AnalysisFurther Analysis The transparent graph paper method was The transparent graph paper method was

not entirely accurate. In order to accurately not entirely accurate. In order to accurately calculate the number of colonies, a more calculate the number of colonies, a more precise method would is needed to count precise method would is needed to count the number of colonies inside an area of a the number of colonies inside an area of a cmcm22..

A more precise method needs to be used to A more precise method needs to be used to count the total area that the fungus covered, count the total area that the fungus covered, which could significantly affect our results.which could significantly affect our results.

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Further AnalysisFurther Analysis

In future experimentation we will more closely In future experimentation we will more closely examine the chemical constituents in cloves in examine the chemical constituents in cloves in order to understand the cellular mechanisms order to understand the cellular mechanisms behind its inhibition of behind its inhibition of S. cerevisiae growth.

To do this we will employ electron microscopy and florescence microscopy techniques.

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ReferencesReferences

Morgan, I. G, and Brown Carter, M.E., Morgan, I. G, and Brown Carter, M.E., Investigating BiologyInvestigating Biology. Benjamin/Cummings . Benjamin/Cummings Publishing Co., Inc. 2002.Publishing Co., Inc. 2002.

Minakshi De, Amit Krishna De, and A. B. Minakshi De, Amit Krishna De, and A. B. Banerjee. (1999). Antimicrobial Screening of Banerjee. (1999). Antimicrobial Screening of Some Indian Spices. Some Indian Spices. Phytotherapy Research, Phytotherapy Research, 13 (7), 616-618. 13 (7), 616-618.

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Producers:

Jason Hinkle, Pandelee Mikroudis, Mike Bohner

SPECIAL THANKS TO:Dr. McLaughlin

Mazin AlbertSamer Moussa