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Anti-fungal Effects of Natural Remedies
Clayton GentilcoreGrade 10
Pittsburgh Central Catholic High School
Problem A very large amount of money is
spent every year on chemical-based remedies, in order to treat athlete’s foot
A more natural and cost-effective method of elimination needs to be determined Sesame Oil Tea Tree Oil
ObjectiveDetermine effectiveness of
natural remedies on survivorship of Saccharomyces
cerevisiae (Yeast)
Fungi Eukaryotic organisms Range from single-celled
yeasts or unicellular spores to multicellular molds, mushrooms, tree brackets, and others
5 phyla of fungi (based on type, or lack, of reproductive spores)
Zygomycetes Zoomycetes Ascomycetes Basidiomycetes Deuteromycetes
Live either as saprophytes or as parasites
Cause diseases called mycoses
Tinea pedis (Athlete’s foot) Caused by dermatophytes
Heterogeneous pathogens Trichophyton, Microsporum,
and Epidermophyton Common and widely
distributed fungal infection Also known as “ringworm” Transmitted through public
showers, swimming pools, etc. Circular lesions in epidermal
layer of skin Peeling and cracking of skin Lesions may release watery
fluid Causes itching and burning
between toes May lead to severe bacterial
infection
Treatments for Athlete’s foot Topical anti-fungal agents
Spray Powder Cream Gel
Anti-fungal drugs Miconazole nitrate Clotrimazole Tolnaftate
Oral anti-fungal agents and antibiotics required in severe cases
Hygiene during treatment Keep infected area dry Changing socks as often as
necessary Prevent spreading infection to
others
Sesame Oil Contains calcium, copper,
iron, magnesium, vitamin B6, vitamin E, and zinc
Contains a high percentage of polyunsaturated fatty acids (omega-6 fatty acids)
Consists of anti-oxidant, and anti-depressant properties
May have a wide range of therapeutic uses
Reducing the effects of anxiety, bone and nerve disorders, lowered immunity, and poor circulation
Insufficient research on medicinal properties
Tea Tree Oil Clear essential oil from the
leaves of the Melaleuca alternifolia plant
Main component for antimicrobial activity is terpinen-4-ol
May also contain antiviral, antibacterial, antifungal, and antiseptic properties
Active against Staphylococcus aureus Includes MRSA, and CA-
MRSA Used to eliminate Candida in
vitro, in order to treat Candidiasis (yeast infection)
Saccharomyces cerevisiae Unicellular fungi (species of
yeast) Ascomycetes
Round to ovoid in shape Grow aerobically on glucose,
maltose, and trehalose Reproduce asexually
Budding Commonly used as a model
organism Similar in structure to
Trichophyton, Microsporum, and Epidermophyton
Utilized as a pathogenic fungi (Athlete’s foot) surrogate in this experiment
Null HypothesisThe different natural remedies will not have a significant effect on the
survivorship of Saccharomyces cerevisiae.Alternative Hypothesis
1. The different natural remedies will have a significant effect on the survivorship of Saccharomyces
cerevisiae.2. Tea Tree Oil will reduce survivorship
more effectively than Sesame Oil.
Materials Sesame Oil Tea Tree Oil Saccharomyces cerevisiae Micropipettes, micro rack, micro tubes Turn table, spreader bars, matches, ethanol, Bunsen burner YEPD media and agar plates (1% yeast extract, 2% peptone,
2% glucose (dextrose), 1.5% agar) 20 mL Sterile capped test tubes with Sterile Dilution Fluid
(SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)
0.22 micron syringe filters + 10 mL syringe Incubator Vortex Sidearm flask, and Klett spectrophotometer
Procedure Saccharomyces cerevisiae 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 incubated at 30°C until a density of 50-60 Klett
spectrophotometer units was reached. (Cell density approximately 107 cells/mL).
The culture was diluted in a sterile dilution fluid to a concentration of approximately 105 cells/mL.
The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL.
0.1 mL. of cell culture was then added to the test tubes, yielding a final volume of 10 mL. and a cell density of approximately 103 cells/mL.
The solution was mixed by vortexing and allowed to sit at room temperature for 15 minutes.
After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on 64 YEPD agar plates.
The plates were incubated at 30°C for 48 hours. The resulting colonies were counted. Each colony is assumed to have
risen from one cell.
Chart of Concentrations0%
Cleaner0.10%
Cleaner1%
Cleaner10%
CleanerS.
cerevisiae 0.1mL 0.1mL 0.1mL 0.1mL
SDF(SterileDilutionFluid)
9.9mL 9.89mL 9.8mL 8.9mL
Remedy(Sesame Oil/
Tea Tree Oil)
0mL 0.01mL 0.1mL 1mL
Total 10mL 10mL 10mL 10mL
Remedy Effects on S. Cerevisiae Survivorship
0.00% 0.10% 1.00% 10.00%0
50
100
150
200
250
300
Sesame Oil
Concentration of Variable
Ave
rag
e N
um
ber
of
Colo
nie
s
1
7.17E-09
2.54E-17
1.41E-07
P-values:
P-values:3.60E-277.31E-34
Sesame OilLD50:7.78%Tea Tree Oil LD50:0.71%
Anova: Single Factor Sesame OilGroups Count Sum Average Variance0% 8 2188 273.5 55.142857140.1% 8 1785 223.125 20.1251% 8 1396 174.5 45.1428571410% 8 1102 125.25 43.35714286
Source of Variation SS df MSBetween Groups 97372.34375 3 32457.44792Within Groups 1146.375 28 40.94196429Total 98518.71875 31
F P-value F crit792.7672373 3.59602E-27 2.946685266
Anova: Single Factor Tea Tree OilGroups Count Sum Average Variance0% 8 2188 273.5 55.142857140.1% 8 1512 189 79.428571431% 8 1085 135.625 35.4107142910% 8 10 1.25 3.642857143
Source of Variation SS df MSBetween Groups 312850.8438 3 104283.6146Within Groups 1215.375 28 43.40625Total 314066.2188 31
F P-value F crit2402.50228 7.30707E-34 2.946685266
Significant Variation (Dunnett’s Test)T-critical value (significant
difference): 2.88Alpha: .05
Tea Tree Oil Concentration T-value Interpretation
0.10% Concentration 14.72 Significant
1% Concentration 30.93 Significant
10% Concentration 71.72 Significant
Sesame Oil Concentration T-value Interpretation
0.10% Concentration 15.75 Significant
1% Concentration 30.94 Significant
10% Concentration 46.34 Significant
Conclusions The results suggest that the
null hypothesis can be rejected.
The results suggest that the alternative hypotheses were supported.
The Tea Tree Oil more effectively eliminated S. cerevisiae than Sesame Oil, as shown through the LD50s.
Limitations and ExtensionsLimitations
Only 2 oils were used.
Only 4 concentrations were used.
S. cerevisiae was the only fungus that was used.
The plating of S. cerevisiae was not fully synchronized.
ExtensionsTest with different types
of bacteria, molds, and algae.
Test with different temperatures, spices, and oils to determine a better method to eliminate S. cerevisiae.
Directly infuse the oil into the agar for a prolonged exposure test.
Perform a “wipe test.”
ReferencesBoyd, Robert and Bryan Hoerl. Basic Medical
Microbiology. Boston: Little Brown Company, 1981.Edmonds, Paul. Microbiology: An Environmental
Perspective. New York: Macmillan Publishing, Inc., 1978.
Ingraham, John L. March of the Microbes. Cambridge, Massachusetts: The Belknap Press of Harvard University Press, 2010.
Nester, Eugene W. The Microbial Perspective. New York: Saunders College Publishing, 1982.
Phaff, Herman J. The Life of Yeasts. Cambridge, Massachusetts: Harvard University Press, 1978.
Rotbart, Harley A. Germ Proof Your Kids. Washington, D.C.: ASM Press, 2008.
0% 0.10%
1%
10%
0% 0.10%
1% 10%
#1.1
259
229 164
124 #1 Total 1086 898 692 513
#1.2
272
219 172
120 #2 Total 1102 887 704 489
#1.3
274
224 179
133 #1 Average 271.5 224.5 173 128.25
#1.4
281
226 177
136 #2 Average 275.5 221.75
176 122.25
#2.1
269
228 167
121 Total 2188 1785 1396
1002
#2.2
283
216 181
128 Total Average
273.5 223.125
174.5
125.25
#2.3
274
221 183
117
#2.4
276
222 173
123
Sesame Oil Data
0% 0.10%
1% 10%
0% 0.10% 1% 10%
#1.1
259
197 137
0 #1 Total 1086 760 526 10
#1.2
272
179 129
2 #2 Total 1102 752 559 0
#1.3
274
182 126
5 #1 Average
271.5 190 131.5 2.5
#1.4
281
202 134
3 #2 Average
275.5 188 139.75
0
#2.1
269
179 142
0 Total 2188 1512 1085 10
#2.2
283
185 139
0 Total Average
273.5 189 135.625
1.25
#2.3
274
192 135
0
#2.4
276
196 143
0
Tea Tree Oil Data