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