Clayton Gentilcore Grade 10 Pittsburgh Central Catholic High School Problem A very large amount of money is spent every year on chemicalbased remedies in order to treat athletes foot A more natural and costeffective method of elimination needs to be determined ID: 774870
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Slide1
Anti-fungal Effects of Natural Remedies
Clayton Gentilcore
Grade 10
Pittsburgh Central Catholic High School
Slide2Problem
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
Slide3Objective
Determine effectiveness of natural remedies on survivorship of
Saccharomyces cerevisiae
(Yeast)
Slide4Fungi
Eukaryotic organismsRange from single-celled yeasts or unicellular spores to multicellular molds, mushrooms, tree brackets, and others5 phyla of fungi (based on type, or lack, of reproductive spores)ZygomycetesZoomycetesAscomycetesBasidiomycetesDeuteromycetesLive either as saprophytes or as parasitesCause diseases called mycoses
Slide5Tinea pedis (Athlete’s foot)
Caused by dermatophytesHeterogeneous pathogensTrichophyton, Microsporum, and EpidermophytonCommon and widely distributed fungal infectionAlso known as “ringworm”Transmitted through public showers, swimming pools, etc.Circular lesions in epidermal layer of skinPeeling and cracking of skinLesions may release watery fluidCauses itching and burning between toesMay lead to severe bacterial infection
Slide6Treatments for Athlete’s foot
Topical anti-fungal agentsSprayPowderCreamGelAnti-fungal drugsMiconazole nitrateClotrimazoleTolnaftateOral anti-fungal agents and antibiotics required in severe casesHygiene during treatmentKeep infected area dryChanging socks as often as necessaryPrevent spreading infection to others
Slide7Sesame Oil
Contains calcium, copper, iron, magnesium, vitamin B6, vitamin E, and zincContains a high percentage of polyunsaturated fatty acids (omega-6 fatty acids)Consists of anti-oxidant, and anti-depressant propertiesMay have a wide range of therapeutic usesReducing the effects of anxiety, bone and nerve disorders, lowered immunity, and poor circulationInsufficient research on medicinal properties
Slide8Tea Tree Oil
Clear essential oil from the leaves of the Melaleuca alternifolia plantMain component for antimicrobial activity is terpinen-4-olMay also contain antiviral, antibacterial, antifungal, and antiseptic propertiesActive against Staphylococcus aureusIncludes MRSA, and CA-MRSAUsed to eliminate Candida in vitro, in order to treat Candidiasis (yeast infection)
Slide9Saccharomyces cerevisiae
Unicellular fungi (species of yeast)Ascomycetes Round to ovoid in shapeGrow aerobically on glucose, maltose, and trehaloseReproduce asexuallyBuddingCommonly used as a model organismSimilar in structure to Trichophyton, Microsporum, and EpidermophytonUtilized as a pathogenic fungi (Athlete’s foot) surrogate in this experiment
Slide10Null Hypothesis
The 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.
Slide11Materials
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
Slide12Procedure
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
10
7
cells/mL).
The culture was diluted in a sterile dilution fluid to a concentration of approximately 10
5
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 m
L.
of cell culture was then added to the test tubes, yielding a
final volume
of 10
mL.
and a cell density of approximately 10
3
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
.
Slide13Chart of Concentrations
0% Cleaner
0.10% Cleaner
1% Cleaner
10% Cleaner
S.
cerevisiae
0.1mL
0.1mL
0.1mL
0.1mL
SDF
(Sterile
Dilution
Fluid)
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
Slide14Remedy Effects on S. Cerevisiae Survivorship
Concentration of Variable
Average Number of Colonies
1
7.17E-09
2.54E-17
1.41E-07
P-values:
P-values:
3.60E-277.31E-34
Sesame Oil
LD50:
7.78%
Tea Tree Oil LD50:
0.71%
Slide15Anova: Single Factor Sesame Oil
Groups Count Sum Average Variance
0% 8 2188 273.5 55.14285714
0.1% 8 1785 223.125 20.125
1% 8 1396 174.5 45.14285714
10% 8 1102 125.25 43.35714286
Source of Variation SS df MS
Between Groups 97372.34375 3 32457.44792
Within Groups 1146.375 28 40.94196429
Total 98518.71875 31
F P-value F crit
792.7672373 3.59602E-27 2.946685266
Slide16Anova: Single Factor Tea Tree Oil
Groups Count Sum Average Variance
0% 8 2188 273.5
55.14285714
0.1% 8 1512 189 79.42857143
1% 8 1085 135.625 35.41071429
10% 8 10 1.25 3.642857143
Source of Variation SS df MS
Between Groups 312850.8438 3 104283.6146
Within Groups 1215.375 28 43.40625
Total 314066.2188 31
F P-value F crit
2402.50228 7.30707E-34 2.946685266
Slide17Significant Variation (Dunnett’s Test)
T-critical value (significant difference): 2.88Alpha: .05
Tea Tree Oil ConcentrationT-valueInterpretation0.10% Concentration14.72Significant1% Concentration30.93Significant10% Concentration71.72Significant
Sesame Oil ConcentrationT-valueInterpretation0.10% Concentration15.75Significant1% Concentration30.94Significant10% Concentration46.34Significant
Slide18Conclusions
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.
Slide19Limitations and Extensions
LimitationsOnly 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.
Extensions
Test 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.”
Slide20References
Boyd, 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.
Slide210%0.10%1%10%0%0.10%1%10%#1.1259229164124#1 Total1086898692513#1.2272219172120#2 Total1102887704489#1.3274224179133#1 Average271.5224.5173128.25#1.4281226177136#2 Average275.5221.75176122.25#2.1269228167121Total2188178513961002#2.2283216181128Total Average273.5223.125174.5125.25#2.3274221183117#2.4276222173123
Sesame Oil Data
Slide220%0.10%1%10%0%0.10%1%10%#1.12591971370#1 Total108676052610#1.22721791292#2 Total11027525590#1.32741821265#1 Average271.5190131.52.5#1.42812021343#2 Average275.5188139.750#2.12691791420Total21881512108510#2.22831851390Total Average273.5189135.6251.25#2.32741921350#2.42761961430
Tea Tree Oil Data