Transcript CCHS Jacob Warden The Effects of Capsaicin on Microbesx

The Effects of
Capsaicin on
Microbes
JACOB WARDEN
FRESHMAN
CENTRAL CATHOLIC HIGH SCHOOL
Capsaicin
Capsaicin is a chemical irritant to all mammals that is found naturally in
chili peppers.
Capsaicin is a naturally occurring chemical found in chili peppers,
especially the flesh around the seeds.
Used as a medicine in diluted forms.
Chemistry of Capsaicin
• Molecular FormulaC18H27NO3
Escherichia coli
 Rod shaped bacteria found in the lower intestines
of mammals
 Some strains beneficial while others are
pathogenic
Serves as a common prokaryotic cell model
Previous Studies
Journal of Ethnopharmacology 1999: Antimicrobial properties
of alkamides present in flavoring plants traditionally used in
Mesoamerica: affinin and capsaicin.
Purpose
To determine what effect
capsaicin extract has on E. coli
survivorship
Hypotheses
Null hypothesis: Capsaicin extract
will not significantly affect the
survivorship of E. coli.
Alternative hypothesis: Capsaicin
extract will significantly affect the
survivorship of E. coli.
Materials
LB agar plates
Sidearm Flask
Escherichia coli
Vortex
Capsaicin Extract (liquid form)
Incubator
Micro and Macro pipettes + Sterile
tips
Gloves/goggles
Spreader bars
LB agar plates (1% Tryptone, 0.5%
Yeast extract, 1% NaCl)
Escherichia coli DH5-alpha (E.Coli)
(Obtained from Doonan Lab, CMU)
Bunsen Burner
Klett Spectrophotometer
SDF (Sterile Dilution Fluid)(100mM
KH2PO, 100mM K2HPO4, 10mM
MgSO4, 1m NaCl)
Sterile Test Tubes
Ethanol
Procedure
1.
Escherichia coli was grown overnight in sterile LB media.
2.
A sample of the overnight culture was added to fresh media in a
sterile sidearm flask.
3.
The culture was incubated at 37°C until a density of 50 Klett
spectrophotometer units was reached. (Cell density approximately
10^8 cells/mL).
4.
The culture was diluted in a sterile dilution fluid to a concentration
of approximately 10^5 cells/mL.
5.
The selected experimental variables were diluted with sterile
dilution fluid to the chosen concentrations to a total of 9.9 mL.
Procedure continued
6. 0.1 mL. of cell culture was then added to the test tubes, yielding a final
volume of 10 mL.
7. The solution was mixed by vortexing.
8. After vortexing to evenly suspend cells, 0.1 mL aliquots were
removed from the tubes and spread on LB agar plates.
9. The plates were incubated at 37°C for 24 hours.
10. The resulting colonies were counted. Each colony is assumed to
have risen from one cell.
Table of Concentrations
Column1
Tube 1
Tube 2
Tube 3
Tube 4
Tube5
Capsaicin Variable X
0ml
0.001ml
0.01ml
0.1ml
1ml
Sterile Fluid
9.9ml
9.899ml
9.89ml
9.8ml
8.9ml
Microbe
0.1ml
0.1ml
0.1ml
0.1ml
0.1ml
Final Volume
10ml
10ml
10ml
10ml
10ml
Final Concentration
X
0%
0.01%
0.10%
1%
10%
The Effects of Capsaicin on
Microbes
Averages
350
P-value: 0.000107
Number of Surviving E. coli Colonies
300
250
200
150
100
50
0
0%
0.01%
0.10%
Concentrations
1%
10%
Dunnett’s Test
T-Critical = 3.90 (significant)
Alpha = .05
Concentration of
Capsaicin Extract
T-Value
Significant?
0.01%
1.91
No
0.1%
0.58
No
1%
10%
2.36
4.73
No
Yes
Conclusion
 The null was rejected for the 0%, 0.01%,
0.1%, and 1% concentrations.
 The null failed to be rejected for the 10%
concentration.
The results and the statistical analyses
indicate that 10% capsaicin extract did
adversely affect E. coli survivorship.
Limitations
 The spread plating was not perfectly synchronized
 One exposure time
 Only one type of bacteria was tested
Limited number of concentrations tested
Extensions
 Different types of bacteria would be tested
 More concentrations of the capsaicin extract would be tested
 Synergistic effects would be tested
Multiple exposure times would be used
References
• CSID:1265957, http://www.chemspider.com/ChemicalStructure.1265957.html (accessed 20:45, Jan 27, 2016) COPY
•"Capsaicin." - Wikipedia, the Free Encyclopedia. N.p., n.d. Web. 22 Jan.
2016.
• "Escherichia Coli." - Wikipedia, the Free Encyclopedia. N.p., n.d. Web.
22 Jan. 2016.
•"Antimicrobial Properties of Alkamides Present in Flavoring Plants
Traditionally Used in Mesoamerica: Affinin and Capsaicin."
Antimicrobial Properties of Alkamides Present in Flavoring Plants
Traditionally Used in Mesoamerica: Affinin and Capsaicin. N.p., n.d.
Web. 27 Jan. 2016.
Data
Replican
ts
Tube 1:
0%
Tube 2:
0.01%
Tube 3:
0.1%
Tube 4:
1%
Tube 5:
10%
#1
#2
#3
#4
Concentrations
Averages
248
300
244
212
0%
251
352
392
180
408
0.01%
333
166
280
174
284
0.10%
226
172
112
284
132
1%
149.5
17
65
28
81
10%
47.75
ANOVA
One-Way ANOVA
SUMMARY
Group
Count
C1
C2
C3
C4
C5
ANOVA
Source
Between
Within
Total
Conclusions
Ho:
Ha
F-Test
F-Critical
Alpha
P-Value
Decision:
Sum
4
4
4
4
4
SS
Average
1004
1332
904
598
191
df
186743
55453.8
242197
μ1=μ2=μ3=...
At least 2 μ's differ
12.6283
3.05557
0.05
0.000107
Reject Ho
Variance
251
1326.67
333
10958.7
226
4194.67
149.5
1091.67
47.75
912.917
MS
4
15
19
Ftest
46685.8
3696.92
P-value
F-Critical
12.6283
0.000107
3.05557