Transcript Antimicrobial Effects of Garlic Extract

Antimicrobial Effects
of Garlic Extract
Nathaniel Barbour
Freshman
Central Catholic High School
3rd Year in PJAS
Garlic
• Scientific Name: Allium Sativum
• Used throughout history
• Used for cooking and as a medicinal agent
• Believed to have healing properties
Chemistry of Garlic
• Contains amino acid alliin
• When garlic is crushed or sliced…
• The product of the reaction is allicin.
Allicin
• Chemical compound diallyl thiosulphinate
• Powerful antibiotic
• Disrupts biofilms and plasma membranes of bacteria
and fungi
• Naturally unstable
• Currently created in synthetic, stabilized form
Previous Studies
• Research suggests that garlic extract is
effective in treating six different types of
antibiotic-resistant bacteria.
• One of these it can affect is MRSA.
E. coli
• Large and diverse group of gram (-) bacteria
• Free living, symbiotes, or pathogens
• Most strains are not pathogenic.
• Serves as a common prokaryotic cell model
Purpose
To determine what effect garlic
extract has on E. coli
survivorship
Hypotheses
• Null hypothesis: Garlic extract will
not significantly affect the
survivorship of E. coli.
• Alternative hypothesis: Garlic will
significantly affect the survivorship of
E. coli.
Materials
• LB agar plates (0.5% yeast •
extract, 1% tryptone, 1% •
sodium chloride)
•
• Escherichia coli (DH5•
alpha)
•
• Sterile Dilution Fluid [SDF]
•
(100mM KH2PO4, 100mM
K2HPO4, 10mM MgSO4, •
1mM NaCl)
•
• Sterile test tubes
•
• Sterile spreader bars
•
• Incubator
Ethanol
Bunsen burner
Vortex
Garlic Extract (liquid)
Micropipettes
Klett Spectrophotometer
Turntable
Labeling tape
Micro rack
Micro tubes
Procedure
1. E. 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 placed in an incubator (37°C) until a density
of 50 Klett spectrophotometer units was reached. This
represents a cell density of approximately 108 cells/mL.
4. The culture was diluted in sterile dilution fluid to a
concentration of approximately 105 cells/mL.
5. The garlic extract was sterile filtered through a 0.22 micron
syringe filter. Garlic extract was mixed with the appropriate
amounts of SDF to create garlic concentrations of 10%, 1%,
and 0.1%.
Table of Concentrations
0% Garlic 0.1% Garlic 1% Garlic 10% Garlic
Microbe 0.1 mL
0.1 mL
0.1 mL
0.1 mL
SDF
9.9 mL
9.89 mL
9.8 mL
8.9 mL
Garlic
Extract
0 mL
0.01 mL
0.1 mL
1 mL
Total
10 mL
10 mL
10 mL
10 mL
Procedure
6. 100 µL aliquots of cell culture was then added to the garlic
solutions, yielding a final volume of 10 mL and a cell density
of approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room
temperature for a 20 minute incubation period.
8. After vortexing to evenly suspend the cells, 100 µL aliquots
were removed from the tubes and spread on LB plates.
9. The plates were incubated at 37˚C for 24 hours.
10. The resulting colonies were counted visually. Each colony
was assumed to have arisen from one cell.
Liquid Exposure
180
P-value
9.4E-09
Average of Colony Counts
160
140
P>0.05
120
100
80
P<0.05
60
P<0.05
40
20
0
0%
0.1%
1%
Concentration of Garlic Extract
10%
Dunnett’s Test
T-Critical = 2.88 (significant)
Alpha = .05
Concentration of
Garlic Extract
T-Value
Significant?
0.1%
1.783877
No
1%
6.564353
Yes
10%
8.027445
Yes
Survivorship Curve for E. coli
180
Average of Colony Counts
160
Approximate LD50: 0.60% garlic extract
140
120
100
80
60
40
20
0
0%
2%
4%
6%
8%
Concentration of Garlic Extract
10%
12%
Conclusion
• The null hypothesis that garlic extract does
not affect E. coli survivorship was rejected for
the concentration of 1% and 10% garlic
extract.
• The null hypothesis could not be rejected for
the lowest concentration of 0.1% garlic
extract.
• The results and the statistical analyses indicate
that 1% and 10% garlic extract did adversely
affect E. coli survivorship.
Limitations
• The plating was not perfectly synchronized.
• Only one species of bacteria was tested.
• Some cells could have had longer or shorter
exposure times.
• The garlic extract contained residual amounts of
alcohol from the extraction process, possibly
affecting the experiment.
Extensions
• More trials to create a better basis for evaluating the
results
• Using pure allicin instead of garlic extract
• Testing a gram (+) species of bacteria, such as
Staphylococcus epidermidis
• Prolonged exposure test using garlic extract infused
into the agar
• Using an antibiotic resistant strain of bacteria for the
test model
References
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http://www.chemistryland.com/CHM107/EarlyChemistry/PreservationChemistry/AlliinStart.jpg (picture)
http://www.3dchem.com/imagesofmolecules/
Allicin.jpg
http://l.thumbs.canstockphoto.com/canstock1
305690.jpg
http://www.garliccentral.com/images/allicin.gif
http://www.marlerblog.com/uploads/image/ecoli(3).jpg