Antimicrobial Effects of Garlic Extract

Nathaniel BarbourFreshmanCentral Catholic High School3rd 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

Procedure1. 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%.

0% Garlic 0.1% Garlic 1% Garlic 10% GarlicMicrobe 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

Table of Concentrations

Procedure6. 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

0.0% 0.1% 1.0% 10.0%0

20

40

60

80

100

120

140

160

180

Concentration of Garlic Extract

Aver

age

of C

olon

y Co

unts

P-value9.4E-09

P>0.05

P<0.05

P<0.05

Dunnett’s TestT-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

0% 2% 4% 6% 8% 10% 12%0

20

40

60

80

100

120

140

160

180

Aver

age

of C

olon

y Co

unts

Concentration of Garlic Extract

Approximate LD50: 0.60% garlic extract

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• Dugdale, David C. “Anaerobic bacteria – Overview.” University of Maryland Medical Center. 21 May 2009. Web. 23

December 2010.• Editors of FC&A Publishing. 999 Little-Known Natural Healing Foods and Proven Home Remedies. Peachtree City, GA: FC&A,

1994. Print.• Horizon Scientific Press and Caister Academic Press. “Gram-negative Bacteria.” Horizon Press. Web. 23 December 2010. <

http://www.horizonpress.com/gateway/gram-negative-bacteria.html>• Kimball, John. “E. coli.” Kimball’s Biology Pages. 22 May 2010. Web. 23 December 2010. <

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Esch.coli.html>.• Loughry, Lyle. Allicin – Beyond Immunity. 2006. Web. 23 December 2010. <

http://www.astrologyzine.com/allicin-beyond-immunity.shtml>.• Marietta College Biology Department. “Symbiosis.” 09 April 2008. Web. 24 December 2010. <

http://www.marietta.edu/~biol/biomes/symbiosis.htm>• Mayo Clinic staff. “MRSA infection.” Mayo Clinic. 29 May 2010. Web. 24 December 2010. <

http://www.mayoclinic.com/health/mrsa/DS00735>.• MedicineNet.com. “GARLIC (Allium sativum L.) - ORAL Side Effects, Medical Uses, and Drug Interactions.” 23 December 2010.

Web. 24 December 2010. <http://www.medicinenet.com/garlic_allium_sativum_l-oral/article.htm>. • Mendham, Trevor. “The Chemistry of Allicin.” Garlic Central. 2003. Web. 24 December 2010. <

http://www.garlic-central.com/allicin-chemistry.html>.• Natural Health Publications Limited. “The Future of Allicin.” Allicin – Facts about Allicin. 2010. Web. 23 December 2010. <

http://www.allicinfacts.com/fallicin.htm>.• Suburban Emergency Management Project. “Who Is Dr. Theodor Escherich?” 2007. Web. 23 December 2010. <

http://www.semp.us/publications/biot_reader.php?BiotID=109>.• Todar, Kenneth. “Pathogenic E. coli.” Todar’s Online Textbook of Bacteriology. 2008. Web. 23 December 2010. <

http://www.textbookofbacteriology.net/e.coli.html>.• Yeager, Selene, et al. The Doctors Book of Food Remedies. Emmaus, PA: Rodale, 1997. Print.• http://www.chemistryland.com/CHM107/EarlyChemistry/PreservationChemistry/AlliinStart.jpg (picture)

http://www.3dchem.com/imagesofmolecules/Allicin.jpg

http://www.garlic-central.com/images/allicin.gif

http://www.marlerblog.com/uploads/image/e-coli(3).jpg

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