1. Antimicrobial Effects of Garlic Extract Nathaniel Barbour Freshman Central Catholic High School 3 rd Year in PJAS

2. Garlic Scientific Name: Allium Sativum Used throughout history Used for cooking and as a medicinal agent Believed to have healing properties

3. Chemistry of Garlic Contains amino acid alliin When garlic is crushed or sliced… The product of the reaction is allicin.

4. Allicin Chemical compound diallyl thiosulphinate Powerful antibiotic Disrupts biofilms and plasma membranes of bacteria and fungi Naturally unstable Currently created in synthetic, stabilized form

5. 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.

6. 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

7. Purpose To determine what effect garlic extract has on E. coli survivorship

8. 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.

9. Materials LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Escherichia coli (DH5- alpha) Sterile Dilution Fluid [SDF] (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 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

10. 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 10 8 cells/mL. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 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%.

11. 0% Garlic0.1% Garlic1% Garlic10% Garlic Microbe0.1 mL SDF9.9 mL9.89 mL9.8 mL8.9 mL Garlic Extract 0 mL0.01 mL0.1 mL1 mL Total10 mL Table of Concentrations

12. 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 10 3 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.

13. Liquid Exposure P-value 9.4E-09 P>0.05 P<0.05

14. Dunnett’s Test T-Critical = 2.88 (significant) Alpha =.05 Concentration of Garlic Extract T-ValueSignificant? 0.1%1.783877No 1%6.564353Yes 10%8.027445Yes

15. Survivorship Curve for E. coli Average of Colony Counts Concentration of Garlic Extract Approximate LD50: 0.60% garlic extract

16. 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.

17. 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.

18. 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

19. 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.chemistryland.com/CHM107/EarlyChemistry/PreservationChemistry/AlliinStart.jpg

20. 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 http://l.thumbs.canstockphoto.com/canstock1 305690.jpg