Antimicrobial Effects of Garlic Extract

Simon Hebert Pittsburgh Central Catholic

Grade 9

Garlic

•Allium Sativum

•Central Asia

•Related to onions, shallots, and leeks

•“Stinking Rose” (distinct odor and layered “petals”)

•Wordwide ingredient

Garlic as Medicine•“Miracle cure:” tumors to heart disease to warts for thousands of years

•Both World Wars to dress open wounds andprevent gangrene (“Russian Penicillin”)

•Supposedly anti-cancer, blood pressure, anti fungal infections, etc.

Garlic as Antibiotic•Louis Pasteur first to scientifically prove antimicrobial effects

•Kills both gram negative and positive bacteria

•Found to be effective against a wide range of pathogenic bacteria including e. coli, staph, salmonella, and more

•Active ingredient is allicin

Allicin•C6H10OS2

•0.3-0.5% of full garlic clove

•Created when enzyme called allinase converts alliin,acid derivative, into allicin (garlic’s pest defense)

•Takes just 10-60 seconds

•Gives garlic its distinct odor

•Extremely unstable: degrades within hours to have no antimicrobial effects, faster with heat or microwaving

Garlic Supplements

•Extremely difficult to make extract, isolate allicin, preserve, and put it into product without degradation

•Diallyl trisulfade is a similar compound to allicin but more stable; some “garlic” supplements contain this

•Allicin causes odor, odorless pills and supplements likely have no antimicrobial effects (unregulated)

•Treatment for many ailments likely placebo effect

Escherichia Coli•Common form of bacteria, found in range of environments

•Gram negative, rod shaped bacillus

•Reproduces rapidly (20 minutes)

•Most harmless or even beneficial, some strains pathogenic, cause food poisoning

•Easily grown, used as model organism for wide range of microorganisms

Past Studies

•5% concentration of garlic killed 80% of staph

•10% killed more than 90% of e. coli

Purpose and Hypothesis•Purpose

Determine the effects of garlic extract on E. coli survivorship

•HypothesisBased on past studies, garlic extract will negatively

affect the survivorship of E. coli

•Null hypothesisGarlic extract will have no effect on the survivorship

of E. coli

Materials•Fresh garlic bulbs•Food processor •Knife •Garlic grinder •Cheesecloth •Rubber band •Tupperware •Sterile filters•Sterile dilution fluid (10 mM KH2PO4, 10 mM K2HPO4,1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

•Micropipettes •Test tubes •Test tube racks •Luria broth agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl)•Spreader bars •Ethanol flame•Safety goggles •Incubator (37° C)•Sharpie •Vortexer •Escherichia coli

Procedure (E. coli)1. E. coli was grown until a density of 50 klett spectrophotometer density was reached. This was approximately 108 cells/mL

2. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL

Procedure (liquid exp.)1. Peeled fresh garlic cloves. Puréed garlic heads in food processor. Pressed out juice with garlic grinder with cheese cloth rubber banded around end. Collected and refrigerated juice. Sterile filtered juice to obtain pure, sterile garlic extract

2. Pipetted amounts indicated on table of garlic juice and sterile dilution fluid into four sterile test tubes. Made two tubes of each concentration. Vortexed solutions to mix thoroughly

Procedure (liquid exp.)3. Added indicated amount of E. Coli. Vortexed final solutions. Incubated solutions at room temperature for 10 minutes

4. Pipetted 0.1 mL of solution from tubes (4 per tube) onto LB agar. Used a new pipette for SDF, extract, and E. coli

5. Spread solution evenly over agar with sterile spreader bar. Used a new spreader for each plate. Made 8 replicate plates per concentration (32 plates in all)

Procedure (liquid exp.)6. Incubated plates for one day in at 37° C. Recorded number of E. coli colonies

Tube 1/1B (Control) Tube 2/2B Tube 3/3B Tube 4/4B

E. coli 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Garlic Juice (100%) 0 mL 0.01 mL 0.1 mL 1 mL

Added Sterile dilution fluid 1 mL 0.99 mL 0.9 mL 0 mL

Sterile Water 8.9 mL 8.9 mL 8.9 mL 8.9 mL

Concentration of Garlic Juice 0% 0.1% 1% 10%

Total 10 mL 10 mL 10 mL 10 mL

Procedure (infusions)1. Performed step 1 of liquid infusion method to obtain pure garlic extract

2. Pipetted 200 uL of sterile dilution fluid onto four plates. Pipetted 200 uL of pure garlic extract onto four plates. Pipetted 180 uL sterile dilution fluid and 20 uL garlic extract onto four plates (12 plates in all)

3. Allowed plates to incubate at 37° C for one hour

Procedure (infusions)4. Pipetted 0.1 mL of tube 1 (control) onto each plate

5. Incubated for one day in at 37° C. Recorded number of E. coli colonies

Results (Liquid)Av

erag

e N

umbe

r of C

olon

ies

0

30

60

90

120

150

180

210

240

270

300

Concentration of Garlic Extract

0% 0.1% 1% 10%

LD50 (Lethal Dose 50%): approx. 4%

P Value: less than 10-6

•P value is negligible, the results were outside of chance

ANOVA (Liquid)

Results (Infusion)Av

erag

e N

umbe

r of C

olon

ies

0

30

60

90

120

150

180

210

240

270

300

Concentration of Garlic Extract

0% 0.1% 1%

LD50 (Lethal Dose 50%): approx. 0.09%

P Value: less than 10-6

•P value is negligible, the results were outside of chance

ANOVA (Infusion)

Dunnett’s Test (liquid)T Crit: 2.88 T Value > T Crit: Significant

T Value < T Crit: Not significant

T Value Conclusion

0.1% 7.0339 Significant

1% 17.835 Significant

10% 26.454 Significant

Dunnett’s Test (infusion)T Crit: 3.03 T Value > T Crit: Significant

T Value < T Crit: Not significant

T Value Conclusion

10% 22.24 Significant

100% 38.45 Significant

Conclusions

•Garlic extract appears to have a strong antimicrobial effect of E. coli. Reject null hypothesis

•The LD50 of the liquid exposure was approximately a 4% concentration

•Garlic extract appears to have a strong antimicrobial effect of E. coli. Reject null hypothesis

•The LD50 of the infusion plates was approximately an 0.09% concentration, likely due to much longer exposure time to E. coli

Liquid Infusion

Limitations

•Only tested one type of bacteria

•Plating, vortexing, pipetting, exposure times slightly unsynchronized

•Allicin degradation before beginning tests

•More concentrations, replicates, etc.

•More precise plating and incubating

•Test other types of bacteria (gram - vs +)

•Make extract minutes before testing

•Isolate allicin

•Test commercial garlic supplements

Future Experimentation

Works Cited•”Allicin." Phytochemicals. N.p., 12 Apr. 2011. Web. 19 Dec. 2015. •”Approximate PH of Foods and Food Products." Food Science. U.S. Food and Drug Administration, 1 Apr. 2007. Web. 19 Dec. 2015. •Higdon, Jane, Victoria Drake, and Larry Lawson. “Micronutrient Information Center." Linus Pauling Institute. Oregon State University, 1 July 2008. Web. 18 Dec. 2015. •Ju Anne, Lim. "Antimicrobial Effect of Garlic against E Coli and Stap.A." Slideshare. Slideshare, 11 Feb. 2011. Web. 19 Dec. 2015. •Kumar, M., and J.S. Berwal. "Sensitivity of Food Pathogens to Garlic." Online Library. Wiley, 2 May 1997. Web. 19 Dec. 2015. •Lokteff, Lana. "Garlic Keeps More Than Vampires Away." Red Ice Creations. Red Ice Radio, 13 Apr. 2011. Web. 18 Dec. 2015. •Palaksha, M. N., Mansoor Ahmed, and Sanjoy Das. "Antibacterial Activity of Garlic Extract on Resistant Bacteria." Journal of Natural Science, Biology, and Medicine. Medknow Publications Pvt Ltd, 12 July 2010. Web. 18 Dec. 2015. •Sardi, Bill. "Garlic and Pharmaceuticals." Knowledge of Health. Knowledge of Health, 27 May 2014. Web. 19 Dec. 2015. •Sivam, G. P. "Protection Against Bacterial Infections by Garlic.” National Center for Biotechnology Information. U.S. National Library of Medicine, 1 Mar. 2001. Web. 19 Dec. 2015. •”What Is Allicin?" AllicinFacts. Allicin Facts, 1 Oct. 2010. Web. 19 Dec. 2015.