Ginger's Effects on Microbial Survivorship By Jonah Duch 9TH GRADE CENTRAL CATHOLIC HIGH SCHOOL

Problem Does ginger have an effect on Escherichia coli and Staphylococcus epidermidis?

Ginger Knobby, fibrous root Smooth light brown skin. Inside of the root is white. Seasons and adds flavor to sweets Excellent natural remedy for nausea, motion sickness, and general stomach upset due to its carminative effect that helps break up and expel intestinal gas.

E. Coli C600/Staph Epidermidis •A gram negative and gram positive model were used as surrogates. •E coli is a gram negative rod shaped bacterium that is normally found in the lower intestine of warm blooded organisms. •Staph is a gram positive bacterium that is normally found on the skin and respiratory tracts of humans, and is a major cause of skin infections.

ECOLI and the WORLD HEALTH •E. coli infections come from contact with feces or contaminated water or food. •Infections can be eliminated if meat is cooked to 160°F (71°C) •Infections associated with poor hygiene in poorer under developed nations. •Symptoms include: Bloody diarrhea. Stomach cramps. Nausea and vomiting. •.

STAPH EPIDERMIDIS and the WORLD HEALTH 2 types of staph infections Cellulitis is a bacterial infection of the skin and underlying fat tissue. Folliculitis is an infection of the pilosebaceous follicle (the hair and oil gland) and is the most common form of staph skin infection. •Infections range from a simple infection to antibiotic- resistant infections to flesh-eating infections. •The Staph used in this experiment is not a pathogen but was used as a surrogate for infection causing Staph species.

Purpose The purpose of this experiment is to test the effects of ginger on Escherichia coli and Staphylococcus epidermidis survivorship.

NULL HYPOTHESIS Alternate Hypothesis The ginger concentrations will not be able to significantly reduce the bacterial survivorship. Alternate Hypothesis The ginger concentrations will be able to significantly reduce the bacterial survivorship.

Materials Escherichia coli Staphylococcus epidermidis Sterile water test tubes Micro-pipettes Micro-pipette tips Test tube rack 100% Ginger juice YEPD Agar Petri Dishes (1% yeast extract, 2% peptone, 2% dextrose) Sterile Dilution Fluid (100mM KH2PO4,100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Sterile Spreader Bars Vortex Bunsen Burner Ethanol Incubator

Procedure I (Liquid Pulse) 1. E. coli and Staph were 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. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. Sterilize ginger juice for use in experiment. 6. Sterilized ginger juice was mixed with the appropriate amount of SDF to create ginger concentrations of 10%, 1%, and 0.1%.

Concentrations 0% .1% 1% 10% Sterile Dilution Fluid 9.9ml 9.89ml 9.8ml Ginger Juice 0ml .01ml .1ml 1ml Microbe (E. coli or Staph) Total Volume 10ml

Procedure I Continued (Liquid Pulse) 6. 100 µL of cell culture was then added to the ginger juice solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on a set of regular YEPD plates. The plates were incubated at 37 C for 24 hours. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

PROCEDURE II (Agar Infusion) 0.1ml of ginger concentrations, 0%, low (20µL Ginger, 180µL SDF) and high (200µL Ginger) were spread on LB agar plates and incubated for 1 hour, allowing the bacteria to absorb the chemicals. 100uL aliquots of bacterial suspensions from the original control tube were spread onto the infused plates. The plates were incubated at 37 C for 24 hours. 4. The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.

Anova/Dunnett's Test Dunnett's Test Equation: ANOVA (Analysis of Variance) Allows for the comparison of multiple means Utilize p-values as measures of significance P>0.05—not significant P<0.05—significant Dunnett's Test ANOVA follow up, localizes source of variation. Uses a t-value and a t-critical value. If t-critical is higher that t-value, variations are considered significant Dunnett's Test Equation:

Effect of Ginger on E. coli P value=0.000113

E. COLI DUNNETT’S TEST T Crit = 2.97 0.1% Ginger 2.42 T Value NOT SIGNIFICANT 1% 3.09 10% 0.86 NOT SIGNIFICANT

Effect of Ginger on Staph P value= 3.6e-05

STAPH DUNNETT’S TEST T Crit = 3.1 0.1% Ginger 3.77 T Value SIGNIFICANT 5.68 10% 6.42

Infusion: Effect of Ginger on E. coli P Value= 0.457811

Infusion: Effect of Ginger on Staph P Value= 0.534824

CONCLUSION For the liquid pulse Staph test, the null hypothesis was rejected, the ginger significantly altered the Staph survivorship. For the E. coli and Staph infusions, the null hypothesis was accepted, the ginger did not significantly alter Staph survivorship. For the liquid pulse E. coli test, the .1% and 10% didn't significantly alter the E. coli survivorship, while the 1% did.

LIMITATIONS and EXTENSIONS Limited number of replicates Limited number of concentrations Slightly desynchronized plating Only one exposure time Extensions Testing with more species of bacteria Using more replicates. Testing more types of concentrations Testing more exposure times

Sources and Acknowledgments Thanks to Mr. Mark Krotec for the laboratory assistance in preparing the colonies. Thanks to Mark Krotec for also agreeing to sponsor the Experiment. http/ Wikipedia.org http/ CDC. Gov/ E. coli O157:H7 Infection http://igentry.blogspot.com/2009/03/interesting- facts-about-ginger.html

E. Coli Anova

Staph Anova

E. Coli Infusion Anova

Staph Infusion Anova