The Effects of Ginseng on Microflora Survivorship Joseph Fantini 11th grade, Pittsburgh Central Catholic High School, 2nd year in PJAS

Problem Ginseng is widely known for its use in folk medicine; medicine that is not always scientifically explicable Developed as folk medicine without understanding effect on microbial life

Potential mutualistic, parasitic, pathogenic, or commensal effects Microbial Flora Internal and external flora include eukaryotic fungi, protists, and bacteria Little is understood about the association between humans and their flora Potential mutualistic, parasitic, pathogenic, or commensal effects Provides nutritional and digestive benefits, secrete vitamins, stimulate antibody production, and protect against pathogenic microbes Ingested substances may have an impact on colony sizes and functions of flora

Escherichia Coli (E. Coli) Large and diverse group of gram (-) bacteria Free living, symbiont, or pathogen Live in the intestinal tract of many mammals Most strains are not pathogenic Serve as a common prokaryotic cell model

Staphylococcus epidermidis Widely studied Gram-positive bacterium Part of microflora found on skin Multiple positive effects on the body Inhibition and killing of nonindigenous species

Ginseng Capsules (Giant Eagle) Components Ginseng being tested Ginseng Capsules (Giant Eagle) Components American Ginseng Extract (Panax quinquefolius) Chinese Red Ginseng Extract (Panax Ginseng) Panax Ginseng (Panaz Ginseng)

Purpose To determine the effects of Ginseng on two different forms of microbial life—Staphylococcus epidermidis and Escherichia coli

Hypothesis Null hypothesis: Ginseng will not have a significant effect on staph and E. coli survivorship. Alternate hypothesis: Ginseng will have a significant effect on staph and E. coli survivorship.

Materials Escherichia coli Bunsen Burner Staphylococcus epidermidis Ginseng Capsules Micropipettes Sterile Water Micro Rack Sterile Pipette Tips Micro Tubes 60 YEPD Agar plates (1% yeast extract, 2% peptone 2% glucose, 1.5% agar) Agar Plates YEPD Media (1% yeast extract, 2% peptone, 2% glucose) Vortex Incubator Sterile dilution fluid (10 mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) Spreader Bars Ethanol

Procedure Microbes were grown overnight in sterile YEPD media A sample of the overnight culture was added to fresh media in a sterile sidearm flask The culture was incubated at 30C until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL The cell culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL Test tubes were made with concentrations of 0%, 0.1%, 1%, and 10% Ginseng. (5 replicates for each of the four concentrations – 20 plates per microbe)

Test Tube Concentrations 0% 0.1% 1% 10% Sterile water 9.9 mL 9.89 mL 9.8 mL 8.9 mL Microbe 0.1 mL Ginseng 0 mL 0.01 mL 1 mL

Procedure continued 6. The tubes were incubated at room temperature for 5 minutes 7. Tubes were vortexed and 0.1 mL aliquots were plated onto YEPD agar 8. Plates were incubated at 30 degrees Celsius for one day and colonies were counted. Each colony was assumed to have arisen from a single cell.

A-Nova Dunnett's Test A-Nova & Dunnett's Test Statistical test that allows for the comparison of means of different groups to determine significant variation Dunnett's Test A follow up test used to find out which variable groups produced significant variation compared to a control

P-Value: 5.91789E-11 P-Value: 1.20143E-14 Cell Colonies Counted Percent Concentration of E. coli and Staph

Dunnett’s Test Results T Value Result (E.COLI) 0% vs. 0.1% Ginseng 4.25 Significant (E.COLI) 0% vs. 1% Ginseng 10.97 (E.COLI) 0% vs. 10% Ginseng 14.95 (STAPH) 0% vs. 0.1% Ginseng 3.81 (STAPH) 0% vs. 1% Ginseng 18.87 (STAPH) 0% vs. 10% Ginseng 24.02 t-crit = 3.239

Interpretation of Results The null hypothesis can be rejected The alternate hypothesis can be accepted Ginseng produced a significant negative effect on E. coli and staph survivorship Statistical analyses supported a significant effect at all concentrations

Limitations Slight positioning differences in the incubation process Slightly desynchronized plating Limited concentration exposures Only one exposure time Only survivorship tested, not growth or other parameters Study does not account for other factors that might affect the microbial flora

Addition of more microbial models such as Yeast Extensions Addition of more microbial models such as Yeast Would give insight on effects on more advanced cells like human cells Different Species of Ginseng can be tested in isolation Different concentrations of the Ginseng could be tested Agar infusion of Ginseng Test for synergistic effects with other chemicals

Bibliography http://davidmlane.com/hyperstat/B112114.html https://www.ncbi.nlm.nih.gov/pubmed/26559705 http://www.ewg.org/research/not-so-sexy https://statistics.laerd.com/statistical-guides/one-way- anova-statistical-guide.php http://textbookofbacteriology.net/normalflora.html http://www.bioquell.com/en-us/resources-and- support/microbiology/staphylococcus-epidermis/

Single Factor A-Nova (E. Coli)

Single Factor A-Nova (Staph)