Conor Flaherty Central Catholic High School

Bacterial Pathogens Diseases and antibiotics. Prescribed antibiotics and medicines: side effects, increased resistance, etc.

Natural Antibiotics  A large variety of Vitamin C-enriched fruits, vegetables, spices, herbs, and garlic all provide organic remedies  Watermelon, not normally recognized as being a curative fruit.  What antimicrobial properties does watermelon have?  Watermelon is a widely available, taste-appealing fruit which tests have shown to exhibit antimicrobial properties. The fruit contains numerous nutritional substances, such as Vitamin C, carotenoids, cucurbatins, and plenty of water.

Watermelon  Originally from Kalahari Desert in Africa, now grown worldwide  Most commonly in warm, tropical climates and seasons.  93% water, hence the name.  Nutritional benefits:  Proposed anti-inflammatory, anti-oxidant, and anti-microbial properties  Possibly cancer-preventing carotenoids  Immune system-strengthening ability  Low-fat, low-calorie traits.  Nutrients:  Vitamin C  Carotenoids lycopene, β-carotene, lutein, β-cryptoxanthin, and L- citrulline  Chemo-preventive cucurbatins  In this experiment, different concentrations of watermelon extract was the variable.

Past Studies/References “Screening of Anti-microbial Effects in Watermelon,” published in 2010 by a group of four Southeast-Asian scientists, found that some components of watermelon extract demonstrated anti-microbial and anti-fungal activity.

Gram-Positive Gram-Negative  Examples:  Staphylococcus  Streptococcus  Corynebacterium  Listeria  Bacillus  Clostridium  Simple cell wall  Antibiotics work against cell wall formation  Examples:  E. coli  Cell wall – thin extra layer of lipopolysaccharide, adds extra level of protection  Outer membrane protects bacteria from several antibiotics Bacterial Pathogens

E. coli  Bacteria found in the lower intestine of warm-blooded animals  Commonly used in biological experiments because of its easy manipulation and adaption to cultivation in a laboratory environment.

Staph. e.  Staph. species consisting harmless skin- dwelling microorganisms  Species of Staph. are commonly used in microbiological experiments because of variety of strain types within species – each strain has different features and toxins.

Purpose  Investigate the antimicrobial properties of watermelon extract.  Test the effects of different concentrations of watermelon extract on the survivorship of gram-positive (E. coli) and gram- negative (Staph.) bacteria

Alternative Hypothesis  Watermelon Extract will reduce the survivorship of E. coli and Staph. e. Null Hypothesis  Watermelon extract will have no effect on bacteria survivorship.

Materials  Watermelon Extract  Incubator  Micro- and Macro- pipettes, Pipette Tips  Klett Spectrophotometer and sidearm flasks  Spreader Bar, Ethanol, Burner  Vortex  SDF (Sterile Dilution Fluid)  Turn-table  Sterile Test Tubes  Escherichia coli  Staphylococcus epidermidis  LB (+agar plates) - (1% Tryptone, 0.5% Yeast Extract, 1% NaCl)  Test Tube Rack  Gloves, Goggles, Safety Equipment  0.22 Micron Syringe Filters

Procedures (1) 1. Bacteria (E. coli and Staph) 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 10 5 cells/mL. 100 µL of cell culture was then added to the silver solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 5. Watermelon Extract and SDF and 0.1mL of cell suspension were transferred to sterile test tubes, creating variable concentrations of: 0, 0.01, 0.1, and 1%

0% Stock Solution 0.01% Stock Solution 0.1% Stock Solution 1% Stock Solution Water8.9 mL Microbial Bacteria 0.1 mL SDF1 mL0.99 mL0.9 mL0 mL Watermelon Extract 0 mL0.01 mL0.1 mL1 mL Total10 mL Concentrations Chart

Procedures (2) 6. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 7. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates. 8. The plates were incubated at 37°C for 24 hours. 9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell µL of cell culture was then added to the silver solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL. 11. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 12. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates. 13. The plates were incubated at 37°C for 24 hours. 14. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Data Table Surviving number of E. coli colonies 1mL Variable, 0mL Sterile Fluid 0.1mL Variable, 0.9mL Sterile Fluid 0.01mL Variable, 0.99mL Sterile Fluid 0mL Variable, 1mL Sterile Fluid Agar Plate Agar Plate Agar Plate Surviving number of Staph. colonies Agar Plate Agar Plate Agar Plate

Results  The results from the data table and graph support the hypothesis, because as the concentration watermelon of watermelon extract increased, the number of surviving bacteria colonies decreased.  This outcome means that the watermelon extract did have a significant effect on the number of bacteria colonies that survived after living in the extract for 24 hours, thereby rejecting the null hypothesis.

Conclusion  The hypothesis was accepted – the watermelon extract effectively decreased the number of bacteria colonies surviving in it.  Watermelon extract reduced survivorship in both species, effect was more pronounced in Staph. e.  The idea that watermelon has antimicrobial properties is supported by this experiment, thus watermelon might actually help fight bacterial infections.

Limitations ---- Extensions  There was a lag time when plating the cells.  Certain bacteria groups experienced slightly varied exposure time while on the agar plates  Only a few concentrations were tested  Only one liquid exposure time was used in the experiment  Test higher concentrations of watermelon extract on bacteria  Test watermelon extract on other bacteria species  Test extract from various watermelon species on bacteria  Use longer liquid exposure times  Directly infuse the extract into the agar for prolonged exposure  Try to determine an LD50 for these species

Works Cited  Thirunavukkarasu, P., T. Ramanathan, N. Ravichandran, and L. Ramkumar. "Screening of Anti-microbial Effect in Watermelon." Science Alert. J. Biological Sciences, Web. 1 Jan  "Watermelon is Recommended for Breast Cancer." Food for Breast Cancer 1. Web. 1 Jan  Maitra, Anjana. "The Luscious Delicious Watermelon." BoloKids.com 4 Mar. 2007: 1-3. Web. 1 Jan  "Health Benefits of Watermelon." iloveindia.com 1-2. Web. 1 Jan