Austin Brugger Grade 10 Pittsburgh Central Catholic High School

 How do the effects of Natural and Processed Apple Juice compare when applied to two different types of bacteria?

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

 Common surface symbiont in many mammals (Human).  Gram (+) bacteria  Most strains considered non-pathogenic.  Pathogenic strains can be life-threatening.

 Most pathogenic bacteria in humans are Gram (+) organisms.  Simple cell wall.  Some antibiotics work against the formation of the cell wall.  Cell was contains an extra layer of lipopolysaccharides for extra protection.  Outer membrane protects bacteria from several antibiotics.

 Solid red apple with a tart taste and tender flesh.  Cultivated in Eastern Canada and New England.  Well-suited for cider, juice, and pies.  Reputation as a healthy snack.

 A flavorful, common processed brand of apple juice.  Contains 50% natural apple juice.  Artificial flavoring and artificial dyes with sucrose.  Lower cost than organic juice.

 To assess the effects of Natural and Processed Apple Juice on the survivorship of Staphylococcus epidermidis and Escherichia coli.

 Null Hypothesis: There will be no effect of Natural and Processed Apple Juice in the survivorship of E.coli and Staph.  Alternative Hypothesis: Both juices will significantly reduce the survivorship of the bacteria, and Processed Apple Juice will reduce survivorship more than Natural Apple Juice.

 McIntosh Apples  Micro and Macro pipettes + Sterile tips  Spreader bars  LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl)  Escherichia coli DH5- alpha(E.Coli) (Obtained from Doonan Lab, CMU)  Staphylococcus epidermidis (Staph) (Obtained from Carolina Science Supply)  Giant Eagle Processed 50% Apple Juice with Preservatives  Burner  Electric Juicer  Sidearm Flask  Turn-table  Vortex  Incubator  Gloves/goggles  Klett Spectrophotometer  SDF (Sterile Dilution Fluid)  20 Sterile Test Tubes  Ethanol  Luria Broth Agar Plates(To be Infused with Agar Plates)  0.22 micron syringe filters and 10mL syringe

1. Bacteria (E.coli and Staph) were grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were 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 cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5. The McIntosh apples were then juiced and sterilized along with the processed apple juice. 6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, and 10%.

µL of cell culture was then added to the apple juice solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 8. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. Concentration Chart 0%0.1%1%10% Sterile Dilution Fluid 9.9 mL9.89 mL9.8 mL8.9 mL Microbe0.1 mL Apple Juice (Natural/Proc essed) 0 mL0.01 mL0.1 mL1 mL Total Volume10 mL

9. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB-agar plates. 10. The plates were incubated at 37 C for 24 hours. 11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

1. Sterilized Apple Juice was infused into the LB agar media in two concentrations, a high10 % (approximately 100mL/L Cranberry Juice) and low 1% (approximately 10mL/L Cranberry Juice), and used to create the LB agar plates. 2. E. coli was grown overnight in sterile LB media uL aliquots of bacterial suspensions from the CONTROL tube (exp 1) were spread onto the infused plates. 4. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. 5.The plates were incubated at 37 C for 24 hours. 6.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Amount of Colonies per Plate (Natural Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean 0% % % % Amount of Colonies per Plate (Processed Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean 0% % % %

Amount of Colonies per Plate (Natural Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean 0% % % % Amount of Colonies per Plate (Processed Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean 0% % % %

P-Value: 2.41E-34 P-Value: 2.41E-34

P-Value: 2.72E-38 P-Value: 2.72E-38 P-Value: 2.02E-33 P-Value: 2.02E-33

T-Critical = 2.88 Alpha= 0.05 Natural Apple Juice Variable ConcentrationT-ValueInterpretation 0.1% Apple Juice30.23 Significant 1% Apple Juice63.58 Significant 10% Apple Juice81.39 Significant Processed Apple Juice Variable ConcentrationT-ValueInterpretation 0.1% Apple Juice36.11 Significant 1% Apple Juice77.37 Significant 10% Apple Juice Significant

T-Critical = 2.88 Alpha= 0.05 Natural Apple Juice Variable ConcentrationT-ValueInterpretation 0.1% Apple Juice37.59 Significant 1% Apple Juice67.87 Significant 10% Apple Juice Significant Processed Apple Juice Variable ConcentrationT-ValueInterpretation 0.1% Apple Juice25.1 Significant 1% Apple Juice44.29 Significant 10% Apple Juice79.27 Significant

LD50’s 0.9% 0.8% 9.7% 9.3%

E. coli Natural Apple Juice 0.1% Apple Juice1% Apple Juice10% Apple Juice Control (0% Apple Juice) 77%49%34% E. coli Processed Apple Juice 0.1% Apple Juice1% Apple Juice10% Apple Juice Control (0% Apple Juice) 75%48%33% Staph Natural Apple Juice 0.1% Apple Juice1% Apple Juice10% Apple Juice Control (0% Apple Juice) 83%70%48% Staph Processed Apple Juice 0.1% Apple Juice1% Apple Juice10% Apple Juice Control (0% Apple Juice) 84%71%49%

Amount of Colonies per Plate (Natural Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean Low 1% High 10% Amount of Colonies per Plate (Processed Apple Juice) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Mean Low 1% High 10%

P-Value: 1.58E-24 P-Value: 1.58E-24 P-Value: 1.97E-21 P-Value: 1.97E-21

T-Critical = 2.88 Alpha= 0.05 Natural Apple Juice Variable ConcentrationT-ValueInterpretation 1% Apple Juice28.94 Significant 10% Apple Juice62.10 Significant Processed Apple Juice Variable ConcentrationT-ValueInterpretation 1% Apple Juice8.45 Significant 10% Apple Juice41.76 Significant

 Reject the null hypothesis that there will be no effect on the survivorship of Staph and E. coli.  The increased concentrations of both processed and natural apple juice correlated with decreasing survivorhip.  The difference between natural and processed apple juice did not play a significant role in the survivorship of the bacteria.

 Plating could have been unsynchronized  Only 3 concentrations of variable tested  Only one exposure time utilized  Use more types of Apples and Apple Juices  Use more Apple Juice concentrations  Perform a Trypan blue exclusion assay to test for dead cells  Vary exposure times  Explore growth curve effects  Synergistic effects?

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