cranberry juice antimicrobial properties zane stiles central catholic high school grade 9
TRANSCRIPT
Cranberry Juice Antimicrobial Properties
Zane StilesCentral Catholic High School
Grade 9
Antimicrobial effects of berries
o Types of berriesRaspberryCranberryBlueberry
o Previous StudiesCancer cellsantioxidant activityStaphylococcus aureus and Escherichia coli
Cranberry Health Benefitso Cancer and disorders
Cardiovascular
Immune Systems
o Dental Plaque
o Kidney Stones
Urinary Tract Infections (UTIs)
o On the Rise
o Frequency
o Cause
o Common Cures
Cranberry Juice and UTIso Shown to prevent or minimize
pathology in vitro
o Inhibit adhesion of E. coli
o Results variable and moderate
Cranberry Juice and UTIso Previous Results Inconclusive
o High Drop-out Rate
o Adverse Effectsgastrointestinal intoleranceweight gain drug-cranberry interactions
Escherichia coli
o Inhabit intestinal tract
o Many different types
o Many are not harmful
o Different types of infections
o Serves as a common prokaryotic cell model
E. coli and Cranberry Juice
o E. coli and other bacteria cause many infections
o Cranberry Juice is claimed to prevent these
o Does cranberry juice affect E. coli populations?
Purpose
To determine if cranberry juice has an affect on E. coli
survivorship in vitro.
Hypotheses
oNull hypothesis: Cranberry juice does not affect E. coli survivorship in vitro.
oAlternative hypothesis: Cranberry juice does affect E. coli survivorship in vitro.
o Labeling tapeo Ethanolo Latex gloveso E. coli DH5 Alphao Micropipetteso Micro racko Micro tubeso LB media (0.5% yeast
extract, 1% tryptone, 1% sodium chloride).
o Luria Broth agar plates (to be infused with cranberry juice)
Materialso Cranberry Juice (natural,
unsweetened)o Sterile Dilution Fluid
(100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
o Turntableo Luria Broth agar plateso Bunsen burnero Spread baro Incubatoro Klett Spectrophotometero Matches
Pulse Liquid Exposure Procedure1. E. coli 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 105 cells/mL.
5. Sterilized Cranberry juice was mixed with the appropriate amount of SDF to create cranberry juice concentrations of 10%, 1%, and 0.1%.
Chart of Liquid Concentration0%
Concentration0.1%
Concentration1%
Concentration10%
Concentration
Microbe 0.1mL 0.1 mL 0.1 mL 0.1 mL
SDF 9.9mL 9.89mL 9.8mL 8.9mL
Cranberry Juice
0mL 0.01mL 0.1mL 1mL
Total 10mL 10mL 10mL 10mL
Pulse Liquid Exposure Procedure
6. 100 µL of cell culture was then added to the cranberry juice solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
8. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on a set of regular LB plates.
9. The plates were incubated at 37 C for 24 hours.
10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Agar Infusion Procedure1. Sterilized Cranberry Juice was infused into the LB
agar media in two concentrations, 10 % (approximately 100mL/L Cranberry Juice) and low (approximately 10mL/L Cranberry Juice), and used to create the LB agar plates.
2. E. coli was grown overnight in sterile LB media.
3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
4. 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.
5. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
Agar Infusion Procedure
6. 100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
7. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB 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.
102 cells103 cells/mL with cranberry juice
108 cells/mL (E. coli)
100 uL
105 cells/mL (E. coli)
100 uL
Col
ony
Cou
ntCranberry Juice Pulse Liquid Exposure Effects on E. coli
Survivorship
Concentration of Cranberry Juice
P-value
1.35E-08
P<0.05
P<0.05
P>0.05
Dunnett’s Test Analysis
T Critical = 3.10 (significant)
Alpha = 0.05
Pulse Liquid Exposure
Variable Concentration
T value Interpretation
0.1% Cranberry Juice
3.00 Not Significant
1% Cranberry Juice
6.18 Significant
10% Cranberry Juice
10.38 Significant
0% 2% 4% 6% 8% 10% 12%0
50
100
150
200
250
300
350
400
450
500
LD 50 = 0.7%
Cranberry Juice Pulse Liquid Exposure Effects on E. coli Survivorship
Col
ony
Cou
nt
Concentration of Cranberry Juice
Col
ony
Cou
ntCranberry Juice Agar Infusion Effects on E. coli
Survivorship
Concentration of Cranberry Juice
P<0.05
P<0.05
P-value
2.73E-13
Dunnett’s Test Analysis
T Critical = 2.67 (significant)
Alpha = 0.05
Agar Infusion Exposure
Variable Concentration
T value Interpretation
1% 11.39 Significant
10% 17.30 Significant
Conclusionso The null hypothesis that cranberry juice
does not affect E. coli survivorship in vitro was rejected for the concentration of 1% and 10% cranberry juice in both types of exposure.
o The null hypothesis could not be rejected for the lowest concentration of 0.1% cranberry juice.
o The results indicate that 1% and 10% cranberry juice did negatively influence E. coli survivorship.
Limitations, Extensions, and Future Studies
o The E. coli plating was not exactly synchronized, possibly contributing to varying colony counts.
o Infused amount of cranberry juice in the LB agar was not exact.
o Does not account for dilution in the body
o Larger sample sizes will be employed in the next round of testing.
o Higher and lower concentrations of cranberry juice will be tested.
o More species of bacteria will be tested.
Limitations
Extensions and Future Studies
Referenceso Kessman, Scott. The Health Benefits of Cranberry Juice. July 2007
http://www.associatedcontent.com/article/34119/the_health_benefits_of_cranberry_juice.html?cat=5
o Author: unknown. Cranberry History. 2010
http://www.oceanspray.com/about/cranberry_history2.aspx
o Author: unknown. E. coli bacteria: what are they, where did they come from, and why are some so dangerous? 2005
http://www.about-ecoli.com/
o Author: unknown. The History of Cranberries November 14, 2006
http://www.kitchenproject.com/history/cranberries/index.htm
o Author: unknown. Growing Cranberries. 2008
http://cranberryjuice.com/about.html
o Author: unknown. SuperFruit: A Rundown on Cranberry Juice. 2008
http://cranberryjuice.com/benefits.html
o Author: unknown. Urinary Tract Infections in Adults. 2005
http://kidney.niddk.nih.gov/kudiseases/pubs/utiadult/
o Heather M.A. Cavanagh, Michael Hipwell, Jenny M. Wilkinson. Journal of Medicinal Food. March 2003,. March 2003, 6(1): 57-61.
o Guay, David. Cranberry and Urinary Tract Infections, May 2009
http://adisonline.com/drugs/pages/articleviewer.aspx?year=2009&issue=69070&article=00002&type=abstract