1. Fruit Juice Suppression of Staph. Biofilm Formation
Project By: Warren Sipe
Central catholic high School
Grade 11

2. Cherry and Cranberry Juices
Pure cherry juice
Cranberry
Cranberry juice (primary ingredient)
Grape juice
Apple juice
Pear juice
Pectin
Vitamin C
Commercially available

3. Biofilms Coherent and generally adherent cells
Extracellular Polymeric Substance (EPS)
80% of human diseases
Phenotypic shift in gene regulation
More resistant- 1000x
Lateral gene transfer
Quorum sensing

4. Biofilm Inhibition Adherence- conditioning films, polysaccharides
Anti-biofilm agents- chemical inhibitors to adherence
Other major methods:
Surface modification (anti-microbial coatings)
Hydrophobicity

5. Model Bacteria (Staph. E)
Staphylococcus epidermidis
Extremely common bacteria
Frequently used for scientific research
Skin flora
Gram positive
Can be pathogenic

6. Importance Biofilms- 80% of human infectious disease
Past studies on anti-microbial effects of juice
Poor understanding of effect on biofilms
Important for hospitals
Catheters
Need for non-toxic, biofilm targeted disinfection

7. Purpose
To determine the effect of cranberry and cherry juices on Staph. E biofilms
To determine the effect of cranberry and cherry juices on Staph. E survivorship
To relate the biofilm effects to the microbiocidal effects

8. Hypothesis
Null hypothesis- Neither juice will significantly effect Staph. E survivorship or biofilm formation
Alternate hypothesis- Both juices will significantly effect Staph. E survivorship and biofilm formation

9. Materials Staph. E culture Klett Spectrophotometer (reading in KU)
Cherry juice
Cranberry juice
LB agar plates
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Sterile dilution fluid (100 mM KH2PO4, mM K2HPO4, 10 mM MgSO4, 1mM NaCl)
Sterile pipette tips
Micropipettes
Vortex
Incubator (37 degrees C)
Sidearm flask
Sterile spreader bars
Ethanol
96 well tissue culture treated microtiter dish
Crystal violet
Acetic acid
Microtiter plate absorbance reader

10. Survivorship Procedure
1. Staph. E was grown overnight in sterile LB Media.
2. The culture was 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 juices were sterilized by means of a 0.2 micron syringe filter 6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 1%, 10%, and 50%.

11. Concentration Chart 0% 1% 10% 50% Sterile Dilution Fluid 9.9 ml 9.8 ml
Staph. E
0.1 ml
Juice
0 ml
1.0 ml
5.0 ml
Total volume
10.0 ml

12. Survivorship Procedure (cont.)
6. The solutions were vortexed and allowed to sit at room temperature for 10 minutes.
µL aliquots were removed from the tubes and spread on LB-agar plates.
8. The plates were incubated at 37°C for 48 hours.
9. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

13. Biofilm Procedure Growing a Biofilm
1. Tubes were prepared according to the dilution chart above.
μL from the tubes was added per well in a 96 well dish. 8 replicates were performed from each tube.
3. The microtiter plate was incubated for 48 hours at 37°C.
Staining the Biofilm
1. After incubation, the cells were gently removed out by turning the plate and allowing to drip dry.
2. The plate was gently submerged in a small tub of water. The plate was allowed to drip dry.
μL of a 0.1% solution of crystal violet in water was added to each well of the microtiter plate.

14. Biofilm Procedure (cont.)
4. The microtiter plate was incubated at room temperature for 10 minutes.
5. The plate was rinsed by submerging in a tub of water as outlined above.
6. The microtiter plate was turned upside down and dried overnight.
Quantifying the Biofilm
μL of 30% acetic acid in water was added to each well of the microtiter plate to solubilize the CV.
2. The microtiter plate was incubated at room temperature for 10 minutes.
3. The absorbance of the microtiter plates was quantified in a microtiter plate reader at 550 nm using 30% acetic acid in water as the blank.
*credit George O’Toole

15. P-Value=
P-Value= E-6

16. P-Value= E-10
P-Value= E-10

18. Analysis and Stats
Did either cranberry or cherry juice significantly effect Staph. survivorship?
Single factor ANOVA- Ps of and E-6, respectively
Significant for both
Reject the null, accept the alternate for both
Did either cranberry or cherry juice significantly effect Staph. biofilms?
Single factor ANOVA- Ps of E-10 and E-10, respectively

19. Conclusions The null hypothesis was rejected
The alternate hypothesis was supported
Both juices showed anti-microbial and biofilm inhibitive effects
Biofilm effect was greater than survivorship effect for both juices

20. Possible Limitations Spread plating was not perfectly synchronized
Only survivorship, and not growth, was measured
Only Staph. E was tested
Only cranberry and cherry juices were tested
Only one growth time was used for the biofilms

21. Extensions
Efforts will be made to achieve more synchronous spread plating
Survivorship and growth will be measured
More species will be tested
Tests will be done at more stages of growth
Other juices will be tested

22. Resources
Special thanks to Dr. Carrie Doonan of CMU for the use of her lab and equipment
Bukhari, Mohammad. "Staphylococcus Epidermidis." Staphylococcus Epidermidis University of Conneticut, Web. 24 Jan
Domenico, Phil. "Natural Anti-Biofilm Agents." The Science of Nutrition. N.p., 22 Sept Web. 31 Dec
Fuente-Núñeza, César De La, Victoria Korolikb, Manjeet Bainsa, Uyen Nguyenc, Elena B. M. Breidensteina, Shawn Horsmand, Shawn Lewenzad, and Lori Burrowsc And. "Inhibition of Bacterial Biofilm Formation and Swarming Motility by a Small Synthetic Cationic Peptide." Antimicrobial Agents and Chemotherapy, 01 May Web Jan
O'Toole, George A. "Microtiter Dish Biofilm Formation Assay." Journal of Visualized Experiments : JoVE. MyJove Corporation, Web. 31 Dec
Romling, U., and C. Balsabore. "Biofilm Infections, Their Resilience to Therapy and Innovative Treatment Strategies." Journal of Internal Medicine (2012): Web Dec

23. Cranberry Survivorship1 10 50
Cranberry Biofilm
190
163
143
157
3.022
1.989
2.155
0.834 2
197
172
170
158
3.184
2.914
2.042
0.828 3
185
193
173
175
3.822
2.724
2.697
1.103 4
182
164
178
166
3.41
1.887
2.365
1.214 5
192
147
3.827
2.343
2.202
1.12 6
188
152
168
3.503
2.66
2.647
0.931
Total
1129
1072
963
981
20.768
14.517
14.108
6.03
Average
160.5
163.5
2.4195
1.005
Cherry Survivorship
Cherry Biofilm
211
169
150
2.832
2.313
1.159
183
140
151
3.231
3.101
1.537
189
154
142
3.558
2.682
1.404
199
165
161
153
3.199
2.123
1.346
200
184
179
156
2.984
2.046
1.327
177
171
3.043
1.994
1.165
1170
1024
943
916
18.847
14.259
7.938
195
2.3765
1.323