Antimicrobial Effects of Apple Juice Austin Brugger Grade
Antimicrobial Effects of Apple Juice Austin Brugger Grade 10 Pittsburgh Central Catholic High School
Problem � How do the effects of Natural and Processed Apple Juice compare when applied to two different types of bacteria?
Escherichia coli (E. Coli) � 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.
Staphylococcus epidermidis (Staph) � Common surface symbiont in many mammals (Human). � Gram (+) bacteria � Most strains considered non-pathogenic. � Pathogenic strains can be life-threatening.
Gram (-) vs. Gram (+) Bacteria Cell was contains an extra layer of lipopolysaccharides for extra protection. � Outer membrane protects bacteria from several antibiotics. � � Most pathogenic bacteria in humans are Gram (+) organisms. � Simple cell wall. � Some antibiotics work against the formation of the cell wall.
Naturally Squeezed Mc. Intosh Apples � 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.
Processed Giant Eagle Apple Juice �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.
Purpose � To assess the effects of Natural and Processed Apple Juice on the survivorship of Staphylococcus epidermidis and Escherichia coli.
Hypotheses � 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.
Materials Mc. Intosh Apples � Micro and Macro pipettes + Sterile tips � Spreader bars � LB agar plates (1% Tryptone, 0. 5% Yeast extract, 1% Na. Cl) � � Escherichia coli DH 5 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 10 m. L syringe �
Procedure (Liquid Exposure) 1. 2. 3. 4. 5. 6. Bacteria (E. coli and Staph) were grown overnight in sterile LB Media. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 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/m. L. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/m. L. The Mc. Intosh apples were then juiced and sterilized along with the processed apple juice. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0. 1%, and 10%.
Procedure (Liquid Exposure continued) Concentration Chart 0% 0. 1% 1% 10% Sterile Dilution Fluid 9. 9 m. L 9. 8 m. L 8. 9 m. L Microbe 0. 1 m. L Apple Juice (Natural/Proc essed) 0 m. L 0. 01 m. L 0. 1 m. L Total Volume 10 m. L 7. 8. 100 µL of cell culture was then added to the apple juice solutions, yielding a final volume of 10 m. L and a cell density of approximately 103 cells/m. L. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.
Procedure (Liquid Exposure Continued) 9. 10. 11. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB-agar plates. The plates were incubated at 37 C for 24 hours. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Agar Infusion Procedure 1. Sterilized Apple Juice was infused into the LB agar media in 2. 3. 4. 5. 6. two concentrations, a high 10 % (approximately 100 m. L/L Cranberry Juice) and low 1% (approximately 10 m. L/L Cranberry Juice), and used to create the LB agar plates. E. coli was grown overnight in sterile LB media. 100 u. L aliquots of bacterial suspensions from the CONTROL tube (exp 1) were spread onto the infused plates. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. The plates were incubated at 37 C for 24 hours. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Data Results (E. coli) 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% 430 426 438 421 436 420 429 433 429. 1 0. 1% 327 320 325 333 330 321 315 317 1% 210 198 217 208 192 218 204 209 10% 144 137 150 139 140 152 151 146 323. 5 207 144. 9 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% 380 391 376 378 382 384 374 383 381 0. 1% 289 291 287 294 286 297 284 285 1% 185 179 180 183 188 190 192 176 10% 127 123 129 126 119 121 130 133 289 184. 1 126
Data Results (Staph) 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% 671 667 661 673 674 670 669 672 669. 6 0. 1% 562 551 557 563 565 568 560 559 1% 474 468 470 476 479 465 463 479 10% 325 320 328 331 338 319 324 315 560. 6 471. 8 325 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% 592 590 588 581 593 600 603 581 591 0. 1% 496 503 487 490 505 487 485 1% 419 423 415 427 409 427 430 410 10% 288 276 292 280 283 294 290 277 494. 1 420 285
Apple Juice Effects on E. coli Survivorship 500 P-Value: 2. 41 E-34 450 P-Value: 7. 3 E-37 Resulting Number of Colonies 400 350 Natural E. coli 300 Processed E. coli 250 200 150 100 50 0 0% 0% 1% Concentration of Apple Juice 10%
Apple Juice Effects on Staph Colonies P-Value: 2. 72 E-38 Resulting Number of Colonies 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 P-Value: 2. 02 E-33 Natural Staph Processed Staph 0% 0% 1% Concentration of Apple Juice 10%
Dunnett’s Test E. coli T-Critical = 2. 88 Alpha= 0. 05 Natural Apple Juice Variable Concentration T-Value Interpretation 0. 1% Apple Juice 30. 23 Significant 1% Apple Juice 63. 58 10% Apple Juice 81. 39 Significant Processed Apple Juice Variable Concentration T-Value Interpretation 0. 1% Apple Juice 36. 11 Significant 1% Apple Juice 77. 37 10% Apple Juice 100. 21 Significant
Dunnett’s Test Staph T-Critical = 2. 88 Alpha= 0. 05 Natural Apple Juice Variable Concentration T-Value Interpretation 0. 1% Apple Juice 37. 59 Significant 1% Apple Juice 67. 87 10% Apple Juice 118. 19 Significant Processed Apple Juice Variable Concentration T-Value Interpretation 0. 1% Apple Juice 25. 1 Significant 1% Apple Juice 44. 29 10% Apple Juice 79. 27 Significant
Survivorship of Staph and E. coli 750 700 E. coli Natural 650 600 E. coli Processed Resulting Number of Colonies 550 Staph Natural 500 450 Staph Processed 400 350 LD 50’s 300 0. 9% 250 200 0. 8% 150 9. 7% 100 9. 3% 50 0 0% 1% 2% 3% 4% 5% 6% 7% Concentration of Apple Juice 8% 9% 10%
Survivorship Percentage Compared to the Control E. coli Natural Apple Juice Control (0% Apple Juice) 0. 1% Apple Juice 10% Apple Juice 77% 49% 34% Staph Natural Apple Juice 0. 1% Apple Juice 10% Apple Juice 83% 70% 48% E. coli Processed Apple Juice Control (0% Apple Juice) 0. 1% Apple Juice 10% Apple Juice 75% 48% 33% Staph Processed Apple Juice 0. 1% Apple Juice 10% Apple Juice 84% 71% 49%
Data Results (Agar Infusion) 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% 341 339 348 347 352 336 345 337 343. 1 High 10% 248 252 246 242 250 239 244 236 244. 6 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% 417 420 413 423 414 406 429 407 416. 1 High 10% 302 300 297 299 305 312 296 308 302. 4
Agar Infusion Effects on E. coli Survivorship P-Value: 1. 58 E-24 500 450 P-Value: 1. 97 E-21 Resulting Number of Colonies 400 350 300 250 Natural 200 Processed 150 100 50 0 0% Low 1% Concentration of Apple Juice High 10%
Dunnett’s Test Agar Infusion T-Critical = 2. 88 Alpha= 0. 05 Natural Apple Juice Variable Concentration T-Value Interpretation 1% Apple Juice 28. 94 Significant 10% Apple Juice 62. 10 Significant Processed Apple Juice Variable Concentration T-Value Interpretation 1% Apple Juice 8. 45 Significant 10% Apple Juice 41. 76 Significant
Conclusions 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. �
Limitations � Plating could have been unsynchronized � Only 3 concentrations of variable tested � Only one exposure time utilized Future Studies � 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?
Works Cited � http: //www. maplevalleyorchards. com/Pages/Apple. Tr ee. Descriptions_M. aspx? page=M � http: //www. webmd. com/a-to-z-guides/e-coliinfection-topic-overview � http: //web. uconn. edu/mcbstaff/graf/Student%20 pres entations/S%20 epidermidis/sepidermidis. html � http: //www. life. umd. edu/classroom/bsci 424/BSCI 22 3 Web. Site. Files/Gram. Posvs. Gram. Neg. htm � http: //www. specialtyproduce. com/produce/Mc. Intosh _Apples_1228. php
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