ANALYSIS OF MICROBIAL COMPETITION Eric Pomper Microbiology 9 Pittsburgh Central Catholic High School Grade 9
Introduction Escherichia coli (E. coli) and Saccharomyces cerevisiae (Yeast) were grown together to determine what, if any, symbiotic relationship existed Models = Escherichia coli and Saccharomyces cerevisiae Variable= Symbiosis
Symbiosis Symbiosis is a close ecological relationship between two or more organisms. The five main types of symbiosis are Mutualism, Commensalism, Parasitism, Competition, and Neutralism. These categories are classified by which organisms benefit from the relationship, if any.
Mutualism, Commensalism, and Parasitism Mutualism Commensalism Parasitism Definition Potential experimental outcome Both organisms benefit when grown together E. coli and S. cerevisiae both have a higher growth rate Examples Sea Anemone / Clownfish One organism is benefitted and the other organism is neither helped nor harmed E. coli or S. cerevisiae has a higher growth rate the other has normal growth Barnacle / Whale One organism is benefitted and the other is harmed E. coli or S. cerevisiae has a higher growth rate the other has less than normal growth Dog / Flea
Competition Symbiotic relationship among living organisms for factors such as resources, food, space, or shelter. Organisms must compete in a variety of ways within their ecosystem to survive: predation, interspecific competition, and intraspecific competition. Experimental outcome: reduced colony number of one species, or reduced colony number of both.
Neutralism Symbiotic relationship in which neither species is affected by the relationship. Experimental outcome: the colony density of yeast and E. coli on mixed plates equal to control plates.
Escherichia coli One of the most common forms of bacteria found in many environments Symbiont in intestinal tracts of many mammals Gram negative, rod shaped bacillus Most non-pathogenic Pathogenic strains can lead to life threatening infections. Photo Source: Wikipedia
Saccharomyces cerevisiae Fungal microorganism famous for use in common types of fermentation Common unicellular eukaryote, most intensively studied cell in molecular and cell biology. Common model used to study cell events in human biology, such as cell division, DNA replication, and the process of the cell cycle Photo Source: Wikipedia
Purpose Purpose = To assess the symbiotic relationship between Escherichia coli (E. coli) and Saccharomyces cerevisiae (Yeast)
Hypotheses Alternate Hypothesis = Competitive symbiosis will occur on both yeast and bacteria growth plates, resulting in a significant reduction in colony number of both species. Null Hypothesis = There will be no significant difference between the number of colonies grown when E. coli and S. cerevisiae are grown together.
Materials 24 LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride) 24 YEPD agar plates( 1% yeast extract, 2% glucose, 2% peptone) Sterile pipette tips Micropipeters Macropipeters Incubator Sidearm flask Spread plate Spreader bar Ethanol 20 ml Sterile capped test tubes E. coli and S. cerevisiae models Sterile dilution fluid Klett Spectrophotometer
Procedure 1. E. coli and S. cerevisaie were grown overnight in sterile LB and YEPD media. 2. Samples of the overnight cultures were added to fresh media in sterile sidearm flasks. 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 10 8 and 10 7 cells/ml. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 4 cells/ml.
Procedure 5. Sterile LB (Bacteria) and YEPD (Yeast) agar plates were labeled. 5 ml solutions using sterile water were made using the following concentrations: - E. coli solution: 4.5 ml sterile water, 0.5 ml E. coli - Yeast solution: 4.5 ml sterile water, 0.5 ml S. cerevisiae - E. coli & yeast solution: 4 ml sterile water, 0.5 ml E. coli, 0.5 ml yeast. 6. The solutions were vortexed to prepare for pipetting. 7. 0.1 ml of the 5 ml solutions were pipetted onto the Bacteria and Yeast agar plates in the following combinations: (48 plates total)
Procedure 8. The plates were incubated at 34 C for 48 hours. 9. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
Questions Does yeast grow equally on YEPD and LB plates? Does E. coli grow equally on YEPD and LB? Competition on YEPD plates? Competition on LB plates?
Colonies (Average of 8 Replicates) Microbial Cell Survivorship 450 400 0.000107 350 0.097723 300 0.005631 250 200 150 100 0.027586 0.000002198 Yeast Bacteria 50 0 0.000501 Y/YEPD Y/LB Y+B/YEPD Y+B/LB B/LB B/YEPD
Data Analysis The data was analyzed using ANOVA. 6 ANOVAs were performed- Yeast grown on LB vs. YEPD. E. coli grown on LB vs. YEPD plates Yeast on YEPD from symbiosis vs. Yeast on YEPD E. coli on YEPD from symbiosis vs. E. coli on YEPD Yeast on LB from symbiosis vs. Yeast on LB E. coli on LB from symbiosis vs. E. coli on LB
ANOVA P Values ANOVA P Value Variation Yeast on YEPD vs. Yeast on LB 0.000002198 Significant E. coli on LB vs. E. coli on YEPD 0.097723 Not Significant Yeast on YEPD vs. Yeast /Symbiosis on YEPD 0.027586 Significant E. coli on YEPD vs. E. coli / Symbiosis on YEPD 0.000107 Significant Yeast on LB vs. Yeast /Symbiosis on LB 0.000501 Significant E. coli on LB vs.. E. coli / Symbiosis on LB 0.005631 Significant
Conclusions Following the results of the ANOVAS, the null hypothesis was rejected. 5 out of the 6 ANOVAS performed produced P values <.05, indicating that there was significant variation among the groups. The null hypothesis was rejected. There is a clear trend that suggests that the E. coli and yeast being grown together caused competition to occur on both the YEPD and LB plates, resulting in a significant reduction in the number of colonies.
Limitations Limited concentrations Limited number of replicates Limited incubation time Limited combinations of media for symbiosis Plating of media solutions could have been better synchronized.
Extensions The area of the plate could have been increased or decreased to make the symbiotic colonies increase or decrease respectively. Perform a growth rate experiment. Another variable could have been used along with symbiosis to see if it had an effect on the symbiosis. Different concentrations of this second variable could have been created to see if these different concentrations had any effect had any effect on the symbiosis.
References www.biology-online.org www.britannica.com/mutualism www.cals.ncsu.edu/course/ent591k/symbiosis.html www.classes.entom.wsu.edu www.dakotayeast.com www.eoearth.org/article/predation www.marietta.eddu/bio/biomes/symbiosis www.medicalnewstoday.com www.ncbi.nlm.nih.gov/pubmed www.sciencedaily.com/articles/parasitism www.tiem.utk.edu/~gross/bioed/bealsmodules/competition.html
Growth Results Repetition Jkkl E. coli on Bacteria Plate Y east on Yeast Plates Y east on Bacteria Plates Sy mbiosis on Yeast Plate Sy mbiosis on Bacteria Plate E. coli on Y east Plates Y east E. coli Y east E. coli 1 354 263 17 348 96 266 0 281 2 338 225 9 354 114 253 0 141 3 373 231 11 285 111 259 0 287 4 287 178 7 342 102 217 0 53 5 365 108 20 377 81 243 0 192 6 263 102 5 476 116 247 0 264 7 315 92 29 481 107 234 0 289 - Yeast growth is not significant on bacteria agar plates 8 399 117 7 467 108 269 0 278 - E. coli growth was more substantial in a symbiotic setting Average 336.75 164.5 13.125 391.25 104.375 248.5 0 223.125
ANOVA Stat Analysis Anova: Single Factor Yeast on YEPD Plates vs. Yeast on LB Plates ANOVA SUMMARY Groups Count Sum Average Variance Column 1 8 1316 164.5 4654 Column 2 8 105 13.125 68.125 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 91657.56 1 91657.56 38.82047 2.1975E-05 4.60011 Within Groups 33054.88 14 2361.063 Total 124712.4 15
ANOVA Stat Analysis Anova: Single Factor E. coli on LB vs. E. coli on YEPD SUMMARY Groups Count Sum Average Variance Column 1 8 2694 336.75 2096.214 Column 2 8 3130 391.25 5450.214 ANOVA rce of Varia SS df MS F P-value F crit Between G 11881 1 11881 3.148774 0.097723 4.60011 Within Grou 52825 14 3773.214 Total 64706 15
ANOVA Stat Analysis Anova: Single Factor Yeast on YEPD vs. Yeast on YEPD from symbiosis SUMMARY Groups Count Sum Average Variance Column 1 8 1316 164.5 4654 Column 2 8 835 104.375 130.5536 ANOVA rce of Varia SS df MS F P-value F crit Between G 14460.06 1 14460.06 6.044477 0.027586 4.60011 Within Grou 33491.88 14 2392.277 Total 47951.94 15
ANOVA Stat Analysis Anova: Single Factor E. coli on YEPD vs. E. coli on YEPD from Symbiosis SUMMARY Groups Count Sum Average Variance Column 1 8 3130 391.25 5450.214 Column 2 8 1988 248.5 298.8571 ANOVA rce of Varia SS df MS F P-value F crit Between G 81510.25 1 81510.25 28.35597 0.000107 4.60011 Within Grou 40243.5 14 2874.536 Total 121753.8 15
ANOVA Stat Analysis Anova: Single Factor Yeast on LB vs. Yeast on LB from Symbiosis SUMMARY Groups Count Sum Average Variance Column 1 8 105 13.125 68.125 Column 2 8 0 0 0 ANOVA rce of Varia SS df MS F P-value F crit Between G 689.0625 1 689.0625 20.22936 0.000501 4.60011 Within Grou 476.875 14 34.0625 Total 1165.938 15
ANOVA Stat Analysis Anova: Single Factor E. coli on LB vs. E. coli on LB from symbiosis SUMMARY Groups Count Sum Average Variance Column 1 8 2694 336.75 2096.214 Column 2 8 1785 223.125 7586.696 ANOVA rce of Varia SS df MS F P-value F crit Between G 51642.56 1 51642.56 10.66674 0.005631 4.60011 Within Grou 67780.38 14 4841.455 Total 119422.9 15