Water Source Influence of Microbial Survivorship By Zachary Poppish Grade 9 Central Catholic High School Pittsburgh

Rationale: Water sources can vary greatly in their quality and properties. Various types of microbes exist in natural water sources. Do river water and creek water vary significantly in their ability to support prokaryotic and eukaryotic microbial life?

Common Creek and River Pollutants Detergents Fertilizers Animal waste Automotive products Pesticides Can runoff products aid or hinder microbial growth?

Allegheny River The sample of the River was taken along the side of a railroad track. Many pollutants in the Allegheny River from: Tires, garbage, styrofoam, plastic, lumber and many other pollutants

Deer Creek The area of Deer Creek where my sample was taken is near a small industrial park This includes: A metal shop that carries petroleum A trucking distribution center. A PennDot station with road salt A Brick yard and Lumber Mill

Microbes Tested Escherichia coli (E.coli) Saccharomyces cerevisiae (Yeast)

Escherichia coli (E.Coli) Gram-negative microflora Found in the intestines of warm-blooded animals as well as excrement Most varieties are harmless, though some can cause illnesses.

Saccharomyces cerevisiae (Yeast) Single celled fungi Most commonly studied organism- similar biochemistry and genes as other Eukaryotes Gram-Positive microflora Most commonly called yeast or baker's yeast

Materials used Sterile pipette tips Micropipettes Vortex Incubator 72 YEPD agar plates(1% yeast extract, 2% peptide, and 2% glucose) Sterile pipette tips Micropipettes Vortex Incubator Spreading turntable Spreader bar Escherichia coli (e.coli) Saccharomyces cerevisiae (yeast) Sample water from Deer Creek Sample water from the Allegheny River Sample of SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, and 1mM NaCl)

Procedure Collected samples from local creek and river in a sterile container Sterilized the 2 water samples using 0.2 micron syringe filter The waters were transferred to sterile 15 mL conical tubes The yeast and E. coli were diluted from their stocks to a tube with a concentration of 10^5 cells per mL 100 microliter samples of the microbes were transferred into the water samples The tubes were incubated at room temperature for 5 min and 1 hr After 5 and 60 minutes 100 microliters of the mixtures were transferred to YEPD agar plates. Plates were incubated (yeast= 37C for 48hr and E. coli 37C for 24hr) The resulting colonies were counted

Variables Independent: Exposure Time/Water sample/Microbe used Dependent: Amount of colonies Control: SDF

Hypothesis Alternative: The natural water bodies will reduce the microbial survivorship. Null: The waters samples will not significantly hinder the microbes survivorship.

E. coli average colonies P-value: 5 min: 0.747061 60 min: 0.002009

Dunnett’s Test-E. coli F-Crit:3.885 Creek River 5 min Unable to dunnett’s test because data was not significant 1 hr -8.026-Significant -1.538-insignificant

Yeast Average Colonies P-value: 5 min: 0.000116 60 min: 0.829171 P-value: 5 min: .0.001233 60 min: .0.587849

Dunnett’s Test-Yeast F-crit:3.885 Creek River 5 min 9.937-significant 1 hr Unable to dunnett’s test because data was not significant

Percent Survivorship

Conclusion The alternative Hypothesis was supported for 2 of the cases. The E. Coli survivorship was reduced after 1 hour in the creek. The creek appeared to enhance yeast growth after 5 minutes. The null was accepted for all other waters/times.

Reason Different pH values? Different minerals and other ions? Different types of pollutants?

Limitations Tested only one river and one creek Only 2 time periods tested Only survivorship and no other health parameters Unknown composition of waters Water was taken from river and creek during winter months

Improvements Test multiple creeks and rivers to test for pollutants/different areas (RESIDENTIAL, AGRICULTURAL AND URBAN) Do a chemical analysis of the waters Test during different times of the year/temperatures Test lakes and ponds for pollutants

Works cited https://about-ecoli.com/ http://www.waterencyclopedia.com/La-Mi/Microbes-in-Lakes-and-Streams.html https://www.lenntech.com/rivers-pollution-quality https://people.uwec.edu/piercech/hml/Common%20sources%20of%20water%20pollution.htm https://redstaryeast.com/science-yeast/what-is-yeast/ https://wiki.yeastgenome.org/index.php/What_are_yeast%3F

Overall Results:I E-coli Sterilized Water 435 421 Creek Water 326 239 5 MINUTES 1 HOUR E-coli Sterilized Water 435 421 Creek Water 326 239 River Water 414 391 Yeast Sterilized Water 62 42 Creek Water 216 38 River Water 64 45 E-coli higher survivorship in 5 min versus 1 hour Reason: Less exposure to chemicals in water

E-Coli: SDF Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 460 452 440 404 432 424 435 1 HR 408 420 400 451 384 464 421 P-Value: 0.367479668 Highest Survivorship: 5 minutes : Test #1 with 460 colonies 1 HR: Test #6 with 464 colonies On Average: E-coli survivorship is higher after 5 minutes

E. COLI SDF (Sterilized Water)

Yeast: SDF Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 87 35 70 55 54 69 62 1 HR 39 46 34 44 34 52 42 P-Value: 0.027479 Highest Survivorship: 5 minutes : Test #1 with 87 colonies 1 HR: Test #6 with 52 colonies On Average: E-coli survivorship is higher after 5 minutes

YEAST SDF (Sterilized Water)

E-Coli: Creek Water P-Value: 0.293216 Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 240 101 540 344 410 320 326 1 HR 400 296 248 136 288 67 239 P-Value: 0.293216 Highest Survivorship: 5 minutes : Test #3 with 540 colonies 1 HR: Test #1 with 400 colonies On Average: E-coli survivorship is higher after 5 minutes

E. COLI CREEK WATER

Yeast: Creek Water P-Value: 0.002736 Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 208 216 424 176 104 168 216 1 HR 28 60 38 43 47 9 38 P-Value: 0.002736 Highest Survivorship: 5 minutes : Test #3 with 424 colonies 1 HR: Test #2 with 60 colonies On Average: E-coli survivorship is higher after 5 minutes

YEAST CREEK WATER

E-Coli: River Water P-Value: 0.586029 Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 308 526 368 524 364 392 414 1 HR 408 400 432 320 360 424 391 P-Value: 0.586029 Highest Survivorship: 5 minutes : Test #2 with 526 colonies 1 HR: Test #6 with 424 colonies On Average: E-coli survivorship is higher after 5 minutes

E. COLI RIVER WATER

Yeast River Water P-Value: 0.121499 Time Test #1 Test #2 Test #3 Test #4 Test #5 Test #6 Average 5 Min 33 51 84 92 38 88 64 1 HR 55 32 50 48 35 49 45 P-Value: 0.121499 Highest Survivorship: 5 minutes : Test #4 at 92 colonies 1 HR: Test #1 at 55 colonies On Average: Yeast survivorship is higher after 5 minutes

YEAST RIVER