John DeSantis Grade 9 Pittsburgh Central Catholic High School

Made mostly of carbon and hydrogen Usually found underground, must be extracted by drilling Used to make gasoline for cars, airplanes, trains, etc. Used as a lubricant for machines. Pennsylvania crude (the type used in this experiment) is highly desired for motor oil refinement.

An estimated 706 million gallons of crude oil enter the ocean every year. Over half of that amount comes from land drainage and waste disposal. Tanker and drilling accidents only account for 8% of the total amount.

Very common Not well publicized Enters in small amounts Will often stay in the water for years

Bacteria found in the intestines of many mammals Prokaryotic cell Gram-negative Cells are rod shaped, usually about 2 micrometers in length Widely used model organism Reproduces rapidly, often within thirty minutes Many different strains, most are non-pathogenic, but pathogenic forms can produce fatal disease

Many components of crude oil have been shown to damage cell membranes. Other components have been shown to cause cancer.

Does crude oil have an effect on the survivorship of E. coli ?

To determine if oil in different concentrations will affect the survivorship of E. coli

Null hypothesis: the oil will not significantly affect E. coli survivorship. Alternate Hypothesis: the oil will significantly affect E. coli survivorship.

 LB agar plates  LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Klett spectrophotometer  Sterile pipette tubes  Micropipettes  Vortex  Incubator  Sidearm flask  Spreading platform, spreader bar  Ethanol, Bunsen burner, Matches  15 mL Sterile conical tubes with Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  Escherichia coli (DH5-Alpha)  0.22 micron syringe filter and 10 mL syringe  (Pennsylvania) Crude Oil

1. Escherichia 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 incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately cells/ml. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%,.1%, 1%, and 10% to total 9.9 ml ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 10 3 cells/ml.

0% Oil0.1% Oil1% Oil10% Oil SDF9.9 mL9.89 mL9.8 mL8.9 mL Oil0 mL0.01 mL0.1 mL1 mL E. Coli0.1 mL Total10 mL

1. Escherichia coli B 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 a shaking water bath until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 or 10 9 cells/ml. 4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%,.1%, 1%, and 10% to total 9.9 ml ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 10 3 cells/ml. Askdlfalsdkfkl;a 7. Every minute the tubes were inverted five times to mix the oil with the cell suspension. 8. The tubes were allowed to incubate at room temperature for 17 minutes. 9. After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on LB agar plates. 10. The plates were left to sit overnight. 11. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

P-value =

Oil ConcentrationT ValueInterpretation 0.1 %1.607Not Significant 1%1.4648Not Significant 10%1.806Not Significant T Critical = 2.88 (Significant variance) Alpha =.05 Dunnett’s Test

The ANOVA suggests that at least two groups varied significantly. The Dunnett’s test suggests that none of the groups varied significantly from the control. The null hypothesis can be accepted, none of the concentrations of crude oil had a significant effect on the survivorship of the E. coli colonies compared to the control.

The oil was somewhat insoluble, and needed to be inverted repeatedly Only one time of exposure was utilized Difficult to synchronize plating Composition of oil? Extensions Test higher concentrations of crude oil Test crude oil’s effects on numerous cell models including a gram positive bacteria and eukaryotic models (yeast, algae) Test the effects of refined motor oil and gasoline Test the effects of oil from different regions Limitations

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Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total