John DeSantis Grade 10 Central Catholic High School

Crude oil pollution enters microorganisms’ habitats.

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. Also effects freshwater and land environments.

Many components of crude oil have been shown to damage cell membranes. Other components, such as benzene, are known carcinogens.

Gram-Negative Bacteria Gram-Positive Bacteria Thin cell wall of peplidoglycan and lipid membrane Outer membrane is a thin extra layer of lipopolysaccharide which adds extra protection for cell Outer membrane protects the bacteria from several antibiotics Simple, thick cell wall Most pathogenic bacteria in humans are gram-positive Antibiotics such as penicillin prevent linking of peptidoglycan and formation of cell wall

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

Common symbiont in mammals; part of the human skin flora Gram-positive Most types are non-pathogenic Pathogenic forms can cause deadly infections Common cause of hospital infection Causes formation of biofilms Commonly used model organism

Used in many cell/biochemical investigations Easy to manipulate and rapidly grows As a eukaryote, it shares similar biochemistry, cell cycle, and genetics with more advanced organisms As a eukaryote, it contains complex structures bound by membranes, including a nucleus

Does crude oil have an effect on the survivorship of Eschericia coli, Staphylococcus epidermidis, and Saccharomyces cerevisiae ?

To determine if oil in different concentrations will affect the survivorship of E. coli, S. epidermidis, and S. cerevisiae.

Null hypothesis: the oil will not significantly affect E. coli, Staph, or Yeast survivorship. Alternate Hypothesis: the oil will significantly affect E. coli, Staph, or Yeast 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)  Staphylococcus epidermidis  Saccharomyces cerevisiae  0.22 micron syringe filter and 10 mL syringe  (Pennsylvania) Crude Oil

1. E. coli and Staph 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 Microbe0.1 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 20 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.

1. Yeast was grown overnight in sterile YEPD 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 Microbe0.1 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 20 minutes. 9. After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on YEPD 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.

1. Sterilized crude oil was infused into the LB agar media in two concentrations, 10 % (approximately 100 mL/L oil) and 0.1% (approximately 1 mL/L oil), and used to create the LB agar plates. 2. E. coli and Staph was grown overnight in sterile LB media. 3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 4. The culture was 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 cells/mL. 5. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

6.100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. 8.The plates were incubated at 37 C for 24 hours. 9.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

1. Sterilized crude oil was infused into the YEPD agar media in two concentrations, 10 % (approximately 100mL/L oil) and 0.1% (approximately 10mL/L oil), and used to create the YEPD agar plates. 2. Yeast was grown overnight in sterile LB media. 3. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 4. The culture was 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 cells/mL. 5. The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL.

6.100 µL of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the solution and spread on the pre-prepared LB plates. 8.The plates were incubated at 37 C for 24 hours. 9.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

P value = Liquid Pulse ExposureAgar Infusion P value =

ConcentrationT ValueInterpretation 0.1 % Not Significant 1% Significant 10% Significant T Critical = 2.88 (Significant) Alpha =.05

P value = Liquid Pulse ExposureAgar Infusion P value = 5.02E-10

ConcentrationT ValueInterpretation 0.1 % Significant 10% Significant T Critical = 2.86 (Significant) Alpha =.05

P value = Liquid Pulse ExposureAgar Infusion P value = 3.88E-24

ConcentrationT ValueInterpretation 0.1 % Not Significant 10% Significant T Critical = 2.86 (Significant) Alpha =.05

The null hypothesis that crude oil does not significantly affect E. coli, Staph, or Yeast survivorship must be rejected for: -E. coli liquid exposure at 1% and 10% -Staph agar infusion at 0.1% and 10% -Yeast agar infusion at 10% The null hypothesis must be accepted for: -E. coli liquid exposure at 0.1% and agar infusion at 0.1% and 10% -Staph liquid exposure at all concentrations and agar infusion at 0.1% -Yeast liquid exposure at all concentrations and agar infusion at 0.1%

The oil was somewhat insoluble, and needed to be inverted repeatedly Difficult to synchronize plating Composition of oil? Extensions Test higher concentrations of crude oil Test the effects of refined motor oil and gasoline Test the effects of oil from different regions Limitations

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P value = Liquid Pulse ExposureAgar Infusion P value =

P value = Liquid Pulse ExposureAgar Infusion P value = 5.02E

P value = Liquid Pulse ExposureAgar Infusion P value = 3.88E