John DeSantis Grade 10 Central Catholic High School

Crude Oil Effects on Microbial Life

John DeSantisGrade 10

Central Catholic High School

• Crude oil pollution enters microorganisms’ habitats.

Problem

• 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.

Petroleum (Crude Oil)

• 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.

Oil Pollution

• Many components of crude oil have been shown to damage cell membranes.

• Other components, such as benzene, are known carcinogens.

Past Studies

Gram Positive and Gram Negative Bacteria

Gram-Negative BacteriaGram-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

Escherichia coli (E. coli)

Staphylococcus epidermidis (Staph)• 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

Saccharomyces cerevisiae• 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?

Question

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

Purpose

• 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.

Hypotheses

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 KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)

Escherichia coli (DH5-Alpha) Staphylococcus epidermidis Saccharomyces cerevisiae 0.22 micron syringe filter

and 10 mL syringe (Pennsylvania) Crude Oil

Materials

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 108-109 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.

6. 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 cells/ml.

Pulse Liquid Exposure Procedure – E. coli and Staph

Liquid Concentrations

0% Oil 0.1% Oil 1% Oil 10% OilSDF 9.9 mL 9.89 mL 9.8 mL 8.9 mLOil 0 mL 0.01 mL 0.1 mL 1 mLMicrobe 0.1 mL 0.1 mL 0.1 mL 0.1 mLTotal 10 mL 10 mL 10 mL 10 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 108 or 109 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.

6. 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 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.

Pulse Liquid Exposure Procedure – E. coli and Staph

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 108-109 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.

6. 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 cells/ml.

Pulse Liquid Exposure Procedure - Yeast

Liquid Concentrations

0% Oil 0.1% Oil 1% Oil 10% OilSDF 9.9 mL 9.89 mL 9.8 mL 8.9 mLOil 0 mL 0.01 mL 0.1 mL 1 mLMicrobe 0.1 mL 0.1 mL 0.1 mL 0.1 mLTotal 10 mL 10 mL 10 mL 10 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 108 or 109 cells/ml.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

5. The petroleum was diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.

6. 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 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.

Pulse Liquid Exposure Procedure - Yeast

Agar Infusion Procedure – E. coli and Staph

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 108 cells/mL.

5. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

Agar Infusion Procedure – E. coli and Staph

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 103 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.

Agar Infusion Procedure - Yeast1. 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 108 cells/mL.

5. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

Agar Infusion Procedure - Yeast

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 103 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.

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

20406080

100120140160180200

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on E. coli Survivorship

P value =0.003480Liquid Pulse

ExposureAgar InfusionP value = 0.62481

Dunnett’s Test AnalysisE. coli Liquid Exposure

Concentration T Value Interpretation

0.1 % 2.8759 Not Significant1% 4.4988 Significant10% 3.511 Significant

T Critical = 2.88 (Significant)Alpha = .05

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

200

400

600

800

1000

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on Staph Survivorship

P value =0.59698Liquid Pulse

ExposureAgar InfusionP value = 5.02E-10

Dunnett’s Test AnalysisStaph Agar Infusion

Concentration T Value Interpretation

0.1 % 13.95 Significant10% 22.91 Significant

T Critical = 2.86 (Significant)Alpha = .05

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

50

100

150

200

250

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on Yeast Survivorship

P value =0.64282Liquid Pulse

ExposureAgar InfusionP value = 3.88E-24

Dunnett’s Test AnalysisYeast Agar Infusion

Concentration T Value Interpretation

0.1 % 1.196446 Not Significant10% 33.21954 Significant

T Critical = 2.86 (Significant)Alpha = .05

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

Conclusions• 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%

Limitations and Extensions• 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

• http://www.waterencyclopedia.com/Oc-Po/Oil-Spills-Impact-on-the-Ocean.html

• http://www.niaid.nih.gov/topics/ecoli/Understanding/Pages/overview.aspx

• http://www.sciencedaily.com/releases/2007/05/070528095321.htm

• http://alaska.boemre.gov/kids/shorts/crude/crude.htm• http://

reinhardtmicrobiology.com/crude-oil-spills-mdash-biological-medical-chemical-dangers.html

References

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

20406080

100120140160180200

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on E. coli Survivorship

P value =0.003480Liquid Pulse

ExposureAgar InfusionP value = 0.62481

147.29167.7 179.17 172.17

153.3 141

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

200

400

600

800

1000

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on Staph Survivorship

P value =0.59698Liquid Pulse

ExposureAgar InfusionP value = 5.02E-10

843.75 838.16776.83 810.17

464

220.3

0.00% 0.10% 1.00% 10.00% 0.10% 10.00%0

50

100

150

200

250

Concentration

Num

ber

of C

olon

ies

Crude Oil Effects on Yeast Survivorship

P value =0.64282Liquid Pulse

ExposureAgar InfusionP value = 3.88E-24

236221.83 226.2

210227.56

1.54