Oil Pollution’s Effect on Microbes

John DeSantisGrade 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.

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.

Ocean Oil Pollution

• Very common

• Not well publicized

• Enters in small amounts

• Will often stay in the water for years

Freshwater Oil Pollution

• 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)

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

• Other components have been shown to cause cancer.

Past Studies

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

Question

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

Purpose

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

• Alternate Hypothesis: the oil will significantly affect E. coli 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) 0.22 micron syringe filter

and 10 mL syringe (Pennsylvania) Crude Oil

Materials

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 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.

Procedure

Procedure (Chart of Concentrations)

0% Oil 0.1% Oil 1% Oil 10% Oil

SDF 9.9 mL 9.89 mL 9.8 mL 8.9 mL

Oil 0 mL 0.01 mL 0.1 mL 1 mL

E. Coli 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Total 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 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.

Procedure (cont.)

0.00% 0.10% 1.00% 10.00%0

25

50

75

100

125

150

175

200

225

250

275

300

Concentration of Oil

Resu

ltin

g N

um

ber

of

Colo

nie

s

269

242

270.75

Crude Oil Effects on E. coli Survivorship

254.875

P-value =0.008878

Statistical Analysis

Oil Concentration

T Value Interpretation

0.1 % 1.607 Not Significant

1% 1.4648 Not Significant

10% 1.806 Not Significant

T Critical = 2.88 (Significant variance)Alpha = .05

Dunnett’s Test

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

• 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.

Conclusions

Limitations and Extensions

The oil was somewhat insoluble, and needed to be inverted repeatedly

Only one time of exposure was utilizedDifficult to synchronize platingComposition of oil?

Extensions

Test higher concentrations of crude oilTest 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 gasolineTest 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

ANOVAAnova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 8 2039 254.875 447.8393Column 2 8 2152 269 76Column 3 8 1936 242 373.7143Column 4 8 2166 270.75 338.5

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 4351.844 3 1450.615 4.694342 0.008878 2.946685Within Groups 8652.375 28 309.0134

Total 13004.22 31