EFFECTS OF FRACKING FLUID ON STAPH. EPIDERMIDIS AND E. COLI

LUKE WEARDEN GRADE 11 CENTRAL CATHOLIC HIGH SCHOOL

PROBLEM

How do fracking chemicals influence the survivorship of Escherichia coli and Staphylococcus epidermidis?

FRACKING BACKGROUND

• Use: accesses natural gas found in subterranean shale

• Fracking is effective in obtaining energy, but can be harmful to the environment

• Many environmentalists groups including the EPA have taken strong anti-fracking stances

• Are living organisms at risk from fracking?

HOW FRACKING WORKS

FRACKING SOLUTION

Chemical

Sodium Chloride

Calcium Chloride

Magnesium Chloride

PotassiumChloride

Barium Chloride

Strontium Chloride

Amount(mg/L)

75.65 16.65 0.475 0.3846 0.1857 0.20

GRAM (-) VS. GRAM (+) BACTERIA

• Cell wall contains an extra layer of lipopolysaccharides for extra protection.

• Outer membrane protects bacteria from several antibiotics.

• Most pathogenic bacteria in humans are Gram (+) organisms.

• Simple cell wall.• Some antibiotics

work against the formation of the cell wall.

STAPHYLOCOCCUS EPIDERMIDIS (STAPH)

Common surface symbiont in many mammals (Human).

Gram (+) bacteria

Most strains considered non-pathogenic.

Pathogenic strains can be life-threatening.

ESCHERICHIA COLI (E.COLI)

Large and diverse group of gram (-) bacteria

Free living, symbionts, or pathogens

Live in the intestinal tract of many mammals.

Most strains are not pathogenic

Serve as a common prokaryotic cell model.

PURPOSE

Assess the effects of fracking chemicals on the survivorship of Staphylococcus epidermidis and Escherichia coli.

HYPOTHESES

Null Hypothesis: There will be no significant effect of fracking chemicals on the survivorship of E.coli and Staph.

Alternative Hypothesis: The fracking chemicals will significantly reduce the survivorship of the bacteria.

MATERIALS• Fracking Chemicals• Micro and Macro pipettes + Sterile tips• Spreader bars• LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl)• Escherichia coli DH5-alpha(E.Coli) • Staphylococcus epidermidis• Burner• Sidearm Flask• Vortex• Incubator• Gloves/goggles• SDF (Sterile Dilution Fluid 10 mm KH2PO4, 1 mm MgSO4, 0.1 mm CaCl2, 100 mm

NaCl)• 20 Sterile Test Tubes• Ethanol • Luria Broth Agar Plates(To be Infused with Agar Plates)• 0.22 micron syringe filters and 10mL syringe• Klett Spectrophotometer

PROCEDURE1. Bacteria (E.coli and Staph) were grown overnight in

sterile LB Media.

2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.

3. The cultures were 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.

4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL.

5. The fracking chemicals were sterilized by means of a 0.2 micron syringe filter

6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.01%, 0.1%, 1%.

CONCENTRATION CHARTIngredients 0% 0.01% 0.1% 1%

Sterile Fluid added

1mL 0.999mL 0.99mL 0.9mL

E.coli/Staph

0.1mL 0.1mL 0.1mL 0.1mL

Fracking Chemicals

0mL 0.001mL 0.01mL 0.1mL

Original Sterile Fluid

8.9mL 8.9mL 8.9mL 8.9mL

Total Volume

10mL 10mL 10mL 10mL

PROCEDURE

7. 100 µL of cell culture was then added to the fracking chemical solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

8. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

9. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB-agar plates.

10. The plates were incubated at 37°C for 24 hours.

11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

ANOVA AND DUNNETT’S TEST

•ANOVA• Statistical test that allows the comparison of means

of different groups

• Dunnett’s Test• Comparison of experimental and control group to

determine significant variation

DATA P-value: 5.472E-28P-value: 2.994E-28

Control 0.01 0.1 10

50

100

150

200

250

300

350

400

450

500

Fracking Fluid Effects of E. coli and Staph

E. ColiStaph

Concentration (percent of Fracking Fluid)

Nu

mb

er o

f S

urv

ivin

g C

olo

nie

s

DUNNETT'S TEST – E. COLI

Concentration T-value Significance

0.01% 2.46 Significant

0.1% 3.77 Significant

1% 4.57 Significant

T-crit = 2.365

DUNNETT’S TEST - STAPH

Concentration T- Value Significance

0.01% 2.69 Significant

0.1% 3.77 Significant

1% 4.58 Significant

T-crit =2.365

CONCLUSIONS

Reject the null hypothesis that fracking chemical will not have a significant effect of Staph and E. coli survivorship. At every concentration for both bacteria the fracking fluid significantly reduced survivorship.

Accept the alternate hypothesis that fracking fluid will have a significant effect on Staph and E. coli survivorship.

An increase in the concentration of fracking fluid will generally correlate with a significant decrease in the survivorship of Staph and E. coli.

LIMITATIONS

Plating was slightly unsynchronized

Only 4 concentrations were tested

Only one exposure time was used

Only one method of exposure used

FUTURE STUDIES

Use a wider variety of concentrations of fracking fluid

Vary exposure times

Use various cell models

Test for synergistic effects

REFERENCEShttps://fracfocus.org/chemical-use/what-chemicals-are-used

http://www.huffingtonpost.com/news/fracking-chemicals/

https://etd.ohiolink.edu/ap/10?0::NO:10:P10_ACCESSION_NUM:osu1366292190

http://onlinelibrary.wiley.com/doi/10.1002/etc.2619/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/

http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview