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
Sodium
Chloride
Calcium
Chloride
Magnesium
Chloride
Potassium Barium
Chloride
Chloride
Strontium
Chloride
75.65
16.65
0.475
0.3846
0.20
Chemical
Amount
(mg/L)
0.1857
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
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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
PROCEDURE
1.
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 CHART
Ingredients 0%
0.01%
0.1%
1%
Sterile
Fluid
added
E.coli/Stap
h
Fracking
Chemicals
Original
Sterile
Fluid
Total
Volume
1mL
0.999mL
0.99mL
0.9mL
0.1mL
0.1mL
0.1mL
0.1mL
0mL
0.001mL
0.01mL
0.1mL
8.9mL
8.9mL
8.9mL
8.9mL
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-28
P-value: 2.994E-28
Fracking Fluid Effects of E. coli and Staph
Number of Surviving Colonies
500
450
400
350
300
250
E. Coli
Staph
200
150
100
50
0
Control
0.01
0.1
Concentration (percent of Fracking Fluid)
1
DUNNETT'S TEST – E. COLI
T-crit = 2.365
Concentration
T-value
Significance
0.01%
2.46
Significant
0.1%
3.77
Significant
1%
4.57
Significant
DUNNETT’S TEST - STAPH
T-crit =2.365
Concentration
T- Value
Significance
0.01%
2.69
Significant
0.1%
3.77
Significant
1%
4.58
Significant
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
REFERENCES
https://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?deniedAccessCustomised
Message=&userIsAuthenticated=false
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/
http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview