Download John DeSantis Crude Oil Effects on Microbial Life

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-Positive
Bacteria
•
•
•
Gram-Negative
Bacteria
•
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
Thin cell wall of peplidoglycan an
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
•
•
•
•
•
•
•
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 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
1.
2.
3.
4.
5.
6.
E. coli and Staph was grown overnight in sterile LB media.
A sample of the overnight culture was added to fresh
media in a sterile sidearm flask.
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.
The culture was diluted in sterile dilution fluid to a
concentration of approximately 105 cells/ml.
The petroleum was diluted with sterile dilution fluid to
concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.
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.
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
Microbe
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Total
10 mL
10 mL
10 mL
10 mL
7.
8.
9.
10.
11.
Every minute the tubes were inverted five times to
mix the oil with the cell suspension.
The tubes were allowed to incubate at room
temperature for 20 minutes.
After vortexing to evenly suspend cells, 0.1 ml.
aliquots were removed from the tubes and spread on
LB agar plates.
The plates were left to sit overnight.
The resulting colonies were counted. Each colony is
assumed to have arisen from one cell.
1.
2.
3.
4.
5.
6.
Yeast was grown overnight in sterile YEPD media.
A sample of the overnight culture was added to fresh
media in a sterile sidearm flask.
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.
The culture was diluted in sterile dilution fluid to a
concentration of approximately 105 cells/ml.
The petroleum was diluted with sterile dilution fluid to
concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.
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.
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
Microbe
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Total
10 mL
10 mL
10 mL
10 mL
7.
8.
9.
10.
11.
Every minute the tubes were inverted five times to
mix the oil with the cell suspension.
The tubes were allowed to incubate at room
temperature for 20 minutes.
After vortexing to evenly suspend cells, 0.1 ml.
aliquots were removed from the tubes and spread on
YEPD agar plates.
The plates were left to sit overnight.
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 108 cells/mL.
5. The culture was diluted in sterile dilution
fluid to a
5
concentration of approximately 10 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 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.
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 108 cells/mL.
5. The culture was diluted in sterile dilution
fluid to a
5
concentration of approximately 10 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 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.
Liquid Pulse Exposure
P value =0.003480
Number of Colonies
200
Agar Infusion
P value = 0.62481
150
100
50
0
0%
0.10%
1%
10%
Concentration
0.10%
10%
T Critical = 2.88 (Significant)
Alpha = .05
Concentration
T Value
Interpretation
0.1 %
2.8759
Not Significant
1%
4.4988
Significant
10%
3.511
Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
Liquid Pulse Exposure
P value =0.59698
Number of Colonies
1000
Agar Infusion
P value = 5.02E-10
800
600
400
200
0
0%
0.10%
1%
10%
Concentration
0.10%
10%
T Critical = 2.86 (Significant)
Alpha = .05
Concentration
T Value
Interpretation
0.1 %
13.95
Significant
10%
22.91
Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
Liquid Pulse Exposure
P value =0.64282
Number of Colonies
250
Agar Infusion
P value = 3.88E-24
200
150
100
50
0
0%
0.10%
1%
10%
Concentration
0.10%
10%
T Critical = 2.86 (Significant)
Alpha = .05
Concentration
T Value
Interpretation
0.1 %
1.196446
Not Significant
10%
33.21954
Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
•
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
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
•
•
•
•
•
•
http://www.waterencyclopedia.com/Oc-Po/Oil-SpillsImpact-on-the-Ocean.html
http://www.niaid.nih.gov/topics/ecoli/Understanding/Pa
ges/overview.aspx
http://www.sciencedaily.com/releases/2007/05/070528
095321.htm
http://alaska.boemre.gov/kids/shorts/crude/crude.htm
http://reinhardtmicrobiology.com/crude-oil-spillsmdash-biological-medical-chemical-dangers.html
Liquid Pulse Exposure
P value =0.003480
200
Number of Colonies
167.7
150
179.17
172.17
147.29
Agar Infusion
P value = 0.62481
153.3
141
100
50
0
0%
0.10%
1%
10%
Concentration
0.10%
10%
Liquid Pulse Exposure
P value =0.59698
Number of Colonies
1000
800
843.75
838.16
776.83
Agar Infusion
P value = 5.02E-10
810.17
600
464
400
220.3
200
0
0%
0.10%
1%
10%
Concentration
0.10%
10%
Liquid Pulse Exposure
Number of Colonies
250
236
P value =0.64282
221.83
226.2
200
210
Agar Infusion
P value = 3.88E-24
227.56
150
100
50
1.54
0
0%
0.10%
1%
10%
Concentration
0.10%
10%