The Effects Of
Temperature and
Antibiotic on E.coli and
Staph epidermidis
James Shaver
Grade 9
Central Catholic High School
Ampicillin
Antibiotic belonging to the amino penicillin group of
penicillin
• Used to treat urinary tract infections, Salmonella,
some Influenza strains, and Meningitis.
• Commonly used in the lab and the medical world.
• Has Gram-positive coverage but limited Gramnegative coverage.
• The Amino group in the antibiotic helps it penetrate
the membrane of Gram-negative bacteria.
The Effects of Ampicillin
- Beta-lactam antibiotic which is part of the
aminopenicillin family.
- Roughly equivalent to its successor,
amoxicillin in terms of spectrum and level
of activity.
The most common side effects include:
-rash
-fever
-diarrhea
-nausea
Bacterial Heat Stress
- Bacterial heat-shock response allows for bacteria to
change (stress) in the environment.
- Bacteria use various response mechanisms for coping
with the stress and for adapting to the new conditions.
Gram (-) Vs. Gram (+) Bacteria
-Cell was 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.
Escherichia coli
- Large and diverse group of gram (-)
bacteria
-Free living, symbiotes, or pathogens
-Live in the intestinal tract of many
mammals.
-Most strains are not pathogenic
-Serve as a common prokaryotic cell
model.
Staphylococcus epidermidis
-Common surface symbiont in many
mammals (Human).
-Gram (+) bacteria
-Most strains considered nonpathogenic.
-Pathogenic strains can be lifethreatening.
Purpose
●
To determine if Ampicillin and temperature have
a synergistic effect on Staphylococcus
epidermidis and Escherichia coli survivorship.
Hypotheses
Null:
1. Ampicillin and Heat individually will not reduce bacterial survivorship.
2. Ampicillin and Heat will not have synergistic effects at reducing bacterial
survivorship.
Alternative:
1. The effects of Ampicillin and Heat individually will significantly reduce
bacterial survivorship.
2. Heat and Ampicillin in synergy will significantly reduce bacterial
survivorship.
Materials
- Escherichia coli DH5-alpha(E.Coli) (Obtained from Doonan Lab, CMU)
- Staphylococcus epidermidis (Staph) (Obtained from Carolina Science Supply)
- Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
- Thermometer
- Latex Gloves
- Micro pipettes
- Micro tubes
- Ampicillin
- 72 LB agar plates
- 72 infused LB agar plates
- Spreader Bar
- Incubator
- Ethanol
- Matches
- Bunsen Burner
- Vortex
- Sharpie
- hot plate
- Luria Broth(LB) Agar Plates ( 0.5% yeast extract,1% tryptone, 1% NaCl)
Procedure for Liquid Pulse
1.Bacteria (E.coli/Staph epidermidis) 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. Ampicillin Stock of AMP 200 (this stock represents 10x or 10 times the
recommended working concentration) was sterile filtered.
6.The Ampicillin Stock was mixed with the appropriate amounts of SDF
to create concentrations of 0x, 0.01x,0.1x and 1x.
Chart of Concentration
Control
0.01x
0.1x
x
E. coli
0.1 ml
0.1 ml
0.1ml
0.1ml
Ampicillin
0 ml
0.01ml
0.1ml
1ml
Sterile Water
9.9 ml
9.89
9.8
8.9
Total Volume
10ml
10ml
10 ml
10ml
Procedures Continued
7. After addition of 100 microliters of bacterial suspension, the tubes
were incubated at the selected temperatures; 40,50,60 degrees F.
8. 100 microliter aliquots from each tube were pipetted on LB agar
plates.
9. The LB agar plates were placed in the incubator overnight and the
number of colonies were then counted.
Procedures for Infused Plates
1.The following volumes of ampicillin were spread onto Lb agar plates
to create the following concentrations; 60 microliters(X), 6 microliters
of sub stock ( 0.01x).
2. The plates were placed in the incubator for one hour to be infused.
3.Each E. coli population subjected to the various temperatures was
then pipetted onto the infused plates.
4.The plates were then incubated for 24 hours at 37 C.
Anova
-Abbreviation for analysis of
variance
-Statistical test comparing
variation within and between
experimental groups
- If the P- value is lower than
the alpha value (.05), then the
result is significant (a result of
the variable influence)
Dunnett’s Test
- A procedure for comparing the mean of each
experimental group individually against the mean of
the control group.
- A Dunnett’s Test allows you to see if there was a
significant difference from the control.
- If the T Value is greater than the T Critical value
than the difference is significant.
The Effects of Temperature on Bacterial Survivorship
Anova
P- Value
Staph- 0.01289
E coli- 4E-07
Dunnett’s Test
T- Crit (3.25)
T- Value 50 C
1.2265
Not Sig
2.259
Not Sig
T- Value 60 C
2.313
Not Sig
14.407
Sig
The Effects of Ampicillin on Bacterial Survivorship 40 C
Anova
Dunnett’s Test
P- Value
T Value of 0.01x
Staph- 0.203322
210
178.8
E coli- 8.52E-08
0.50861
Not Sig
T-Crit (3.25)
3.88418
Sig
T- Value of 1x
2.14252
Not Sig
17.2157
Sig
Synergistic Effects on Staph Survivorship(Infusion)
Number of Bacteria Colonies
Interaction P- Value
0.000414
0x
0.1x
Concentration of Ampicillin
1x
Synergistic Effects on E coli Survivorship(Infusion)
P-Value Interaction
Number of Bacteria Colonies
3.75E-11
0x
0.1x
Concentration of Ampicillin
1x
Effects of Temperature on E coli Survivorship
Anova
P-Value
5.29E-05
Dunnett’s Test
T-Crit-2.86
50 C
1.14947(Not Sig)
60 C
3.31763(Sig)
Effects of Ampicillin on E coli Survivorship
Anova
P-Value
4E-06
Dunnett’s Test
T- Crit(3.10)
0.01x
3.680174 Sig
0.1x
11.771 Sig
1x
11.8156 Sig
Ampicillin Effects on E coli (Liquid Pulse)
Number of Bacteria Colonies
P-Value Interaction
5.59E-09
0x
0.01x
Concentration of Ampicillin
0.1x
1x
Percent Decrease In Survivorship
Compared to Control
E coli
Staph
Epidermidis
0.1x
1x
40 C
17%
77%
50 C
32%
85%
40 C
36%
71%
50 C
18%
63%
Conclusion
Experiment 1(Liquid Pulse)
- 1st null hypothesis is rejected because temperature and ampicillin
individually had effects on bacterial survivorship.
- 2nd null hypothesis is rejected because temperature and ampicillin
had synergistic effects on bacterial survivorship.
Experiment 2(LB Agar Infusion)
- 1st null hypothesis is rejected because temperature and ampicillin
individually had effects on bacterial survivorship.
- 2nd null hypothesis is rejected because the temperature and
ampicillin had synergistic effects on bacterial survivorship.
Limitations And Future Studies
- Plating could have been
unsynchronized.
- Only three
concentrations of the
variable were tested.
- Only survivorship
examined.
- Use more Antibiotics
- Use a greater range of
temperatures including
those that are closer to
eachother
-Perform a Trypan blue
exclusion essay to test
for dead cells
- Explore growth curve
effects
References
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidi
s/sepidermidis.html
-http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con20032105
-http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0000105/
Data Infused Plates
Infused Plates E coli
Control
Low Concentration High Concentration
40 degrees
224,228,236,204
168,176,204,189
54,44,39,68
50 degrees
144,156,168,224
104,110,123,131
24,16,31,29
60 degrees
0,0,0,0
0,0,0,0
0,0,0,0
Data Infused Plates
Infused Plates
Staph epidermidis
Control
Low Concentration
High Concentration
40 degrees
,256,360,224
,236,256,220
96,88,92,99
50 degrees
252,234,208,204
156,176,193,207
81,96,78,76
60 degrees
0,0,0,0
0,0,0,0
0,0,0,0
Data Liquid Pulse
Regular Plates E coli
0mg Stock
0.01x
0.1x
1x
40 degrees
208,352,284,333,
305,252
421,532,327,554,366,
496
276,175,180,189,2 184,184,304,209,258,
01,222
196
50 degrees
176,208,304,222,
258,202
240,288,311,225,233,
316
240,220,176,220,2 211,144,156,202,178,
28,248
198
60 degrees
0,0,0,0,0,0
0,0,0,0,0,0
0,0,0,0,0,0
0,0,0,0,0,0
Data Liquid Pulse
Regular Plates E coli
0mg Stock
0.01x
0.1x
1x
40 degrees
,352,284,333,
305,252
421,327,366,
,175,180,189,201,
222
184,184,209,196
50 degrees
176,208,304,222,
258,202
240,288,311,225,233,
316
240,220,176,220,2 ,144,156,202,178,198
28,248
60 degrees
0,0,0,0,0,0
0,0,0,0,0,0
0,0,0,0,0,0
0,0,0,0,0,0