Transcript Mike Fantini Pjas Presentation (1)x

Hydrogen Peroxide
Anti-Microbial Effects
Michael Fantini
Grade 9
Central Catholic High School
Hydrogen Peroxide
• Commonly used as a household wound disinfectant
• Highly damaging to tissue
• Used at high concentration 3%
• Has peroxide group
• Powerful oxidizer
• Corrosive at higher concentrations
Model Bacteria
◆ Escherichia coli
– gram negative
– rod-shaped
– intestinal mammalian symbiont
– widely studied
– easy to maintain, high growth rate, simple nutritional
requirements
Model Bacteria Cont.
◆ Staphylococcus epidermidis
– gram positive
– round shaped
– form clusters
– exterior human flora
– easy to grow in a lab setting
– can produce biofilms
–form clusters
Purpose
•Investigate the effects of hydrogen
peroxide at various concentrations on
E. coli and Staph
Rationale
•Can hydrogen peroxide be used as a
disinfectant at lower concentrations?
Hypotheses
Null:
Hydrogen Peroxide will not have a significant
effect on the survivorship of the bacteria
Alternative:
Hydrogen peroxide will have a significant negative
effect on the survivorship of the bacteria
Materials
• LB agar plates
• LB media (0.5% yeast extract, 1%
tryptone, 1% sodium chloride)
• Sterile Dilution Fluid
• Sterile pipette tips
• Micropipettes
• Staphylococcus epidermidis
• Escherichia coli
• Vortex
• Incubator
• Sidearm flask
• Spreader bars
• ethanol
• Sterile capped test tubes with sterile
distilled water
• Hydrogen peroxide
• SDF(100mM KH2PO, 100mM
K2HPO4, 10mM MgSO4, 1m NaCl)
Procedures
Liquid Exposure
1. Both bacteria were grown until a density of 50 klett spectrophotometer
density was reached. This was approximately 10^8 cells/mL
2. Both cultures were diluted in sterile dilution fluid to a concentration of
approximately 10^5 cells/mL
3. The stock was added to tubes containing 8.9 mL of sterile water to create
concentrations of [3*10^-4], [3*10^-3], [0.03], [0.15], and [0.3]
4. Sterile water was added to the tubes to bring all of the quantities of the
tubes up to 9.9 mL
5. 0.1 mL of bacteria were added to their respective tubes
6. The tubes were allowed to stand at room temperature for 10 minutes
7. 0.1 mL of each tube were plated onto LB agar, 6 plates per tube
Procedures Cont.
8. The plates were grown overnight at 37 C and resulting colonies
counted
9. Stat tests were completed
Total
Percent in
Tubes
Sterile
Water
Hydrogen
Peroxide
Bacteria
Total
Volume
0%
3*10^-4% 3*10^-3%
0.03%
0.15%
0.3%
9.9 ml
9.899 ml
9.89 ml
9.8 ml
9.4 ml
8.9 ml
0 ml
0.001 ml
0.01 ml
0.1 ml
0.5 ml
1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
10 ml
10 ml
10 ml
10 ml
10 ml
10 ml
Hydrogen Peroxide Effects on Microbial Survivorship
Bacterial Survivorship
250
LD50
~ [0.01]
~ [0.02]
Colonies
200
150
P-Values
3.39E-24
9.95E-17
100
50
0
0%
3*10^-4%
3*10^-3%
0.03%
Concentration
Staph
E. coli
0.15%
0.30%
Dunnett’s Test Results: Staph
Concentration
T-Value
T-Critical
Results
3*10^-4%
15
3.39
Significant
3*10^-3%
27
3.39
Significant
0.03%
28
3.39
Significant
0.15%
29
3.39
Significant
0.3%
29
3.39
Significant
Dunnett’s Test Results: E. Coli
Concentration
T-Value
T-Critical
Results
3*10^-4%
7
3.39
Significant
3*10^-3%
14
3.39
Significant
0.03%
15
3.39
Significant
0.15%
15
3.39
Significant
0.3%
15
3.39
Significant
Procedures Cont.
Direct Infusion
1. 200uL of hydrogen peroxide was added to 2 LB agar plates per
bacteria
2. 20uL of hydrogen peroxide and 180uL of water were added to 2 LB
agar plates per bacteria
3. The LB agar plates were put in an incubator for one hour
4. 0.1 mL of each bacteria were added to their respective infused
plates
5. The plates were incubated at 37 C and the resulting colonies were
counted
Direct Infusion
Staph Survivorship Data
0 ul
20 ul
E. Coli Survivorship Data
200 ul
420
0
0
378
0
0
P-Values
0.000266
0.000121
0 ul
20 ul
200 ul
273
0
0
296
0
0
Conclusion
• The null hypothesis was rejected for all
concentrations
• H2O2 had a significant negative effect on the
survivorship of bacteria
• H2O2 was observed to be very lethal even at
low concentrations
Possible Limits and Extensions
• Slight variance in spread
plating synchronization
• Only survivorship assessed
• Only two species used
• Possible non-uniform
spreading of hydrogen
peroxide
• One exposure time
• More trials and exposure
times
• Different microbes
• Different concentrations of
hydrogen peroxide
• Synergistic effects of
hydrogen peroxide and
other variables
Works Cited
"E. Coli Bacteria Infection Symptoms, Causes, Treatments." WebMD. WebMD, n.d. Web. 06
Jan. 2016.
"E. coli (Escherichia Coli)." Centers for Disease Control and Prevention. Centers for Disease
Control and Prevention, 21 Dec. 2015. Web. 06 Jan. 2016.
"Escherichia Coli." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016.
"Hydrogen Peroxide | Chemical Compound." Encyclopedia Britannica Online. Encyclopedia
Britannica, n.d. Web. 06 Jan. 2016.
"Hydrogen Peroxide." Hydrogen Peroxide. N.p., n.d. Web. 06 Jan. 2016.
"Hydrogen Peroxide." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016.
"Staphylococcus Epidermidis." - MicrobeWiki. N.p., n.d. Web. 06 Jan. 2016.
"Staphylococcus Epidermidis." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Jan. 2016.
Anova: Single Factor
Staph Liquid Pulse
SUMMARY
Groups
Count
Sum
Average
Variance
0
6
1397
232.8333
442.1667
0.0001
6
689
114.8333
374.9667
0.1
6
109
18.16667
225.3667
1
6
41
6.833333
109.3667
5
6
0
0
0
10
6
0
0
0
ANOVA
Source of Variation
SS
df
MS
F
Between Groups
267768.2
5
53553.64
Within Groups
5759.333
30
191.9778
Total
273527.6
35
P-value
278.9575
F crit
3.39E-24
2.533555
Anova: Single Factor
E. Coli Liquid Pulse
SUMMARY
Groups
Count
Sum
Average
Variance
0
6
1202
200.3333
2420.267
0.0001
6
663
110.5
359.5
0.1
6
87
14.5
115.5
1
6
29
4.833333
48.56667
5
6
0
0
0
10
6
0
0
0
ANOVA
Source of Variation
SS
df
MS
F
Between Groups
206453.8
5
41290.76
Within Groups
14719.17
30
490.6389
221173
35
Total
P-value
84.15713
F crit
9.95E-17
2.533555
Anova: Single Factor
Staph Direct Infusion
SUMMARY
Groups
Count
Sum
Average
Variance
0
2
798
399
882
20
2
0
0
0
200
2
0
0
0
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
F
212268
2
106134
882
3
294
213150
5
P-value
361
0.00026618
F crit
9.552094496
Anova: Single Factor
E. Coli Direct Infusion
SUMMARY
Groups
Count
Sum
Average
Variance
0
2
569
284.5
264.5
20
2
0
0
0
200
2
0
0
0
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
107920.3333
2
53960.16667
264.5
3
88.16666667
108184.8333
5
F
P-value
612.0245747
0.00012089
F crit
9.552094496