Download Chlorine Bleach on Microbial Resistance

Chlorine Bleach on
Microbial Resistance
Daniel Crawford
Grade 11
Central Catholic High School
Antibacterial Agents
• Well-documented evidence that bacteria are
able to develop resistance to antibiotics
Are Bacteria able to develop resistance to
household Antibacterial Agents?
• Different antibacterial agents commonly used
Does initial exposure to one antibacterial
agent affect resistance to another?
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 (E. coli)
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Large and diverse group of gram (-) bacteria
Free living, symbionts, or pathogens
Live in the intestinal tract of many mammals
Most strains are non-pathogenic
Serve as a common prokaryotic cell model
Staphylococcus epidermidis (Staph.)
• Common surface symbiont in many mammals
(Human).
• Gram (+) bacteria
• Most strains considered non-pathogenic.
• Pathogenic strains can be life-threatening.
Chlorine Bleach
• Sodium Hypochlorite, NaOCl
• Forms hypochlorous acid (HOCl) with water
– NaOCl (s) + H2O (l) → HOCl (aq) + NaOH- (aq)
• Hypochlorous acid divides into hydrochloric
acid (HCl) and oxygen (O).
• Strong Oxidizer
– effective at killing bacteria, fungi, and viruses.
– Denaturization, Low pH
Hydrogen Peroxide, (H2O2)
• Standard [3%] of H2O2
• Clear liquid
• H2O2 readily becomes H + HO2 or 2OH.
– Free radicals, very reactive
– Powerful oxidizing agent
• Hydrazine fuel, First Aid, Bleaching Agent
Purpose
To determine if Escherichia coli or
Staphylococcus epidermidis
initially exposed to Chlorine Bleach
Are able to develop resistance to
Hydrogen Peroxide
Hypotheses
• Null Hypothesis: Initial exposure WILL NOT
increase E. coli and Staph. survivorship, when
exposed to Hydrogen Peroxide
• Alternative Hypothesis: Initial exposure WILL
increase E. coli and Staph survivorship, when
exposed to Hydrogen Peroxide
Materials
• Clorox Brand Bleach (8.75%
NaOCl)
• Standard Hydrogen Peroxide
([3%])
• 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) (Obtained from Doonan
Lab, CMU)
• Staphylococcus epidermidis
(Staph) (Obtained from Ward’s
Scientific)
•
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•
Burner
Sidearm Flask
Vortex
Incubator
Gloves/goggles
Klett Spectrophotometer
SDF (Sterile Dilution
Fluid)(100mM KH2PO, 100mM
K2HPO4, 10mM MgSO4, 1m
NaCl)
• Sterile Test Tubes
• Ethanol
Procedure (Liquid Exposure)
Steps 1-4 referred to a Dilution Process
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.
Procedure (Pt. 1)
5. Tubes were mixed, with the amounts shown
on the table below, to result in
*[10 %] was
concentrations of Chlorine Bleach of*: also used
-3.5
[10-5%]
[0%]
[10-4%]
[10-3%]
[10-2%]
SDF
9.9 mL
9.8 mL
8.9 mL
9.8 mL
8.9 mL
Microbe
0.1 mL
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Chlorine
Bleach
0 mL
0.1 mL @
1 mL @
0.1 mL @
1 mL @
[10-3%]
[10-3%]
[10-1%]
[10-1%]
Total
Volume
10 mL
10 mL
10 mL
10 mL
10 mL
pH
7
7
7
7
7
Procedure (Pt. 1)
Steps 6-8 referred to a Plating Process
6. The solutions were mixed by vortexing and
allowed to sit at room temperature for 3
minutes.
7. After vortexing to evenly suspend cells,
0.1mL aliquots were removed from the tubes
spread on LB agar plates.
8. The plates were incubated at 37 C overnight
and the resulting colonies were counted.
Procedure (Pt. 1)
9. The Dilution Process was completed and the
following concentrations of Hydrogen Peroxide,
at the following concentrations, and Plating
Process was performed
[0%]
[10-2%] [10-1%]
[1%]
SDF
9.9 mL
9.8 mL
8.9 mL
0 mL
Microbe
0.1 mL
0.1 mL
0.1 mL
0.1 mL
Hydrogen
Peroxide
0 mL
0.1 mL @
1 mL @
[1%]
[1%]
9.9 @
Total
Volume
10 mL
10 mL
10 mL
10 mL
pH
7
7
6
5
[1%]
Number of Colonies
Results (Pt. 1)
Bleach Effects on Bacterial Survivorship
700
p-value < 0.05 E. coli
611.5
600
537 p-value < 0.05
Staph.
489.25
500
400
433.5
From test with
comparable control
378.5
364
300
t-value
45.85
56.38
200
100
0
6.333
[0%]
0
[10-5%]
-5
[10-4%]
-4
[10-3.5%]
-3.5
Bleach Concentration
0 7
[10-3%]
-3
0 0
[10-2%]
-2
Results (Pt. 1)
Number of Colonies
600
500
400
Hydrogen Peroxide Effects on
Bacterial Survivorship
489.25
463.6666667
414
364
t-value
35.14 15.34
300
200
144.75
90.5
100
0
E. coli
Staph.
p-value < 0.05
p-value < 0.05
0
0
[0%]
[10-2-2%]
[10-1-1%]
1
[1%]
Hydrogen Peroxide Concentration
Procedure (Pt. 2)
10. Cloning: 1 colony transferred to sterile LB
media and incubated for 2 days
– Colonies From:
E. coli exposed to [10-3.5%] Bleach
Staph. exposed to [10-3%] Bleach
Procedure (Pt. 2)
11.The Dilution Process was performed with:
– E. coli Strain exposed to Bleach (now, “Exposed”)
– Staph. Strain exposed to Bleach (now, “Exposed”)
– E. coli Strain NOT exposed to Bleach (“Not Exposed”)
– Staph. Strain NOT exposed to Bleach (“Not Exposed”)
• (both from original strain)
Procedure (Pt. 2)
12. Each strain (Exposed and Not-Exposed) was exposed
to appropriate concentration, based on species
survivorship:
E. coli :
Staph.:
• [0%] (Control)
• [0%] (Control)
• [10-1%] Hydrogen Peroxide • [10-1%] Hydrogen Peroxide
13.The Plating Process was completed, statistical
tests were performed, and conclusions drawn
Hydrogen Peroxide Effects on
E. coli survivorship
500
Number of Colonies
450
E. coli, Not Exp.
E. coli, Exp.
438.25
400
350
300
250
200
301.33333
Percent
Survivorship
1.83%
4.31%
ANOVA p-value
4.30*10-7
1.40*10-6
150
100
50
8
13
0
[0%]
[10-1%]
Hydrogen Peroxide Concentration
Hydrogen Peroxide Effect on
Staph. Survivorship
350
Staph., Not Exp.
Staph., Exp.
Number of Colonies
316.667
300
250
200
150
183.25
Percent
Survivorship
1.74%
0%
ANOVA p-value
1.48*10-8
7.28*10-7
100
50
0
5.5
0
[0%]
[10-1%]
Hydrogen Peroxide Concentration
Conclusion
• Initial Exposure to Bleach appeared to
NOT INCREASE resistance of
Staph. to Hydrogen Peroxide effects
Null Hypothesis Accepted
• Initial Exposure to Bleach appeared to
INCREASE resistance of
E. coli to Hydrogen Peroxide effects
Null Hypothesis Rejected
Limitations
• Plating slightly
unsynchronized
• Only Liquid Pulse
Procedure utilized
• Only one clone: more
“generations” may be
needed to develop
resistance
Extensions
• More variables,
concentrations, exposure
times, etc.
• Different Exposure
methods– agar infusion
• Study growth, over time
• More cloning; more
“generations”
• Develop Resistance to
Bleach
Bleach Effects on E. coli Survivorship
500
Number of Colonies
450
400
350
300
250
438.25
Percent
Survivorship
101.39%
91.68%
301.3333333
444.3333333
E. coli, Not Exp.
E. coli, Exp.
ANOVA p-value
0.82
276.25
0.14
200
150
100
50
0
[0%]
[10-4%]
Bleach Concentration
Bleach Effects on Staph. Survivorship
350
316.67
Staph., Not Exp.
Number of Colonies
300
Staph., Exp.
250
200
150
183.25
Percent
Survivorship
0.09%
0.95%
ANOVA p-value
1.24*10-6
8.14*10-7
100
50
0
0.25
1.25
[0%]
[10-3%]
Bleach Concentration
Resources
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http://housekeeping.about.com/od/environment/tp/Ph-Levels-For-Common-Cleaning-Supplies.htm
http://www.epa.gov/kidshometour/products/bleach.htm
http://science.howstuffworks.com/question189.htm
http://www.lenntech.com/processes/disinfection/chemical/disinfectants-sodium-hypochlorite.htm
http://www.clorox.com/products/clorox-concentrated-regular-bleach/?gclid=CMHDlqjO7b8CFStgMgodYSwAMg
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=516902&loc=ec_rcs
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=516902&loc=ec_rcs
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=14798&loc=ec_rcs
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=4068533&loc=ec_rcs
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=5234&loc=ec_rcs
http://www.whatsinproducts.com/files/brands_pdf/1335704518.pdf
http://www.ehow.com/facts_6760567_toilet-cleaner-acid-base_.html
http://www.epa.gov/ttn/atw/hlthef/hydrochl.html
http://scorecard.goodguide.com/chemical-profiles/html/hydrochloric_acid.html
http://www.chemicalbook.com/ChemicalProductProperty_EN_CB11211808.htm
http://www.pinoychem.com/2008/09/thick-toilet-bowl-cleaner-using.html
http://oregonstate.edu/dept/larc/sites/default/files/pdf/chlorine-fact-sheet.pdf
http://www.elmhurst.edu/~chm/vchembook/184ph.html
http://www.ehow.com/info_8196650_hydrogen-ions.html
http://brainstretch.net/introChem/makingCompounds/hydroxide/hydroxideFrame.htm
http://www.globalw.com/support/ph-measurement.html
http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_1.html
http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_2.html
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html
pH
• Measure of Acidity or Basicity of aqueous
solution
• Hydrogen ions (H+) and hydroxide ions (OH-).
– More H+, More Acidic
– More OH-, More Basic
• measured on a scale of 0-14
• pH
< 7: Acidic
> 7: Basic
= 7: Neutral
Anova: Single Factor Bleach on Staph
SUMMARY
Groups
Count
Sum
Average
Variance
0%
2
728
364
50
10-5%
4
1734
433.5
777
10-4%
4
1514
378.5
91
10-3%
4
28
7
10
10-2%
4
0
0
0
ANOVA
Source of
Variation
Between
Groups
Within Groups
Total
SS
df
MS
699258.4
4
174814.6
2684
13
206.4615
701942.4
17
F
846.7176
T-value
35.13692
P-value
1.44E-15
F crit
3.179117
Dunnett’s test
• Dunnett’s test
– comparison of two groups to determine significant
variation.
– t-crit. ≈2.7
– t-value
• > t-crit → SIGNIFICANT
• < t-crit → NOT SIGNIFICANT
Percent Survivorship
E. coli
Not Exp.
Exp.
Bleach
101.39%
91.68%
Hydrogen Peroxide
1.83%
4.31%
Staph.
Not Exp.
Exp.
Bleach
0.09%
0.95%
Hydrogen Peroxide
1.74%
0%
[10-3.5%]
SDF
6.74
Microbe
0.1
Chlorine Bleach 3.16 mL @ [10-3%]
Total Volume
10
pH
7