Transcript The Effect of Clorox Disinfecting Wipes on E.Coli

Eric Carnivale
Grade 9
Pittsburgh Central Catholic High School
Prevention and Treatment of
Bacterial Infection
 Washing hands
 Thorough cooking of food
 Washing food
 Food employees washing hands
and wearing gloves
 Antibiotics
 Clorox® Disinfecting Wipes
Uses of Clorox® Disinfecting
Wipes
 Used on household
surfaces to kill
bacteria
 Claims to kill the flu
virus, H1N1 ,and
common bacteria
Clorox® Disinfecting Wipes
 Claims to kill 99.9% of all
germs.
 Contains Dimethyl Benzyl
Ammonium Chloride
 Destroys, neutralizes, or
inhibits growth of bacteria
 Ruptures lipid bilayer of
bacterial membrane
Escherichia coli
 Major cause of food-borne illness
 70,000 cases/year in U.S.
 Consumption of undercooked meats
 Consumption of contaminated
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vegetables or unpasteurized milk
Also resides in fecal matter.
Most studied bacteria
Commonly found in the digestive
tract of animals.
Can survive outside of host organism
Not all strains of E.coli can result in
sickness or disease
Purpose
To test the effect of Clorox®
Disinfecting wipes on the
survivorship of Escherichia
coli bacteria.
Hypotheses
Null hypothesis: The Clorox® disinfecting
wipes will have no significant effect on the
survivorship of E. coli.
Alternative hypothesis: The Clorox®
Disinfecting wipes will have a significant effect on
the survivorship of E. coli.
Materials
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E.coli
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride).
Petri dishes with LB agar
Clorox® Disinfecting Wipes
4 Sterile 15 ml conical tubes
Peanut butter jar lid (to fit inside of Petri dish)
Scissors
Vortex
Forceps
Incubator
Spreading bar
Ethanol
Bunsen burner
Micro Pipette and sterile tips
SDF (Sterile Dilution Fluid) 100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM
NaCl
 Test tube rack
 Klett Spectrophotometer
Procedure (Surface Contact)
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E. coli was grown overnight in a sterilized lb media
A sample of the overnight culture was added to fresh LB media in a
sterile sidearm flask.
The cultures were incubated at 37°C until a density of 50 Klett
spectrophotometer units was reached. These represent cell densities
of approximately 108 cells/mL.
The cultures were diluted in sterile dilution fluid to a concentration
of approximately 103 cells/mL.
Prior to addition of E.coli Clorox® Disinfecting Wipes were cut to the
size of a Petri dish.
A Clorox Disinfecting Wipe was held down on the surface of the
plate for 0,10,20,30,40 ,or 60 seconds (then removed).
100µl of E.coli was pipetted onto each plate.
The plates were incubated at 37 degrees Celsius for 1 day.
The resulted colonies were counted. Each colony is assumed to have
risen from 1 cell.
Procedure (Elutant Effects)
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A 5cm by 5cm cut piece of the disinfecting wipe was placed in
SDF.
The tube with wipe and SDF was vortexed for1 minute.
The wipe was removed and disposed of.
Different concentrations were made by pipetting 0, 0.1, 1, and
5 ml’s of the wipe-SDF mixture and combining it with the
appropriate amount of E.coli to make a final volume of 10mls.
100 µl of the 0, 0.1,1 5 and ml concentration was pipetted onto
6 plates.
The spreading bar was sterilized and the E.coli was spread
onto the plates.
Plates were Incubated at 37 degrees Celsius for 1 day.
The resulted colonies were counted. Each colony is assumed to
have arisen from 1 cell.
Procedure Con’t (Elutant Effects)
Tube
E.Coli (mL)
Wipe-SDF
mixture (mL)
Final volume
(mL)
1
10
0
10
2
9.9
0.1
10
3
9
1
10
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5
5
10
ANOVA/Dunnetts’ Test
The ANOVA is a statistical test used to compare the
variation within the groups to the variation between
groups.
The Dunnetts’ test allows a further pair-wise comparison
of the means of each group versus the control group.
This is used to determine the source of the variation.
Surface wipe surface effects
P-Value= 2.27E-06
350
Number of Colonies
300
250
200
150
100
50
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0
10
20
30
Exposure Times (seconds)
40
60
Statistical Analysis for wipe effects experiment
Dunetts’ Test
T-critical = 3.10
Exposure Time(sec)
T- Value
Interpretation
10
7.41
Significant
20
8.67
Significant
30
9.66
Significant
40
9.93
Significant
60
10.03
Significant
Surface wipe elutant effects on E.coli survivorship
P-Value= 4.3E-10
Number of Colonies
350
300
250
200
150
100
50
0
0
0.1
1
mL’s of wipe-SDF mixture
5
Statistical analysis for wipe elutant experiment
Dunetts’ test
T-Critical = 3.10
Ml’s of stock
added
T-value
Interpretation
0.1
9.90
Significant
1
11.01
Significant
5
11.37
Significant
Conclusion
The statistical analysis allows for a
rejection of the Null hypothesis.
 Surface wiped plates and wipe elutant
appeared to have resulted in a
significant reduction in E.coli
survivorship.
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Extensions and Limitations
 Using different bacteria and different
brands of anti-bacterial wipes to determine
which wipe is the most effective against
common bacteria.
 The wipe was not directly exposed to the
E.coli on the plate.
 It was difficult to sychronize plating time.
References
 http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi
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.html
http://familydoctor.org/online/famdocen/home/common/
digestive/disorders/242.html
http://www.about-ecoli.com/
http://www.clorox.com/products/overview.php?prod_id=c
dw
http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id
=PC37016
http://en.wikipedia.org/wiki/Benzalkonium_chloride
http://en.wikipedia.org/wiki/Escherichia_coli