Transcript Ziccarrelli Disinfectant Concentration Effects on Bacteriax

Joey Ziccarelli
Grade 9
Central Catholic High School
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Many disinfectant manufacturers claim that
their product effectively kills microbes.
These disinfectants are commonly used in
society to either sterilize a wound or to
reduce the concentration of pathogens on
surfaces.
How effective are these products?
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To determine the effects of common
antiseptic solutions on the survivorship of a
gram positive and gram negative bacteria:
specifically Staphylococcus Epidermidis and
Escherichia coli
To assess the antibacterial properties of
common household disinfectants
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Iodine was discovered by the French chemist Barnard Courtois in
1811. Courtois was extracting sodium and potassium compounds
from seaweed ash.
Iodine is used as an indicator test for starch and turns a deep blue
when it comes in contact with it.
Potassium iodide (KI) is used to make photographic film.
When mixed with iodine in alcohol, as an antiseptic for external
wounds.
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Vinegar is a liquid substance consisting mainly of acetic acid
and water, the acetic acid being produced through the
fermentation of ethanol by acetic acid bacteria.
Produced either by fast or slow fermentation processes.
Types of vinegar: Malt, Wine, Sherry, Apple Cider, Fruit,
Balsamic, Rice, Wine vinegar, Distilled vinegar
Any type of vinegar may be distilled to produce a colorless
solution of about 5% to 8% acetic acid in water.
This is variously known as distilled spirit, or white vinegar,
and is used for medicinal, laboratory, and cleaning purposes,
as well as in cooking, baking, meat preservation, and
pickling.
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Louis Jacque Thenard discovered hydrogen peroxide in 1818.
Hydrogen peroxide consists of oxygen and hydrogen atoms. In the
environment, hydrogen peroxide can be found in very low
concentrations.
Gaseous hydrogen peroxide is produced by photo chemical
reactions in the atmosphere surrounding the earth. It can also be
found in water in small quantities.
The peroxide ion consists of a single bond between two oxygen
atoms: (O-O)2-. It is a strong oxidizer. Hydrogen peroxide has the
chemical formula H2O2 and the following structural formula: H-OO-H
3% H2O2 - One of the most common disinfectants for minor cuts
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Null Hypothesis: Hydrogen Peroxide,
Antiseptic Iodine, Vinegar will have no effect
on E. coli and Staph e survivorship.
Alternative Hypothesis: Hydrogen Peroxide,
Antiseptic Iodine, Vinegar will significantly
reduce survivorship of E. coli and Staph e.
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Glass Test Tubes with
SDF (100mM KH2PO4,
100mM K2HPO4, 10mM
MgSO4, 1mM NaCl)
Macropipettes
Micropipettes + tips
Disinfectants (Iodine
Antiseptic, Vinegar,
Hydrogen Peroxide)
Vortex
Ethanol, Spreaders
LB Agar Plates
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LB media (0.5% yeast
extract, 1% tryptone, 1%
sodium chloride)
Incubator
Escherichia coli (DH5
Alpha)
Staphylococcus
epidermidis
Test tube rack
Side Arm Flask
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Most commonly studied historical microbe
Gram negative
Found in animal intestines (including humans)
Necessary to keep body healthy
Sometimes found in food as a result of cross
contamination (many times during the
slaughtering process)
Symptoms: Diarrhea, Abdominal cramping,
Nausea and vomiting
 Cell wall is thin
extra layer of
lipopolysaccharid
e which adds
extra level of
protection.
 If the toxin enters
the circulatory
system it causes
a toxic reaction.
 This outer
membrane
protects the
bacteria from
several
antibiotics.
 Professor Duman
Lab at CMU
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Often found as an external symbiont on
human skin
Gram positive
Round (Coccid)
Forms grape-like clusters
Symptoms: Boils, Impetigo, Cellulitis
Staphylococcal scalded skin syndrome
 Most
pathogenic
bacteria in
humans are
gram-positive
organisms.
 Simple cell
wall.
 Antibiotics
such as
penicillin work
against the
formation of
the cell wall.
 Carolina
Company
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A: Liquid Exposure
Escherichia coli and Staphylococcus epidermidis were 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 108109 cells/ml.
The culture was diluted in sterile dilution fluid to a concentration of
approximately 105 cells/ml.
The disinfectants were 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.]
The pH of each disinfectant was recorded during the experiment.
The pH of antiseptic iodine was 6. The pH of Hydrogen Peroxide was 6.
The pH of distilled vinegar was 3.
Stock Solution
0%
0.1%
1%
10%
SDF
9.9 mL
9.89 mL
9.8 mL
8.9 mL
Disinfectant
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
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The tubes were vortexed and allowed to incubate at room
temperature for a 20 minute time period.
After vortexing to evenly suspend cells, 0.1 ml. aliquots were
removed from the tubes and spread on LB agar plates.
B: Infusion Test
200 microliters of disinfectant (or SDF) were spread onto LB agar
plates. The plates were inverted and incubated for two hours at
37 C for infusion.
As in the liquid experiment, bacteria was spread onto the infused
plates.
The plates were left to sit overnight.
The resulting colonies were counted. Each colony is assumed to
have arisen from one cell.
It is assumed that the 200 microliters diffused into the agar
plate’s 20 milliliters evenly at a 1/100 variable concentration in
the agar.
Disinfectant Effects on E. coli Survivorship
80
Resulting Number of Colonies
70
2.91E-08
60
50
3.31E-20
40
E-coli Antiseptic
E-coli Vinegar
30
E-coli Hydrogen Peroxide
3.75E-19
2.59E-21
1.31E-21
20
10
0.392186
0
0%
0.10%
1%
Concentration of Disinfectant
10%
Disinfectants Effect on Staph Survivorship
80
1.48E-26
70
1.64E-26
Resulting
Number of Colonies
60
50
40
Staph Antiseptic
Staph Vinegar
30
Staph Hydrogen Peroxide
1.89E-39
7.34E-39
1.9E-27
20
10
0.020846
0
0%
0.10%
1%
Concentration of Disinfectant
10%
T-critical = 2.76
Alpha value= .05
Vinegar
Concentration
T-Value
E. coli
Staph
E. coli
Staph
0.1%
1.35
58.26
Not Significant
Significant
1%
8.32
59.25
Significant
Significant
10%
17.32
59.5
Significant
Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
Interpretation
T-critical = 2.76
Alpha value= .05
Antiseptic Concentration
T-Value
Interpretation
E. coli
Staph
E. coli
Staph
0.1%
8.9
60.25
Significant
Significant
1%
18.62
60.76
Significant
Significant
10%
18.75
61.01
Significant
Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
T-critical = 2.76
Alpha value= .05
Hydrogen Peroxide
Concentration
T-Value
E. coli
Staph
E. coli
Staph
0.1%
9.04
.91
Significant
Not Sig.
1%
19.52
5.15
Significant Significant
10%
19.55
31.5
Significant Significant
𝑡𝑑 =
𝑀𝑖 − 𝑀𝑐
2𝑀𝑆𝐸
𝑛ℎ
Interpretation
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Overall, the three disinfectant variables used
in this experiment had a significant effect on
the survivorship of Escherichia coli and
Staphyloccocus Epidermidis.
Therefore, the null hypothesis was rejected.
Limitations
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The disinfectants were somewhat insoluble and needed
to be inverted repeatedly
Only one time of exposure was utilized
Difficult to synchronize plating
Future Experimentation
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Test various concentrations of disinfectants
Test the disinfectants effects on numerous cell models
(yeast, algae)
Test the effects of different disinfectants
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http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC1057892/
http://cmr.asm.org/content/12/1/147.short
http://www.academicjournals.org/ajpp/pdf/%
20pdf2009/May/ElMahmood%20%20and%20%20Doughari.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC88971/
http://aem.asm.org/content/10/5/458
Anova
• Abbreviation for analysis of variance
• Statistical test to see variance between and
within groups
•If the P- value is
larger than the
alpha value (.05),
then the result is
significant
Sample ANOVA used in experiment
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
721
72.1
26.32222
Column 2
10
1
0.1
0.1
Column 3
10
4
0.4
0.266667
Column 4
10
10
1
1.111111
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
SS
df
MS
F
38453.4
3
12817.8
250.2
36
6.95
38703.6
39
P-value
1844.288
1.89E-39
F crit
2.866266
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
721
72.1
26.32222
Column 2
10
16
1.6
2.044444
Column 3
10
10
1
0.666667
Column 4
10
7
0.7
0.455556
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
SS
df
MS
37811.7
3
12603.9
265.4
36
7.372222
38077.1
39
F
1709.647
P-value
7.34E-39
F crit
2.866266
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
721
72.1
26.32222
Column 2
10
741
74.1
46.98889
Column 3
10
608
60.8
32.4
Column 4
10
31
3.1
9.877778
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
SS
df
MS
33599.68
3
11199.89
1040.3
36
28.89722
34639.98
39
F
387.5768
P-value
1.9E-27
F crit
2.866266
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
698
69.8
213.0667
Column 2
10
644
64.4
71.82222
Column 3
10
365
36.5
34.72222
Column 4
10
5
0.5
0.277778
ANOVA
Source of
Variation
Between
Groups
Within Groups
Total
SS
df
MS
30245.4
3
10081.8
2879
36
79.97222
33124.4
39
F
126.0663
P-value
3.75E-19
F crit
2.866266
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
698
69.8
213.0667
Column 2
10
431
43.1
57.87778
Column 3
10
9
0.9
0.766667
Column 4
10
4
0.4
0.266667
ANOVA
Source of
Variation
Between
Groups
Within Groups
Total
SS
df
MS
34702.1
3
11567.37
2447.8
36
67.99444
37149.9
39
F
170.1222
P-value
2.59E-21
F crit
2.866266
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
10
698
69.8
213.0667
Column 2
10
376
37.6
40.48889
Column 3
10
3
0.3
0.233333
Column 4
10
2
0.2
0.177778
ANOVA
Source of
Variation
Between
Groups
Within Groups
Total
SS
df
MS
33753.28
3
11251.09
2285.7
36
63.49167
36038.98
39
F
177.2058
P-value
1.31E-21
F crit
2.866266