Jackson Kaib
CCHS
Toilet Water Microbial
Survivorship Analysis
Escherichia coli (E.coli)
•
A common bacteria, found in many
environments
•
E.coli gram negative, rod shaped
bacillus
•
Mostly non-pathogenic
•
Pathogenic strains can lead to life
threatening infections
•
Most widely studied Prokaryotic cell
Gram Negative (E.coli)
•
Cell wall is a thin layer of
lipopolysaccharide which adds an extra
level of protection
•
If the toxin enters the circulatory system
it can cause a toxic reaction
•
This outer most membrane protects the
bacteria from several antibiotics
Staphylococcus epidermidis (Staph)
•
A round-shaped, gram
positive bacteria, found on the
skin on the skin or mucous
membranes of animals
•
Most strains are not harmful
•
Pathogenic strains can be life
threatening
•
Common bacteria model
Recent studies
•
A study was performed on the bacterial amounts
and growth rates between various restaurants
•
The results suggested that the ice water had
higher bacterial amounts and could sustain
bacterial better than that of the toilet water
•
This experiment was conducted in the same light
and used some of the same procedures therefore
this experiment could show similar results given
the restaurants tested were in the same state
Purpose
•
The purpose of this experiment was to
determine in what solution E.coli and Staph
could grow and survive in restaurant ice
water or toilet water
Hypothesis
Null hypothesis: The
bacterial environments of ice
and toilet water survivorship
rate will not vary significantly
•
•
Alternative hypothesis:The
bacterial environments of ice
and toilet water will vary
significantly
Materials
•
LB agar plates
•
Sterile test tubes
•
LB media
•
Sterile bags
•
Escherichia Coli
•
Incubator
•
Staphylococcus epidermidis
•
Sterile filter
•
Restaurant (1 and 2)Toilet water
•
Vortex
•
Restaurant (1 and 2) Ice
•
Test tube rack
•
Ethanol
•
Micro burner
•
Sidearm flask
•
•
•
Spreader bar
Micropipette
Sterile pipette tips
Procedure
•
Bacteria (E.coli and Staph) was grown overnight in sterile LB media.
•
A sample of the overnight culture was added to fresh media in a sterile
sidearm flask.
•
The cultures were placed in incubators at 37°C until a density of 50 Klett
spectrophotometer units were reached.
•
The cultures were diluted in sterile dilution fluid to a concentration of
approximately 10^5 cells/mL
•
Tubes were made up of 9.9 ml sterile filtered variable water
•
.1 ml of microbe was placed in the water
Procedure (cont)
•
7. The solutions were mixed by vortexing and allowed to sit
at room temperature for 12 minutes.
•
8. After vortexing to evenly suspend cells, 0.1 ml was
removed from the tubes and spread on LB plates.
•
9. The plates were incubated at 37°C E.coli 24 hours and
staph for 48 hours .
•
10. The resulting colonies were counted. Each colony is
assumed to have arisen from one cell.
Tube makeup
C
STP
C
EC
R1
EC
T
R1
EC
I
R2
EC
T
R2
EC
I
R1
STP
T
R1
STP
I
R2
STP
T
R2
STP
I
Toilet
water
0.0
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
0.0
ml
Ice
water
0.0
ml
0.0
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
0.0
ml
9.9
ml
SDF
9.9
ml
9.9
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
STP
0.1
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
EC
0.0
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.1
ml
0.0
ml
0.0
ml
0.0
ml
0.0
ml
Staph survivorship
#
S
U
R
V
I
V
I
N
G
C
O
L
O
N
I
E
S
500
386
400
208.5
300
144.5
200
166.5
152.5
100
0
Control
R1 T
R1 I
Source
R2 T
R2 I
E.coli survivorship
#
S
U
R
V
I
V
I
N
G
625
C
O
L
O
N
I
E
S
250
490
500
461
343
387.75
375
125
2.25
0
Control
R1 T
R1 I
Source
R2 T
R2 I
Percent of change
E.coli
Staph
•
R1 toilet = - 30%
•
R1 toilet = - 63%
•
R1 ice = - 21 %
•
R1 ice = - 57 %
•
R2 toilet = - 99.5 %
•
R2 toilet = - 60%
•
R2 ice = - 6 %
•
R2 ice = - 46 %
Anova results
location
Single factor Anova
Restaurant 1
E.coli
P-value=0.097
Non Significant
Restaurant 2
E.coli
P-value=2.42-06
Significant
Restaurant 1
Staph
P-value=1.75-07
Significant
Restaurant 2
Staph
P-value=6.37-06
Significant
DUNNETT’S TEST RESULTS T-CRIT=2.78
Location
T-Value
Significance
Restaurant 2
E.coli
Ice water=-0.64
Toilet water=10.28
Nonsignificant
Significant
Restaurant 1
Staph
Ice water=-13.67
Toilet water=-15.04
Significant
Significant
Restaurant 2
Staph
ice water=-7.96
toilet water=-10.47
Significant
Significant
Conclusion
Location
Significance
The null hypothesis was
rejected/accepted
Restaurant 2
E.coli
Nonsignificant
Significant
Accepted
Rejected
Restaurant 1
Staph
Significant
Significant
Rejected
Rejected
Restaurant 2
Staph
Significant
Significant
Rejected
Rejected
Restaurant 1
Ecoli
Nonsignificant
Nonsignificant
Accepted
Accepted
Limitations
•
Delay time in stop watch going off and plating.
•
Limited number of replicates
•
Limited incubation time
•
Time in-between restaurant sample gathering
Extensions
•
Examine more restaurants at varying price ranges
•
Add more replicates
•
Different bacteria
•
More plates
Sources
• http://www.homeinspector.org/resources/journals/water-analysis2.pdf
• http://www.dailymail.co.uk/news/article-2334533/Ice-restaurants-bacteria-water-toilets.html
• http://os.care2.com/all/ice-is-dirtier-than-toilet-water-in-these-fast-foodrestaurants?oswbuild=b0.37&mediaKey=care2&ref=https%3A%2F%2Fwww.google.com%2
F&origin=http%3A%2F%2Fwww.care2.com%2Fgreenliving%2Fice-is-dirtier-than-toiletwater-in-these-fast-foodrestaurants.html&oswts=1422907193345&width=1024&height=672&size=large&olcts=14229
07193949
• http://www.inquisitr.com/684608/restaurant-ice-is-dirtier-than-toilet-water-says-disgustingnew-report/
• http://www.scientificamerican.com/article/drinking-from-the-toilet/
E.coli Anova
Restaurant 1
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
4
1551
387.75
19650.92
Column 2
4
1369
342.25
2295.583
Column 3
4
1958
489.5
363.6667
ANOVA
Source of Variation
SS
df
MS
F
Between Groups
45474.5
2
22737.25
Within Groups
66930.5
9
7436.722
Total
112405
11
P-value
3.057429
F crit
0.097
4.256495
E.coli anova
restaurant 2
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
4
1844
461
11574
Column 2
4
9
2.25
6.916667
Column 3
4
1958
489.5
363.6667
ANOVA
Source of Variation
SS
df
MS
F
Between Groups
598235.2
2
299117.6
Within Groups
35833.75
9
3981.528
Total
634068.9
11
P-value
75.12633
2.42E-06
F crit
4.256495
Staph anova
restaurant 1
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
4
666
166.5
843.6667
Column 2
4
578
144.5
349.6667
Column 3
4
1544
386
354
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
142648.7
2
71324.33
4642
9
515.7778
147290.7
11
F
P-value
138.285
1.75E-07
F crit
4.256495
Staph anova
restaurant 2
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
4
1544
386
354
Column 2
4
833
208.25
2310.917
Column 3
4
610
152.5
321.6667
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
F
118967.2
2
59483.58
8959.75
9
995.5278
127926.9
11
P-value
59.7508
6.37E-06
F crit
4.256495