Transcript E coli survivorship in ice Herbst FINAL PJAS Powerpoint

Survivorship of E. coli in Ice cubes
Cameron Herbst
Pittsburgh Central Catholic High School
The Problem
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Bacterial infection of water is a concern in all parts
of the world.
Many people ask for no ice cubes in drinks due to
possible contamination.
A commonly studied bacteria that afflicts water is
Escherichia coli, which makes a good test
specimen.
It is thought that ice cubes might contain viable
pathogenic bacteria.
Related Studies
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Department of Food Science, University of
Manitoba, Bollman, Ismond, and Blank tested
the survivorship of E. coli in frozen foods.
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US Department of Agriculture, Juneja, Snyder,
Jr. and Marmer tested thermal destruction of E.
coli in beef and chicken.
E. coli
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One of the most common forms
of bacteria; Free living,
symbiotic or pathogenic.
Has been utilized as the most
studied prokaryote.
There are many of different
strains of E. coli, most of which
are non-pathogenic. However,
there are strains which can
produce fatal disease.
E. coli Background Information
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Contaminates water whenever manure or sewage
comes into contact with potable water.
Doubles its cell count within a time span of twenty
minutes.
Infection caused by undercooked beef is known as
Hemolytic Uremic Syndrome (HUS).
Symptoms manifest within a 1-8 day span, but
usually are visible between 2-5 days of infection.
Purpose
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To investigate whether microbes such as E.
coli can remain viable in ice cubes, possibly
leading to contaminated drinking and
cooking water.
Hypotheses
 Null
- E. coli survivorship in ice will not
vary significantly from the control (20C
sterile dilution fluid).
 The alternative hypothesis is that all of
the freezing durations will significantly
reduce E. coli survivorship.
Materials
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Ethanol (for sterilization of instruments)
Latex gloves
E. coli DH5 alpha
Micropipettes
Micro rack
Ten microtubes
-20C freezer
SDF (per 1 liter) (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Turn table
LB agar plates
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Bunsen burner
Spreader bar
Matches
Sterile pipette tips
Incubator
Vortex
Klett spectrophotometer
Procedure
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E. coli was 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 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.
The culture was diluted in sterile dilution fluid to a
concentration of approximately 103 cells/mL.
The following ingredients were transferred to 1.5 mL
sterile microtubes and their replicates; 0.9 mL of
sterile dilution fluid and 0.1 mL of 103 cells/mL E. coli
solution.
Procedure
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The microtubes were placed in a -20C freezer for their
allotted times.
Immediately after thawing, 100 µL aliquots were
removed from the tubes and spread on LB plates. (10
replicates)
The plates were incubated at 37 degrees Celsius for 24
hours.
The resulting colonies were counted. Each colony is
assumed to have arisen from one cell.
E. coli Ice Cube Survivorship
Number of Colonies
p=1.88E-21
300
250
200
150
100
50
p<0.01
p<0.01
60
3 days
0
0
5
30
Freezing Time (minutes)
Dunnett’s Test
α = 0.01
t-critical= 4.55
Variable Comparison
t-value
Interpretation
5 versus 0 (control)
1.5
Not Significant
30 versus 0 (control)
2.7
Not Significant
60 versus 0 (control)
23.9
Significant
3 days versus 0 (control)
28.4
Significant
Interpretation
 There
appeared to be a trend of a direct
correlation between freezing time and
survivorship. Longer freezing times
resulted in less survivorship.
Conclusion
 The
null hypothesis can be rejected
for 60 minutes and 3 days of freezing
duration.
 60 minutes and three days of freezing
appeared to significantly reduce E.
coli survivorship.
Limitations and Extensions
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Five minutes of freezing may not have resulted in solid
cubes.
In future studies, E. coli may be frozen in different
concentrations to observe if concentration and freezing
affects survivorship.
E. coli can be frozen even longer than 3 days for continued
data.
A different species of bacteria could be used in the
experiment.
Plating was not exactly synchronized, which could have
resulted in extra time for bacterial replication. A team of
students could remedy this technical problem.
Cited Websites
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http://www.cbc.ca/newsinreview/Sep2000/walkerton
/facts.htm
http://www.cdc.gov/nczved/dfbmd/disease_listing/ste
c_gi.html
http://www.epa.gov/safewater/contaminants/ecoli.ht
ml
http://e-colibasics.com/