survivorship of e. coli in ice cubes cameron herbst pittsburgh central catholic high school
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Survivorship of E. coli in Ice cubes
Cameron Herbst
Pittsburgh Central Catholic High School
The Problem
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
Department of Food Science, University of Manitoba, Bollman, Ismond, and Blank tested the survivorship of E. coli in frozen foods.
US Department of Agriculture, Juneja, Snyder, Jr. and Marmer tested thermal destruction of E. coli in beef and chicken.
E. coli
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
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
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
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
1) E. coli was grown overnight in sterile LB media.2) A sample of the overnight culture was added to
fresh media in a sterile sidearm flask.3) 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.
4) The culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/mL.
5) 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
6) The microtubes were placed in a -20C freezer for their allotted times.
7) Immediately after thawing, 100 µL aliquots were removed from the tubes and spread on LB plates. (10 replicates)
8) The plates were incubated at 37 degrees Celsius for 24 hours.
9) The resulting colonies were counted. Each colony is assumed to have arisen from one cell.
E. coli Ice Cube Survivorship
0
50
100
150
200
250
300
0 5 30 60 3 days
Freezing Time (minutes)
Num
ber o
f Col
onie
s
p=1.88E-21
p<0.01 p<0.01
Dunnett’s Test
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
α = 0.01t-critical= 4.55
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
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
http://www.cbc.ca/newsinreview/Sep2000/walkerton/facts.htm
http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html
http://www.epa.gov/safewater/contaminants/ecoli.html
http://e-colibasics.com/
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