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 KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 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 10 8 cells/mL. 4) The culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 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 10 3 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.

p=1.88E-21 p<0.01

Dunnett’s Test Variable Comparisont-valueInterpretation 5 versus 0 (control)1.5Not Significant 30 versus 0 (control)2.7Not Significant 60 versus 0 (control)23.9Significant 3 days versus 0 (control) 28.4Significant α = 0.01 t-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.

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