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/