1. Aquatic Thermal Tolerance of E.coli
J.R. Stragar
Grade 9
Pittsburgh Central Catholic High School

2. E.coli
Escherichia coli (E.coli) is one of the most common forms of bacteria found in many environments including the intestinal tracts of many mammals.
E.coli has also been utilized as the most studied prokaryote in biological research.
There are many of different strains of E.coli, most of which are non-pathogenic. However, there are strains which can produce fatal disease in humans and other mammals.

3. E.coli Recent Outbreaks
One of the more recent outbreaks of E.coli has been the Taco Bell lettuce E.coli outbreak.
 As of Wednesday, December 13, 2006, 71 persons have become ill with Escherichia coli 0157:H7 associated with eating contaminated lettuce at Taco Bell restaurants in the northeastern United States.
Cases were reported to the CDC from five states: New Jersey (33), New York (22), Pennsylvania (13), Delaware (2) and South Carolina (1).
Of the 71 persons with illness, 48 have been confirmed as cases.

4. E.coli in Different Temperatures
Aside from using drugs to treat an E.coli infection or preservatives to prevent E.coli contamination, many people have sought simpler remedies for reducing exposure to potentially pathogenic microbes.
E.coli is often transmitted in solution (water).
QUESTION: How does the temperature of the aquatic environment effect the survivorship of E.coli?
E.coli can grow in temperatures ranging from 7°C to 50°C, with an optimum temperature of 37°C.
It is destroyed by thorough cooking of foods until all parts reach a temperature of 70°C or higher.
2 methods utilized to reduce the amount of bacteria in foods have been heating and cooling (refrigeration).

5. Purpose
To examine the effect of water environment temperature on E.coli survivorship.

6. Hypotheses The 50° C water temperature will kill the most E.coli.
The null hypothesis is that temperature will have no effect on the survivorship of E.coli.
Increasing exposure time will result in a decrease in survivorship in all temperatures.
The other null is that incubation time will have no effect on survivorship of E.coli.

7. Materials
60 LB agar plates( 1 % tryptone, 5 % yeast extract, 1% NaCl, 1.5 % agar)
LB media (1 % tryptone, 5 % yeast extract, 1% NaCl)
Klett spectrophotometer
Sterile pipette tips
Micropipettors
4 one liter beakers
Freezer
Vortex
Hot and cold tap water
Incubator
Sidearm flask
Spread plate
Spreader bar
Ethanol
20 mL Sterile capped test tubes
E.coli B
Sterile dilution fluid

8. Procedure
Four 1 liter beakers were filled with 500 ml of tap water. The temperatures were adjusted to 5, 10, 30 and 50 degrees C and maintained at these temperatures for the duration of the experiment.
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 cultures were placed in a shaking water bath (37 degrees C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 109 cells/ml.
The culture was diluted in sterile dilution fluid to a concentration of approximately 103 cells/ml.
The solution was mixed by vortexing and allowed to sit at each selected temperature (0, 5,10,30,50° C) for 15 and 30 minutes.
At the end of each time period the solutions were vortexed, and .1 ml aliquots were removed from the tubes and spread on LB agar plates.
The plates were incubated at 37° C for 24 hours.
The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

10. Conclusion
The evidence (p<.05) strongly supports the alternative hypothesis. The null hypothesis is rejected. All tested temperatures appeared to significantly reduce E.coli survivorship.
The second hypothesis that increased exposure time to a given temperature would adversely affect survivorship is also strongly supported.

11. Limitations
-20° C Freezing Temperature is hard to maintain throughout vortexing and pippetting
Number of trials

12. Extensions Wider range of temperatures Longer exposure times
More Trials

13. Sources United States Department of Agriculture (USDA)
Centers for Disease Control and Prevention (CDC)
Food and Drug Administration (FDA)
World Health Organization (WHO)