1. The Freezing/Thawing effects of E.coli
Nolen Paul
9th Grade
Pittsburgh Central Catholic high school

2. Temperature
Temperature effects can be influenced by the chemical environment of an organism
Some microorganisms tolerate a low temperature stressed environment
Organisms vary in the resistance with temperature change

3. Salt Sodium Chloride (NaCl) Salt is a natural mineral
Plays critical point in nerve and muscle regulation
Helps establish tonicity interstitial blood fluids

4. Escherichia coli (E.coli) model Microbe
Highly common bacteria
Consistently used in scientific research
A rod-shaped microbial flora, ~2 micrometers in diameter
Select strains derived from E.coli can be deadly to humans and animals
Optimal reproduction rate: 20 minutes
Gram negative

5. Main Questions
How does a low temperature stress effect a common contaminant?
Is E.coli’s response to low temperature stress altered by environmental salt concentration?

6. Hypothesis Null Alternative
The low temperature stress will have no significant effect on the survivorship of E.coli
The low temperature stress will significantly effect the survivorship of the E.coli
A salty environment will improve the survivorship of low temperature stressed E.coli

7. Materials x9 sterile 15 mL conical tubes Micro pipettes + sterile tips
Sterile distilled water
Vortex
0.2 Micron syringe filters
LB agar plates (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
10% NaCl Stock
Refrigerator + Freezer
Sterile spreader bars
E.coli (DH5-alpha)
Incubator (37°C)

8. Procedure
A 10% sodium chloride stock was created by passing through a 0.02 micron syringe filter
9 sterile 15mL conical tubes were used for following concentrations
Three tubes with 9.9mL sterile fluid and .1mL of E.coli
Three tubes 9.8mL of sterile water, .1mL of stock, and .1mL of E.coli
Three tubes with 8.9mL of sterile water, 1mL of stock, and .1mL of E.coli
1 of each concentration (1x 0%, 1x .1%, 1x 1%) were placed in a rack located in a room- temperature area
1 of each concentration were placed in a rack in the refrigerator
The last three tubes were replaced in a freezer
All 9 tubes were left in the designated temperature for approximately 30 minutes
8 helpers were gathered and was assigned a tube with a specific concentration and temperature and were given 6 plates each(total of 54 plates including my own)
The solutions were spread the solution evenly over the plates
After the low temperature stressed environment, 100 micro liters of solution from each tube and plated on agar the plates were incubated overnight at 37°C and the resulting colonies were grown.

9. Concentration Chart Concentration 0% 0.1% 1% E.coli 0.1mL
Sodium Chloride Stock (10%)
0mL
1mL
Sterile Fluid
9.9mL
9.8mL
8.9mL
Total Volume
10mL

10. Procedure (Agar Infusion)
Plates were infused with the salt solutions According to the chart below
Plates were allowed to incubate for 1 hour, to promote infusion
100 μL sample from the control tube to the plates then spread.
The resulting colonies were counted visually, each colony being assumed to have arisen from one cell.
High
Low
None
Sterile dilution fluid
0μL
180μL
200μL
Variable
20μL

11. Data
A Two-Factor Anova revealed a significant interaction between salt and E.coli.

12. P-Value: 4.5E-13

13. P-Value: 3.9E-10

14. P-Value: 2.5E-14

15. Dunnett’s Test 0% .1% 1% 20°C (Room temp) No significant change
(Refrigerator)
-10°C
(Freezer)

16. Conclusion The P-value is greater than .05 so the null was rejected.
The Dunnett’s test revealed that only salt had a significant effect
As the concentrations were increased the amount of colonies increased greatly.
The temperature didn’t any effect on the survivorship of E.coli

17. Possible Limitations
Survivorship was the focus, growth rate was not examined
Only one species  
Only 3 temperatures tested
There were only 3 different concentrations of variable tested
The tubes were only in a designated temperature for 30 minutes

18. Extensions
Steps will be taken in order to insure the synchronicity for spread plating
Test the growth rate as well as survivorship
More of a variety of species will be tested
A higher number of concentrations will be tested
A wider ranger temperature could be tested

19. Anova

20. Resources
"Escherichia Coli."MicrobeWIki.Kenyon.edu, n.d. Web. 20 Dec. 2016
"E. Coli (Eschericia coli)." CDC.gov. US Center for Disease Control and Prevention, 29 December Web. 12 December 2016
Cherche, C., and A.Z. Gu. Effect of Bacterial Growth stage resistance to Temperature. Diss. Northwestern University, Web 15 December 2016

21. Statistical Tests ANOVA Statistical Dunnett’s Test
Analysis of Variance - Multiple mean comparison, allows you to compare different groups of statistical data
P-value is smaller than the Alpha Value (0.05)= variation
Dunnett’s Test
This is a follow up statistical test, which examines the individual variation between each of the concentrations compared to the control