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

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

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

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

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?

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

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)

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.

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

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

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

P-Value: 4.5E-13

P-Value: 3.9E-10

P-Value: 2.5E-14

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

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

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

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

Anova

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 2013. Web. 12 December 2016 Cherche, C., and A.Z. Gu. Effect of Bacterial Growth stage resistance to Temperature. Diss. Northwestern University, 2011. Web 15 December 2016

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