Date: Monday, November 26th, 2018

Title: Titer Calculation and Plaque Assay

Rationale: The purpose of today’s lab is to calculate the titer of the lysate using a previous plaque assay and then to attempt to web a plate using the titer calculations.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plates from last lab were evaluated.
3. The plaque assay from last lab had 133 plaques present, equivalent to 133 plaque forming units (pfu).
4. Diameters of the plate and the plaques were measured using a ruler and a microscope.
   1. Plate Diameter: 85 mm
   2. Average Plaque Diameter: 1.5 mm
5. The area of the plate and the plaques were calculated using A=πr^2
   1. Plate area: A=πr^2 =π(42.5)^2 =5674.5 mm^2
   2. Plaque area: A=πr^2 =π(.75)^2 =1.767 mm^2
6. The area of the plate was divided by the area of the plaques in order to calculate how many plaques would be necessary to web a plate.
   1. (5674.5 mm^2)/(1.767 mm^2) =3211 pfu
7. The titer of the lysate was determined using the formula Titer=(pfu x dilution factor x 1000 μL)/(μL lysate used x 1 mL)
   1. Titer =(133 pfu x 10^8 x 1000 μL)/(10 μL x 1 mL) =1.33 x 10^12 pfu/mL
8. The volume needed to web a plate was determined using the following math:
   1. (3211 pfu x 1 mL x 1000 μL)/(1.33 x 10^12 pfu x 1 mL) =2 x 10^-6 μL lysate needed to web a plate
9. Since the lysate was diluted by factors of 10, 2 x 10^-6 μL of 10^0 lysate equates to 20 μL of 10^-7 lysate.
10. A plaque assay and top agar control were set up using the following formula:
    1. 4 mL LB broth
    2. 45 μL 1M CaCl2
    3. 5 mL 2x Top Agar
11. 20 μL of the 10^-7 lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
12. 4.5 mL of top agar solution was added to a control plate.
13. 4.5 mL of top agar solution was added to the culture tube solution and pipetted to mix before being transferred to a plate.
14. The plates were left for 15 minutes to solidify, inverted, and placed in an incubator for the next 48 hours.

Observations: The top agar control from last experiment was free of contamination. The solution in the plate made during this lab session was touched by accident and therefore might have contamination. The arthrobacter may out-compete the contaminants. This will be evaluated next lab. The titer of the lysate is very high, at 1.33 x 10^12. A highly diluted sample will need to be used to web plates.

Results: This lab yielded a plaque assay that will be webbed if successful and a top agar control to be evaluated next lab.

Next Step: The next step is to evaluate the plates from this experiment and flood a plate if it is webbed. If the plate is not webbed, a titer calculation might need to be reevaluated and a new plaque assay will be made with a different volume of lysate in order to web one.