Date: Wednesday, November 7th, 2018

Title: Titer Calculations and Plaque Assay

Rationale: The purpose of today’s lab is to calculate the titer of plates with plaque and attempt to web a plate.

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 and plaque assays were evaluated.
2. The plates yielded negative results so the experiment was combined with a partner’s who had positive results.
3. Plaques on the 10° and 10^-1 plates were counted.
   1. The 10° plate had 465 plaques present.
   2. The 10^-1 plate had 21 plaques present.
4. The following math was performed in order to determine the titer of each sample:
   1. For the 10° plate: (465 pfu ÷ 30 microliters) * (1000 microliters ÷ 1 mL) * (1, the dilution factor) = 15,500 pfu/mL.
   2. For the 10^-1 plate: (21 pfu ÷ 30 microliters) * (1000 microliters ÷ 1 mL) * (10, the dilution factor) = 7,000 pfu/mL.
5. A microscope and ruler were used in order to determine the diameter of the plates and the average diameter of plaques on each plate.
   1. The plate was determined to have a diameter of 8.5 cm.
   2. The plaques on the 10° plate were determined to have an average diameter of .1 cm.
   3. The plaques on the 10^-1 plate were determined to have an average diameter of .1 cm.
6. Using A=πr^2, the area of the plaques and the area of the plates were determined:
   1. Plates: A=πr^2=π(4.25 cm)^2=56.745 cm^2.
   2. Plaques:  A=πr^2=π(.05 cm)^2=.00785 cm^2.
7. The number of plaques needed to web a plate was determined to be 7.225×10^3 by dividing the area of the plate by the area of the plaques.
8. The amount of lysate needed to web a plate was determined for each dilution using the following math:
   1. 10° plate: (7.225×10^3 pfu) * (1 mL ÷ 15,500 pfu) = 482 microliters 10° lysate.
   2. 10^-1 plate: (7.225×10^3 pfu) * (1 mL ÷ 7,000 pfu) = 1.032 mL 10^-1 lysate.
9. A plaque assay was made for the 10^-1 dilution using the following formula:
   1. 4 mL LB broth
   2. 5 mL 2x Top Agar
   3. 45 microliters 1M CaCl2
10. 60 microliters of 10^-1 lysate was added to .5 mL arthrobacter and left for 15 minutes to infect in a culture tube.
11. 4.5 mL top agar solution was added to a control plate and 4.5 mL was added to the culture tube, pipetted, and added to a plate.
12. The plates were left for 15 minutes to harden before being incubated.

Observations: Below are the results of the last experiment. The contamination is unlike contamination seen before. It has spots throughout the plate as well as tendril-like patterns throughout the plate. Most of the LB broth and top agar in the autoclave are contaminated and it’s unclear why. It’s possible the autoclave wasn’t turned on or simply didn’t kill everything. The middle plate is the 10^-1 plate borrowed from a lab partner.

Results: This experiment yielded a plaque assay that will hopefully be close to webbed so that it can be flooded.

Next Step: The next step is to flood the plate in order to get the phage from the plate into phage buffer.