Date: Monday, November 19th, 2018

Title: High Titer Plaque Assay

Rationale: The purpose of today’s lab is to make a plaque assay using a dilution that will form plaques without lysing a plate completely.

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 10^-8 dilution was the only spot that had formed plaques that were countable, so that dilution was selected.
4. Top agar for a plaque assay was setup using the following formula:
   1. 4 mL LB Broth
   2. 5 mL 2x Top Agar
   3. 45 microliters 1M CaCl2
5. A culture tube with .5 mL arthrobacter was mixed with 10 microliters of 10^-8 lysate and left to infect for 15 minutes.
6. 4.5 mL of the top agar solution was added to a top agar control plate and left to sit for 15 minutes.
7. 4.5 mL of the top agar solution was added to the culture tube, pipetted, and added to a plate.
8. The plates were inverted and left to incubate over night.

Observations: The majority of the dilutions tested on the spot test were too high of a titer to make individual plaques, so the 10^-8 lysate was used in order to make a plate that a titer calculation can be made with. There was contamination on the top agar control from last experiment.

Results: This experiment yielded a plaque assay and a top agar control that can be evaluated in the future.

Next Steps: The next step is to calculate the titer and volume needed to web a plate using the new plaque assay.