Date: Monday, October 8th, 2018

Title: Third Attempt of Purification: Second Passage Soil Sample B

Rationale: The purpose of today’s lab is to go back to an older plaque assay with more promising plaques since new plates are yielding negative results.

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. Plaque assays from the first purification passage were taken back out and a third plaque was chosen to be picked.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into the chosen plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for two plates was made using the following recipe in a 50 mL conical vial:
   1. 3.8 mL LB broth (Note: less LB broth was used due to an increase in lysate used)
   2. 5 mL 2x Top Agar
   3. 45.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 100 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL microliters arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 5 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.4 mL of the top agar solution was added to a top agar control plate.
12. 4.4 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: The plaque assay from last lab yielded negative results. It’s interesting that the plaques have disappeared when they should be becoming more prevalent. It’s possible that the “plaques” picked so far have been bubbles or that the plaques have been picked incorrectly. Either way, more lysate was used this time so that more phage would be available to infect the arthro lawn.

Results: This experiment yielded a new plaque assay that should have a higher titer that can either be passaged again or flooded for amplification.

Next Step: If the plaque assay yields positive results, the next step is to passage the sample for the third time. If the plaque assay yields negative results, the next step is to work with Soil Sample C.