Date: Wednesday, October 3st, 2018

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

Rationale: The purpose of today’s lab is passage the same lysate from the previous lab using a larger sample of phage in order to raise the titer of the 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.
2. Plaque assays from the third purification passage were taken back out and evaluated. Since there was only one plaque on the plate, the plate was stored for future experimentation.
3. Agar for 3 plates was made using the following recipe in a 50 mL conical vial:
   1. 8.0 mL LB broth
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
4. The LB broth and 1M CaCl2 were added to the 50 mL conical.
5. 50 microliters of the phage and phage buffer solution from the same microcentrifuge tube used for the third passage were transferred to a culture tube containing .5 mL arthrobacter.
6. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
7. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
8. 4.5 mL of the top agar solution was added to a top agar control plate.
9. 4.5 mL was added to the culture tube containing the arthro and phage sample.
10. This solution was added to an agar plate and moved around to cover the plate with solution.
11. The plates were left sitting to allow the agar to harden.
12. The plates were left to incubate without being inverted since the agar didn’t harden.

Observations: The plaque assay from the third passage only had one plaque on it. Since the sample had such a low titer and was expected to have more plaques on it, 50 microliters of lysate was used instead of the usual 10 in order to amplify results. Also, the top agar was left to sit for over 20 minutes and still did not harden. This could have been because of the temperature of the plates.

Results: This experiment yielded a new plaque assay that should have a higher titer than before.

Next Step: The next step is to evaluate the plaque assay from this third passage and begin amplification of the phage. If the plaque assay doesn’t yield positive results, the next step is to pick a different plaque from an earlier plate and begin the process of purification again.