Date: Wednesday, September 26th, 2018

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

Rationale: The purpose of today’s lab is to repeat the procedure from the last lab day and passage the phage for a second time.

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

Situation: It was determined by experimentation that what had been used during the last lab day was not arthrobacter ATCC 21022. This was determined by trying to infect a bacterial lawn with a high titer bacteriophage. The results were negative, meaning the bacteria was not arthro. Because of this, every procedure from Monday involving arthrobacter has to be repeated. Below is an image of my second passage plaque assay with no plaques (the small dot was determined to be the location of a bubble and therefore not a plaque).

Procedure:

1. An aseptic zone was set up.
2. Plaque assays from the first purification passage were taken back out and a second 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 four plates was made using the following recipe in a 50 mL conical vial:
   1. 8.4 mL LB broth (Note: more LB broth was used due to a decrease in available arthrobacter)
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing 400 microliters arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 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 day wasn’t usable since the bacteria in the top agar wasn’t arthrobacter. By keeping our old plaque assays, there were more plaques available to pick without having to totally repeat the whole purification process from the first passage.

Results: This experiment yielded a new plaque assay that can be evaluated and either passaged again or begin to web a plate.

Next Step: The next step is to either further passage the phage, try to web a plate and prepare for amplification, or begin the purification process again with either another plaque on my first plaque assay or with new soil.

Date: Monday, September 24th, 2018

Title: Purification: Second Passage Soil Sample B

Rationale: The purpose of today’s lab is to further passage the bacteriophage in order to purify the phage sample.

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 were evaluated and plaques were marked on the plate.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into a 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 four plates was made using the following recipe in a 50 mL conical vial:
   1. 8 mL LB broth
   2. 10 mL 2x Top Agar
   3. 90.0 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 10 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 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: This plate yielded a significantly smaller number of plaques. This is probably due the purification leaving out other phage species as the plaque assays and dilutions isolate the phage more and more.

Results: This lab yielded a plaque assay that can be further evaluated in order to perform a third passage on the phage sample.

Next Steps: The next step is to evaluate the plaque assay during the next lab. If the results are positive, the next step is to passage the phage for a third, possibly final, time. If the results are negative, then the next step is to either pick a different plaque from the first passage or to start with new soil (soil sample C).