Date: Monday, November 5th, 2018

Title: Plaque Picking and Serial Dilutions with Adopted Plate

Rationale: The purpose of today’s lab is pick a plaque from a lab partner’s plaque assay and set up serial dilutions in order to get plaques.

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 previous week were evaluated and found to yield inconclusive results.
3. 100 microliters of phage buffer were added to a microcentrifuge tube.
4. A pipette tip was touched into a plaque from an adopted plate and swirled in the microcentrifuge tube to add phage to solution. This was marked as the 10° serial dilution.
5. The 10° tube was shaken to mix phage with buffer.
6. 90 microliters of phage buffer were added to each of two other microcentrifuge tubes.
7. 10 microliters of the 10° dilution were added to one of the tubes, marked as the 10^-1 dilution. This tube was then shaken.
8. 10 microliters of the 10^-1 dilution were added to the last tube marked as the 10^-2 dilution. This tube was also shaken.
9. 10 microliters of each dilution were added to different culture tubes with .5 mL ATC 21022 each.
10. Agar was made using the following recipe:
    1. 8 mL LB broth
    2. 10 mL 2x Top Agar
    3. 90 microliters 1M CaCl2
11. 4.5 mL of the TA solution was added to each culture tube.
12. The culture tubes with bacteria, phage, and top agar were pipetted to mix the solution.
13. The contents of the culture tubes were added to their corresponding plates based on their dilution number.
14. The plates were left for 15 minutes to harden before being inverted and incubated.

Observations: The plates from last week did not fully harden and a layer of liquid solution was on top of the agar. Below are the results from the last experiment compared to the results from the partner the lysate was adopted from. It’s possible that the plates were positive for plaques and the plates were fully lysed over the weekend.

 

Results: This lab yielded three plaque assays and a top agar control that will hopefully allow plaques to form without fully lysing a plate.

Next Steps: The next step if the serial dilutions come back positive is to web a plate and further explore the phage. Alternatively, the next step could be to pick a second plaque from the positive plaque assay and perform serial dilutions on it if the plates yield negative results.