Date: Wednesday, September 19th, 2018

Title: Plaque Picking and Serial Dilutions

Rationale: The purpose of today’s lab is pick a plaque from the plaque assay and set up serial dilutions that can be plaque assayed further.

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 added to a microcentrifuge tube.
4. A pipette tip was touched into a plaque and swirled in the microcentrifuge tube to add phage to solution. This is 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. 20 mL LB broth
    2. 25 mL 2x Top Agar
    3. 225 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 briefly vortexed 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 control for the plaque assays was contaminated, much like the control for the spot tests before. It’s still unclear what is causing the contamination, but it’s likely that arthro is somehow getting into the TA control.

Results: The plaque assay from before yielded positive results, with upwards of 20 plaques. This was confirmed not to be contamination, and hopefully this yields actual phages that can be isolated and sequenced.

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