10/8/18 Plaque Assay and Plaque Picking

Objective:

The goal of this procedure is to do anything possible to try to find a plauqe and confirm that I do in fact have phage. This is necessary as my re-do of passage 2 yielded no plaques, and my spot test did not yield great results. The plaque assay I did most recently also yielded negative results, but after looking at older plates under the dissecting scope, I decided to try to pick to promising looking spots to see if I could make one last attempt to find phage.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for Plaque Assay:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

Materials for Plaque Picking:

• Agar plates with plaques of interest
• Micropipette tip
• Phage buffer
• Microcentrifuge tubes (incorrectly referred to as pipette caps in previous entries)

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table
2. 70% Ethanol was also applied

Then a phages were picked

1. 100 µL of phage buffer was allocated into two microcentrifuge tubes.
2. A pipette tip was used to collect the chosen plaques (see photo)
3. The tip was swirled in the phage buffer and vortexed

Then a plaque assay was performed

1. Four agar plates were labeled and set aside
2. 20 µL from each microcentrifuge tube was transferred into a culture tube containing .5 ml of Arthrobacter
3. The culture tube was set aside for 20 minutes.

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

1. The agar was prepared according to the following recipe (makes four plates):
2. 4.5 ml of this mixture was transferred to the plate labeled “TA control” and set aside
3. 4.5 ml of the contents were transferred to each culture tube and poured into corresponding agar plates
4. The agar was allowed to solidify for 10 minutes before being inverted and incubated

Results:

As can be seen from the images shown above, there were no plaque. The plaque assay that was performed on my P1 plaque was contaminated, and the plaque assay that was performed on my P2 plaque yielded negative results. These results suggest that there is no Arthrobacter phage present in my soil sample 2, or if there are, none were in the lysates I created using my second sample. I can assert this because I have done relatively exhausted testing and have continually gotten negative results, leading me to believe that they are accurate.

Analysis:

The results from this lab are somewhat confusing at first because I originally thought I had plaque and therefore phage. When I went back to old plates, there were spots that looked like plaque, and it my group members found similar spots. All of this led me to believe that it was highly unlikely that I did not have phage; however, after the negative results of this lab, I will conclude I do not have phage.

When considering why I originally thought I had plaques several explanations come to mind. The first is that in our inexperience my group members and I mistook air bubbles for a plaque. This could have caused us to pick them, and in our excitement, we could have overlooked signs that our plaques were not in fact not plaques but air bubbles. This explanation is possible and even probable, but it does not explain why other, more experienced lab workers, namely our TAs, also thought there was a possibility we had plaques. It is possible that everyone was wrong, but it is also possible that along the way we somehow managed to kill our phage. This could have occurred through mishandling or bad luck, and it will be impossible to confirm. Regardless of how this mistake happened, it necessitates testing soil 3, so that is what I will do.

Finally, these results will help us answer the overaching question our table seeks to answer; while we do not have enough data to difintevley say the Live Oak isn’t conducive for phage, we do now have one negative data point. Depending on the results of future testing, we may be able to make conclusions based off of this data.

Future:

My next steps will be to wash and enrich soil 3 so I will be able to run plaques assays using the enriched lysate. I will also collect soil metadata in an attempt to help me answer the big question. During my next lab period, I will perform the washing and enrichment, and I will start the metadata collection.