11/5/18 Flooding a Plate and Titer Test Plaque Assay

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of flooding a phage heavy (but not webbed) plate in order to create more lysate because while we failed to web a plate, Lucy P. is almost out of lysate. After flooding the plate we decided to run a plaque assay in order to asses the titer of the new lysate so that can be used to attempt to web a plate next lab. While this procedure was being done, another group was also trying to web a plate with the existing 10^0 lysate by using 30 µL lysate.

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 Flooding a Plate:

• Phage buffer
• Incubator/Shaker
• Syringe Filter
• 15 ml conical vial

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

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

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

Them the previously created 14.l µL agar plate was flooded.

1. 5 ml of phage buffer was pipetted onto the agar plate.
2. The plate was left in the incubator, set to room temperature, to slowly shake for an hour.
3. At an hour, a syringe filter was used to filter the resulting lysate
4. ~3 ml of the lysate was transferred it into a vial for future testing.

Then a plaque assay on the new lysate was performed.

1. Two agar plates were labeled
2. 10 µL of the new lysate were transferred into a culture tube containing .5 ml of Arthrobacter
3. The culture tubes were set aside for 15 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 two plates):
2. 4.5 ml of the agar was transferred to the plate labeled “TA control”
3. The plate was swirled and set aside
4. 4.5 ml of the agar was transferred into the culture tube
5. The resulting mixture was poured into the corresponding plates
6. The was set aside for 10 minutes to allow agar to solidify.
7. Plates were left to incubate until nest class

Results:

The results of this lab will not be visible until Wednesday’s lab, but I can say that all appeared to go well in both the flooding of the plate and the creation of a titer test plaque assay.

Update:

Our titer test webbed the plate, our control was very contaminated, and the 30 µL plaque assay on the old lysate did not web the plate. (pictured below in order mentioned)

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay. However, because Lucy P. and I were unable to create a webbed plate, and we were running out of lysate we needed to make more. The solution to this problem was addressed in two ways, Lucy P. and I flooded the 14.1 µL plate because it had more phage on it so that we could create more lysate. While we were doing this, two others used the last of the remaining old lysate to try to web a plate. This gives us two possible ways to get a high titer lysate and ensures that we won’t run out of lysate for testing. Once we succeed in webbing a plate, we will flood the plate in order to create a higher titer lysate.

Future:

During our next lab, we will likely calculate the titer of our new lysate based on the results of the plaque assay. We will then try to web a plate again so that we can flood and create a higher titer lysate.

Update:

Because our titer test plate was webbed we will flood that webbed plate, and then will perform serial dilutions in Friday’s lab.