11/27/18 Flooding Webbed Plates

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 the webbed plates previously created during the last lab.

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
• Refrigerator
• Syringe Filter
• 15 ml conical vial
• 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 seven webbed plates were flooded.

1. 8 ml of phage buffer was pipetted onto each agar plate
2. The plates were placed in the refrigerator to sit for ~22 hours

Results:

The results of this lab will not be visible until Wednesday’s lab, but I can say that flooding the plate seemed to occur without incident.

Update: The plates flooded successfully and the lysate was filtered and further testing was run.

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 or spot test. This appeared to be a success which we will confirm with a titer test on Wednesday.

Future:

During our next lab, we will perform serial dilutions to determine the titer of the resulting lysate from this lab and because we are crunched for time we will also likely to TEM and start DNA extractions.