11/14/18 Titer Calculations, Flooding Plates, Webbing 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 calculating the titer of the lysate created last lab in addition to flooding plates from the serial test titer dilutions, and making more webbed plates to flood later.

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 a 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
   

The titer from the previous serial dilutions was calculated (also included under the updated results section of the 11/12 lab)

The 10^-1 and 10^-2 plates were flooded

• 8 ml of phage buffer was pipetted onto the agar plate.
• The plate was left on the shaker at room temperature, to slowly shake for an hour.
• At an hour, a syringe filter was used to filter the resulting lysate into a conical vial to create ~11 ml of lysate for future testing

Then plaque assays on the new lysates were performed.

1. Five agar plates were labeled
2. 10 µL of the 10^-2 dilution were transferred into each of the four 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 5 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 each 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:

Update: While it wasn’t possible to see the results of the webbed plates when the lab period was finished on 11/14, it is clear that on 11/15 the plates were close to webbed, suggesting that the procedures detailed above were a sucess.

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 testing gave us a medium titer lysate that we will continue to amplify, we hope that by flooding all of our plates we will create enough high titer lysate to move on to TEM.

Future:

During our next lab period, we will flood our four webbed plates. We will also need to perform a titer test on the two new lysates we have created.