11/12/18 Titer Test Serial Dilutions and Plaque Assays

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 previously created lysate through serial dilutions.

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 Serial Dilution:

• Phage Buffer
• Microcentrifuge Tubes
• Vortex Machine
• Pipette

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
   

Then the serial dilutions were performed.

1. Five levels of dilution were created: 10^-1, 10^-2, 10^-3, 10^-4, 10^-5
2. Five microcentrifuge tubes were filled with 90 µL of phage buffer
3. 10 µL of the previously created lysate (10^0) were transferred to the 10^-1
4. The tube was vortexed to mix
5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution

Then plaque assays on the new lysates were performed.

1. Six agar plates were labeled
2. 10 µL of each dilution were transferred into a culture tube containing .5 ml of Arthrobacter (1 dilution per tube)
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 six 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:

As can be seen in the image above, the serial dilutions produced the desired results. Our 10^-1 and 10^-2 plates were almost lysed and our 10^-3 plate was webbed. We calculated the following titer:

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.

Future:

During our next lab period we will flood our 10^-1 and 10^-2 plates to try to get a high titer lysate, in addition, we will likley also create more webbed plates that we can flood so that we will have a large quantity of high titer lysate.