9/19/18 Plaque Picking and Serial Dilution

Objective:

The goal of this procedure is to use one of the plaques discovered after Monday’s procedure to preform plaque assays at different dilutions. This will help confirm the presence of phage and will contribute to isolating them. Our goal is to isolate a phage. We are focusing on avoiding the contamination that occurred on our controls of both our spot test and plaque assay. We are also seeking to address the following questions every 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 a 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 weather or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for 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 Serial Dilution:

• Microcentrifuge tubes
• Phage Buffer
• A lysate that requires diluting (10^0)

Materials for Phage 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 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 a phage was picked *Note: Each group member picked one plaque from their respective plates for a total of 3 picked plaques*

1. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description “10^0”
2. A pipette tip was used to stab the center of the chosen plaque on each plate (the chosen plaque is indicated my the red arrow on the image below) *Note: My hands shake and it is possible I contaminated my pipette tip with the surrounding agar when I tried to stab my plaque* 
3. The (hopefully) phage-infected tip was swirled in the phage buffer and then the solution was vortexed and set aside.

Then the serial dilutions were preformed.

1. Three levels of dilution were created: 10^0, 10^-1, 10^-2
2. The microcentrifuge tube labeled “10^0” created in the procedure above served as the first dilution
3. Two more microcentrifuge tubes were gathered and labeled with initials, date, and the descriptions of “10^-1” or “10^-2” respectively
4. Each of these tubes was then filled with 90 µL of phage buffer
5. 10 µL of solution was taken the 10^0 tube and transferred to the tube labeled 10^-1
6. It was vortexed to mix
7. 10 µL of solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
8. It was vortexed to mix and set aside

Then the multitude of plaque assays were preformed.

1. Ten agar plates were labeled. An agar plate was labeled with initials, date, and description of dilution (10^0, 10^-1, or 10^-2) for each group member, and one agar plate was labeled with data and “TA control”
2. The dilutions that were created in the last procedure were gathered
3. 10 µL of each dilution was aseptically transferred into a culture tube containing .5 ml of Arthrobacter using a Serological pipette
4. The culture tube was capped and set aside for 15 minutes. This process was repeated eight more times

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 ten plates):
2. Under aseptic conditions, 20.o ml of LB broth was transferred into a 50 ml tube.
3. Under aseptic conditions, 225.0 µL of 1 M CaCl2 was transferred into the same 50 ml tube.
4. Under aseptic conditions,  25.o ml of 2X TA was transferred into the same 50 ml tube
5. The mixture was pipetted several times to mix it

When agar preparations were finished the bacteria and lysate had been allowed to sit for 15 minutes

1. 4.5 ml of the contents in the 50 ml tube was transferred into the TA Control plate, capped, and set aside
2. 4.5 ml of the contents in the 50 ml tube was transferred into the culture tube containing lysate and bacteria
3. The mixture was pipetted or occasionally vortexed several times to mix it
4. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description of the appropriate dilution
5. The plate was capped and set aside for 10 minutes to allow agar to solidify. This procedure was repeated eight more times, once for each group member and each dilution.
6. Once the labeled plaque assay had solidified, the plate was inverted and placed in the incubator *Note: One of my plates refused to solidify so I put it in the incubator not inverted*
7. Plates were left to incubate until nest class

Results:

There are no immediate results from this procedure. The results will be made clear on Monday and recorded appropriately. It will be important to note whether or not contamination occurred in our control plate though, because if it did this can still cast potential doubt about the existence of phage in our soil sample #2. All results will be recorded after Monday’s lab.

Analysis:

Based on the appearance of what we assume to be plaques on our plaque assay, it seems reasonable to assert that there are phage in our soil sample #2. This will be ether confirmed or denied by the results of our dilutions. In addition, we were extremely careful in conducting our procedures during this lab in order to avoid contamination, so if it appears, we will need to take more serious action. I will be able to assert a lot more once I see the results of my dilution assays.

Future:

Depending on the results of the dilution assays, I will ether move forward to try to web a plate and further explore my phage, or I will have to start over. If necessary I will wash the that soil that was collected (soil sample #3) and create lysates in order to run spot tests and plaque assays.