10/31/18 Plaque Assay to Web a Plate

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 how a plate was webbed in order to be flooded during the next lab. It is important to note that two slightly different agar recipes were used in order to ensure a webbed plate, as will be explained below, it was calculated that only 14 µL of lysate was needed to web a plate, but we made plates with 15 µL and 14.1 µL.

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 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 amount of 10^0 lysate required to web a plate was calculated.

*Note: We initially miscalculated to think we needed 14.01 µl of lysate, and because our pipettes weren’t accurate to the .01, we decided to do one plaque assay with 14.1 µl and one with 15 µl just to ensure we got a webbed plate*

Then a plaque assay on the 10^0 purified lysate was performed.

1. Three agar plates including a control were
2. 14.1 µL of the 10^0 lysate was transferred into a culture tube containing .5 ml of Arthrobacter
3. 15 µL of the 10^0 lysate was transferred into a culture tube containing .5 ml of Arthrobacter
4. 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 three 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 mixtures were poured into the corresponding plates
6. The was set aside for 10 minutes to allow agar to solidify. *Note: the 15 µL agar plate had a small hair in it that was removed with a pipette tip – this contamination could impact results*
7. Plates were left to incubate until nest class

Results:

The results of this lab will not be visible until Monday’s lab, but I can say that all appeared to go well with the exception of the mystery hair in the 15 µL agar plate, if this causes something odd to occur, that will be recorded here on Monday.

Analysis:

The idea behind this procedure is to enable us to create larger quantities of high titer lysate. This can be done after a plaque has been purified by creatting 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. This procedure was the first step in that process.

Future:

My next steps will depend on the results of the plaque assay. If all goes according to plan and this procedure creates two lysed plates, then my next steps will be to work with Lucy P. to flood these plates and create more high titer lysate. If this does not succeed in creating webbed plates, then we will try to web again.