9/17/18 Plaque Assay of Soil #2 Enriched Lysate and Collection of Soil Sample #3

Objective:

The goal of these procedures is to use the previously created lysate to preform a plaque assay to test for phage presence in our previously collected soil (soil sample #2). In addition, during this lab period we also collected a third soil sample in case the plaque assay on soil sample two yielded a negative result. Our goal is to isolate a phage, and because our spot test yielded negative results, we did a plaque assay to confirm while also preparing to take next steps if soil sample 2 is negative for phage. We are also trying to avoid the contamination that occurred on our controls (see below). 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.

In addiction to our guiding questions, for this lab we were asked to consider the following:

1. Group 4 all had plaques on their plaque assays. Justin had the most and well defined plaque (but all 3 got plaque). They each did a spot test in addition to their plaque assays, but only Justin had a plaque on his spot test. What do you think is going on?
   • After considering this question we determined that Justin’s sample likely had the highest titer and therefore resulted in more clearing and better defined spots. It is also possible that the way he cared for his sample was more conducive to phage survival (ether during soil transport or enrichment).
2. Lathan checked a purified lysate by a plaque assay using 10 µL of a 10^-3 lysate. He got 14 plaques. How many µL of Lathan’s lysate should he add to web a plate (8 cm in diameter) if his average plaque diameter is 1 mm.
   • 4.6 µL of 10^0 lysate (work below)
     

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 Soil Collection:

• Sharpie
• Tape Measure
• Plastic Bags
• Digging tools
• 15 ml Tubes

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 plaque assay on the enriched lysate from soil sample #2 was preformed.

1. Four agar plates were labeled. An agar plate was labeled with initials, date, and description for each group member, and one agar plate was labeled with data and “TA control”
2. The remaining enriched lysate from last procedure (spot test), stored in a pipette cap, was gathered (like cap picture below)
3. 10 µL of the remaining enriched lysate 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 twice more (once for each group member).

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 four plates):
2. Under aseptic conditions, 8.o ml of LB broth was transfered into a 50 ml tube.
3. Under aseptic conditions, 90 µL of 1 M CaCl2 was transferred into the same 50 ml tube. *Note: the first time we attempted to pipette the calcium, we accidentally drew too much out so we had to discard the pipette tip full of calcium and try again*
4. Under aseptic conditions,  5.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 culture tube containing lysate and bacteria
2. The mixture was pipetted several times to mix it
3. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description
4. The plate was capped and set aside for 10 minutes to allow agar to solidify. This procedure was repeated twice more, once for each group member.
5. The remaining contents in the 50 ml tube was poured onto the TA control agar plate
6. The TA control plate was allowed to sit for about 10 minutes before being placed into the incubator
7. Once the labeled plaque assay had solidified, the plate was inverted and placed in the incubator
8. Plates were left to incubate until nest class

Soil collection was preformed as follows:

1. The species of tree that soil should be collected from was identified *Note: in this case we choose live oak as we did in soil sample 2*
2. The provided bags were labeled with name, date, and description
3. A tree of this species was loacted on campus
4. The GPS coordinates were noted
5. The tree’s trunk diameter was measured and recorder
6. The tree’s height was measured and recorded using shadow length of a known height and then measuring the shadow of the tree and using trig
7. The tree’s average canopy diameter was measured and recorded by taking measurement of the shortest and longest diameters and averaging them together (results in the table in results column)
8. A digging instrument was used to clear debris and dig down several centimeters into the soil
9. The provided plastic bags were filled with soil and a tree leaf (for confirmation of species identification)
10. The bags were brought back to lab
11. Refrigerate Tube and bag until next class

Results:

The results immediately gathered from these procedures are seen in the table above. The results of the plaque assay on soil #2 will be recorder here when available. It will not be possible to ascertain weather or not phage are present in soil sample #3 until the soil can be washed and then assays and spot tests can be preformed.

Update:

There are visible plaques on all three plates (see images below for my plates), but their is also contamination in the control. We are unable to determine how this contamination came to be, but we are operating under the assumption that it is not the cause of plaques forming. We will test this assumption in the future.

Analysis:

Based on the appearance of the tree and the measurements we took, we would assert that the tree is most likely healthy. Should we find phage in this soil and in the soil of trees nearby, this information could become important in determining if tree health is a factor for phage presence. It is also important to note that this was a live oak tree and it could become clear that live oak trees do not readily have phage in their soil after further sailing and analysis of soil sample #2’s plaque assay. The opposite could also be true and we will be more equipped to analyse our data once we test for phages.

Update:

Based on the results of visible plaques we can assert that there is likely phage present in the soil from sample #2. However, because the control plate was contaminated we cannot be certain. The results of further testing of plaques should reveal if there are in fact phages in soil sample #2, or if my group simply managed to contaminate everything

.

Future:

If necessary I will wash the that soil was collected (soil #3) and create lysates in order to run spot tests and plaque assays.

Based on the update, this will not be immediately necessary. On Wednesday I will pick a plaque and preform serial dilutions. Based on the results of that I will likely try to web a plate or be forced to begin testing soil sample #3.