10/15/18 Soil Washing and Enrichment of Soil Sample #3 Attempt 2

Objective:

The goal of this procedure is to wash and enrich the soil for later testing, with the ultimate goal being to find a phage. I am redoing this procedure because there was bacterial contamination in my original enriched lysate. I am also finishing my metadata collection for %sand, silt, clay because I was unable to finish it previously and the bacterial contamination would not have effected these results.

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 Soil Washing:

• Syringe filter
• .5 ml Arthrobacter
• 50 ml conical vial
• 15 ml conical vial
• LB Broth
• refrigerator
• Incubator
• Centrifuge
• Pipette
• Test tube stand

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table
2. 70% Ethanol was also applied

The soil was washed and enriched according to the following procedure:

1. ~2 ml of soil was added into a 15 ml conical vial
2. 11.5 ml of LB broth was added
3. The vial was vortexed for 15 minutes
4. The vial was then centrifuged for 10 minutes
5. ~ 2ml were pipetted into a fresh vial for a direct lysate and placed in the fridge
6. a .22 micron syringe filter was used to filter ~8 ml of lysate into a 50 ml conical
7. .5 ml of Arthrobacter was added to the 50 ml conical and put in the incubator

Results:

The majority of these procedures will not have results until the next lab and this entry will be updated when results are available. That said, the % sand, silt, clay results are detailed in the table below.

In addition, the soil washing seems to have gone well with addition caution taken for it to be completed aseptically and has resulted in both enriched and direct lysate for future testing.

Update:

As can be seen from the comparison pictures, this soil enrichment was a success because it was not contaminated.

Analysis:

The procedures I’ve conducted over the last few labs were meant to allow me to learn more about our new soil samples and get us prepared for future testing. Based on my results as a whole I can assert that my soil sample is very dry. I can also assert that my soil sample was slightly acidic based on the results of my pH test. And, based on the conclusion of my sand, silt, clay analysis I can also say my soil is very sandy, to the point that it would be classified as sand according to the table below. This will help my group address our question further because having a collection of soil metadata will help us determine whether or not other factors besides tree species determine phage presence.

(https://samanthaapes.weebly.com/apes-in-a-box-soil-pyramid.html)

Future:

This entry has been updated to reflect the results of the metadata testing and soil enrichment. During my next lab testing period, I will begin PCR to test for phage DNA.