10/10/18 Soil Washing, Enrichment, and Metadata Collection of Soil Sample #3

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 will also be collecting metadata in order to help adress the overarching questions we are trying to answer as a class and as a table group.

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
• Scale
• Weigh Boat
• Test tube stand

Materials for % Water Analysis:

• Scale
• Weigh Boat

Materials for % Sand, Silt, Clay Analysis:

• Falcon Tube
• Dispersion Fluid
• Deionized (DI) water

Materials for pH Test:

• pH vial
• DI water
• pH Paper
• pH comparison color scale

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 ml of LB broth was added
3. The vial was shaken and vortexed intermittently for 15 minutes
4. The vial was then centrifuged at 10,000 g 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

% Water analysis was performed according to the following procedure:

1. A weigh boat was weighed and the mass was recorded
2. Then a small amount of dirt was poured into the weigh boat and the combined weight was recorded
3. The weigh boat was labeled with initials and date and allowed to sit in the fume hood until next lab

% Sand, silt, clay analysis was performed according to the following procedure:

1. 10 ml of soil was transferred into a falcon tube
2. DI water was added until the total contents of the tube was 15 ml (the photo is an example from another lab)
3. 3 drops of dispersion fluid were added to the tube
4. The tube was covered with a hand and shaken for 30 seconds
5. The liquid was poured into a second falcon tube
6. Both tubes were then placed under the fume hood until next lab

The pH of the soil was collected according to the following procedure:

1. A small amount of soil was scooped into a pH vial
2. The rest of the vial was filled with DI water
3. The vial was shaken for 10 seconds
4. Then the contents of the vial was allowed to settle for 2 minutes
5. A strip of pH paper was put in the vial for 45 seconds and then compared to the pH color scale

(The image shows the resulting color change of the DI water after the pH slip was removed)

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 pH of the tested soil sample was around 6.0, leading us to conclude that our soil was slightly acidic.

In addition, the soil washing seems to have gone well and has resulted in both enriched and direct lysate for future testing.

Update:

After checking on the results during open lab, I collected data for the percent water analysis (see chart), but I decided to allow the %sand, silt, clay falcon tubes sit until Monday to settle more.

Analysis:

These procedures were meant to allow us to learn more about our new soil samples and get us prepared for future testing. Based on my current results 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. 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.

Future:

This entry has been updated to reflect the results of the metadata testing; however, when the procedures stated above were completed the initial future procedures were to simply check on the results of the experiments after the appropriate amount of time had elapsed. Now that this is completed a plaque assay will be performed using the enriched lysate created. Depending on the results of that plaque assay, I may chose to pick a plauqe and begin enrichment, or I may need to try yet another soil.