Rationale:

Today’s lab goal was to set up the lysate for a plaque assay and spot test as well as begin the process of gathering soil metadata.

Materials Used:

• 15-mL Conical Vial
• Weigh Plate for soil
• Serological Pipette
• DI Water
• Soil Dispersion Liquid
• LB Broth
• 0.5 mL of Artho
• pH Paper

Procedure:

• The experiment began with gathering the soil samples previously collected the week prior .
• After gathering soil and a 15-mL conical vial, 2-mL of soil was added to the bottom of the conical vial along with 10 mL of LB broth.
• After the LB Broth was added, one of my lab partners put my sample through the vortex for 15 minutes, while this was occurring I added 10-mL of soil to a 50-mL tube along with 20-mL of DI water and soil dispersion liquid to gather soil composition metadata.
• I shook the soil for 30 seconds and let it sit for 30 seconds, I left the soil to sit for 48 hours for the particles to separate.
• Once the soil composition experiment was finished, my sample was finished with its vortex my vial lid broke and I had to transfer my liquid to another conical vial before sending it off to centrifuge at 10,000 g for 5 minutes.
• While my soil was in the centrifuge, I started the experiment to calculate my percent water of the soil sample.
• To begin, I weighed my empty petri dish to get a weight of 6.90 g.
• I added my wet soil to the empty Petri dish and got a mass of 11.60 g.
• Then, using subtraction, I found the mass of the wet soil which is 4.7 g.
• After calculating the mass of the wet soil, I left the petri dish out for 48 hours as well to let the water evaporate from the soil.
• After my vial was retrieved from the centrifuge, my lysate was still extremely cloudy. To try and make up for this, I split my lysate and put it in the centrifuge again for another 5 minutes at 10,000 g.
• After removing the vial from the centrifuge, my lysate was significantly clearer. After examining it, I filtered the enrichment through a syringe filter.
• After filtration, I split the lysate in 2 vials. One was my direct isolation which had 2-mL and the other was my enriched with 10-mL with 0.5-mL of Arthobacter.
• After splitting the two, I put the vials up for 48 hours to use for the next lab.
• At the end of the lab, I added a small amount of soil to a vial, and filled it up with D.I water and shook vigorously for 10 seconds.
• After shaking, I waited for 2 minutes for the soil to set and inserted a piece of pH paper no longer than an inch into the vial for 45 seconds. After 45 seconds I removed the paper to examine the pH.

Data/Observations

• The lysate after the second centrifuge was still slightly cloudy despite the second centrifuge.
• The soil separated very well for the metadata, there appears to be a lot of sand with very little to no silt or clay present in the soil.
• pH paper came from the soil a blue, the closest color to match was a navy blue

Conclusions:

• The soil pH was approximately 7.5
• Soil appears to have a large amount of sand and little silt and clay present.
• Lysate appeared slightly dark, not entirely clear throughout, despite the second centrifuge. Must be due to the switching the tubes after the lid cracking. Hopefully that does not affect the phage present in the plaque assay and spot test.

Next Steps:

• The next steps will be to test for the presence of phage in the lysate with a spot test and a plaque assay. Also I will need to examine the soil composition and calculate the percent of water present in the soil.