Rationale: Soil washing and enrichment on soil samples collected from Oak trees around Baylor University’s Campus. Without adding Arthrobacter, we had to create a direct lysate sample for the experiment from the soil. Our particular sample was found at 31*32’40 N 97*7’9” W near Waco Hall.

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• .5mL Arthrobacter
• 15mL conical vial
• Pipette
• 50mL tube

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments.

1. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution.
2. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate.
3. We then got an ethanol burner, and our aseptic zone was created.

Description of Procedure: 

• As we were utilizing the aseptic technique, my lab partner opened my 50mL vial (that had the soil), and he placed the top on the table away from the aseptic zone for about five seconds before picking it back up.
  • note possible contamination.
• I filled the tube with LB broth that Dr. Adair had provided us. As I filled it up to the required mL, Ramen closed my cap, and he sat the tube down over the carriages.
• I then brought my tube over to the weight, in which it read 51.64g.
• I then started to shake my tube for the next 15 or so minutes. After the class spinning, we had to find a partner that had a +/- .1mL mass of our broth.
• I didn’t find anyone, but someone around the range. So I had to add water to my solution. The new mass of my solution read 52.56g.
• After this, one of the TAs took my solution, and he gave it to Dr. Adair, where she put the many solutions in a centrifuge at 3000g for three minutes.
• After the three minutes, we went back to the lab with our solutions, where we were told to come back Friday 8/24/18 to finish our findings.
• Friday- I filtered lysate came out to 16ml Lucy pipetted 6ml into the small 15ml vial for the direct sample and added the Arthrobacter  to the remaining 10ml

Observations:

• Transferring the first set of LB broth was not done in an aseptic zone.
• The addition of water before the centrifuge spin was not done in an aseptic zone.

Results: 

The procedure form Wednesday was completed with the direct lysate sample having now been obtained.

Analysis:

The process was difficult, from making sure everything was done in an Aseptic zone (not everything but as in, the opening of our vials, to transferring the LB broth) to the precise measurements, it was a lot the first day. Time aspect played a big part since I was unable to actually finish this step, due to the fact I had a class after. My lab partner did this step and the extraction. Note, after the enriched solution was gathered from Friday, 8/24, my lab partner did not label my solution, but she did record the volumes of the solutions.

Interpretations/Conclusions/Next Steps: 

The procedure was now complete. Friday I filtered lysate came out to 16ml. I will need to be sure that I use the Aseptic technique, and not let my lab partners do my lab in a non-aseptic environment. Next step Monday 8/27/18, I will prep the dish/dishes that I will need in order to run a spot test, and hopefully, I will have bacteriophage.