Rationale:

Separate the phages and bacteria from an environmental sample with the purpose of obtaining phages for experimentation

Procedure:

• To prevent contamination, wipe the tables with CiDecon and ethanol (70%) and set up an aseptic zone by using an ethanol lamp
• Near the lamp, pour the LB Broth up to the 35 mL mark in the 50 mL conical vial containing the soil sample and then close the sample as quickly as possible
• Shake the sample vigorously for 15 minutes.
• After shaking for 15 minutes or more, place the sample in a centrifuge for five minutes
• Using the tube top vacuum filter under the vacuum hood, filter the supernatant. Use a bulb pipette to transfer the supernatant to the filter.
• Filter supernatant until reaching 20 mL of lysate.
• After reaching 20 mL, carefully unscrew the filter from the vial filled with lysate and quickly screw on the lid to prevent contamination
• Separate the lysate evenly; half of the sample will stay in the vial while the other half will go into a new one (15 mL conical vial).
• The lysate in the new vial, labeled “Direct Isolation”, will go into the fridge to be filtered later.
• Combine the other half of the lysate with 0.5 mL of Arthrobacter (the host) near the lamp and label it “Enriched 1.”
• Close the lid and place the lysate in a test tube rack to be placed in the refrigerator.
• Place all equipment back to their proper place and wipe the table with CiDecon and ethanol (70%).

Results and Analysis:

• Rather than placing the sample in the bag, I placed it in the vial and sealed it shut. Over the course of two days, the vial remained in my room with cold temperatures (68 F). During this time, soil particles and perspiration lined the walls.
• Due to the large spaces between the clusters of soil, I tapped the vial against the table to fill those spaces. In doing so, my soil sample went from 17 mL to 10 mL; 2 mL less than the minimum requirement. Due to this problem, I did not produce as much lysate as I should have to create my Direct Isolation and Enrichment.
• The rate at which the supernatant flowed through the filter was incredibly slow and I lightly tapped the vacuum filter against the table.
• After pouring the Arthrobacter in the lysate, I noticed that there was still some left in the vial.
• The soil sample combined with the LB Broth reached a mass of 51.4 g.
• The Direct Isolation had a mass of 13.8 g.

Conclusion and Future Plans:

• The procedure was carried with little mistakes but none that can cause much contamination. Despite having less soil than what was required, I have enough to carry on for further experimentation.
• In the future (Friday- 8/24/18), I will filter the Direct Isolation using the tube top vacuum filter.