8/22/18 Enrichment One Part One

Objective:

The goal of this procedure was to preform soil washing and enrichment on soil samples collected from oak trees around Baylor University’s campus in order to isolate bacteria-phages. The particular sample used in this procedure was found at 31*32’40” N 97*7’9″ W near Waco Hall.

Procedures and Protocols:

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• 1 50 ml tube
• ~ 20 ml LB Broth
• Centrifuge

In order to accomplish this goal two procedures had to occur. The first was the creation of an aseptic zone.

1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

The second procedure was soil washing and enrichment (see photo below).

1. Collect soil from designated oak tree by digging a few centimeters below the grass
2. Place soil in a 50 ml tube up to the 15 ml line
3. Add LB broth up to the 35 ml line under aseptic conditions
4. Shake the tube for 15 minutes (during this 15 minutes the tube was dropped on the floor, potentially disturbing the soil and broth, but then the shaking resumed)
5. Place tube in centrifuge for 5 minutes on 3,000 X g in order to separate the soil (pellet) and supernatant
6. Place tube in the fridge to complete the procedure on a later date

Results:

This procedure yielded a tube of centrifuge separated supernatant and pellet as seen in the image. The solids separated by mass in the centrifuge with the most dense on the bottom. The centrifuge was spun on a low enough power to leave any existing phages in the supernatant.

Analysis:

The procedure had to be stopped halfway through so the results were incomplete and will be detailed in the next entry. Based on the results I do have I can make the assumption that the centrifuge didn’t fully separate my sample because it appears to be murky and there is debris on the top of the supernatant.

Future:

My plans for the future are to complete the procedure I started during Friday’s open lab in order to have an enriched sample and direct sample.