Date: Wednesday, August 22, 2018

Title: Soil Washing

Rationale: The purpose of today’s lab was to isolate potential bacteriophages from dirt, bacterium, and other particles in the soil sample I collected (Soil A*) by filtering the sample into a direct and enriched lysate.

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating our equipment or samples from above since the flame creates a circulating air current.
3. I transferred the sample of Soil A from a plastic storage bag to a 50 mL conical vial in the aseptic zone until the soil reached the 15 mL mark.
4. I transferred LB broth to the 50 mL conical vial in the aseptic zone until Soil A and the LB broth together reached the 35 mL mark.
5. I shook the 50 mL conical vial with LB broth and Soil A for 15 minutes (15:11-15:26). This was done in intervals switching between my hand and a vortex machine.
6. During the mixing process, I weighed my 50 mL conical vial, finding it to be 52.01 g. The weight of the vials are important so that the centrifuge can be balanced without a risk to the rotor being posed by uneven weights.
7. My 50 mL conical vial was placed in the centrifuge along with other students’ samples at 3,000 revolutions per minute for five minutes. This was done to separate possible bacteriophages from heavier particles such as dirt.
8. Some students at this point filtered their supernatant to isolate their lysate; however, due to time constraints, my centrifuged 50 mL conical was placed in the fridge to be worked on later in the week.

Observations:

• The mixed solution of LB broth and Soil A was dark and larger particles were unevenly distributed throughout.
• After being centrifuged, the dirt was all at the bottom of the 50 mL conical. It was tightly packed and smoothed over.
• The resulting supernatant was mid-to-dark yellow in coloration after being centrifuged, similar to a slightly dehydrated urine sample.

Results:

• This experiment yielded me a 50 mL conical with a separated supernatant to be filtered at a later date.

Next Steps:

• My next step is to filter my supernatant before examining my lysate for bacteriophages.

*Soil A is my first sample of soil, found near a red oak with a few dying branches outside of the Dawson residence hall at Baylor University.