Date: Monday, September 10th, 2018

Title: Soil Filtration and Metadata

Rationale: The purpose of today’s lab was to filter out our soil, get an enriched and direct sample, and record soil metadata.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

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 the equipment or samples from above since the flame creates a circulating air current.
3. Next I filled a 50 mL conical with my soil up to the 2 mL mark.
4. I filled the same conical up to the 12 mL mark with LB broth and shook for 14 minutes.
5. I massed my conical vial and recorded it to weigh 19.79 g. I spun this for 5 minutes at 10,000 g.
6. I filtered the supernatant with a 22 micrometer syringe filter to a new 50 mL conical.
7. I added .5 mL ATC 21022 to my lysate, labelled it BJR and E for “enriched,” and set it in the incubator at 3:50 pm.
8. I then filtered 1 mL of my supernatant to a 15 mL vial to use as my direct isolation, storing it in the fridge.
9. While waiting for my soil to soin, I added soil to a Falcon tube to the 10 mL mark.
10. I added DI water to the 30 mL mark, added 2 drops of dispersion fluid, shook it for 30 seconds, and left it to disperse.
11. I set some soil on a plastic boat to let it dehydrate to find the percent water. The boat weighed 2.479 grams, weighing 9.738 grams after adding soils. This means I had 7.259 grams of wet soil in the boat.

Observations:

• The soil in the Falcon tube already began to separate by the time I left, showing different layers of different sized particles.

Results:

• This project yielded me a direct isolation, an enriched isolation, soil metadata, and a dispersed Falcon tube allowing me to record soil composition.

Next Step

• My next step is to use my enriched lysate, direct isolation, and a phage buffer to make a spot test. I will also finish recording metadata.