Date: Friday, August 24th, 2018

Title: Supernatant Filtration and Lysate Enrichment

Rationale: The purpose of today’s lab was to filter my supernatant from my 50 mL conical vial to two new vials in order to examine them and use them for a Spot Test next week.

Procedure:

1. I 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. I lit a burner in the middle of the table in order to keep falling particles from contaminating my equipment or sample from above since the flame creates a circulating air current.
3. I used a pipette in the aseptic zone to transfer my supernatant from the 50 mL conical to two 15 mL vials.
4. The two smaller vials filled with supernatant were then spun for five minutes in a small centrifuge in order to separate any heavier particles that may have drifted up since 8-22-18 when I originally centrifuged my 50 mL conical.
5. Under the fume hood, I used a pipette to transfer the supernatant from my two smaller vials to a 22 micrometer filter in order to separate potential bacteriophages from bacteria too large to fit through the filter.
6. I transferred 10.5 mL of the filtered lysate to a 15 mL vial and kept 10 mL of filtered lysate in another 50 mL conical.
7. I then added .5 mL arthrobacter (ATC 21022) to the Enriched sample (50 mL conical vial) in the aseptic zone.
8. The direct sample was then refrigerated, while the enriched sample was incubated.

Observations:

• Soil and other particles were present in the supernatant after the day spent out of the lab, meaning it’s important to filter out supernatant soon after being centrifuged in order to keep an uncontaminated sample as well as one that won’t clog the filter.

Results:

• This experiment yielded me both an enriched and a direct isolation.

Next Step:

• My next step is to set up a spot test next week to test for plaques and therefore bacteriophages.