Soil Washing Part 2 8.24.18

Rationale)

To continue the soil washing of Soil Sample A from 8.22.18 and achieve a direct isolation and an enriched isolation from the lysate produced, which can then be used in both a spot test and a plaque assay to determine the potential presence of phage in Soil Sample A.

Procedures)

1. Retrieve the two vials labeled Soil A from 8.22.18, which had been centrifuged by the TAs in the time between 8.22.18 and 8.24.18, and an additional .22μ filter.

2. Setup the filter under the fume hood and attach the vacuum, once setup open both vials of Soil A and using a pipette transfer the supernatant from the vials to the aperture of the filter apparatus and let filter into the 50mL conical vial attached.

3. Once filtration is complete and all the lysate is in the 50mL conical vial turn off the vacuum and open the sterile packaging of the lid to the vial. Remove the filter apparatus and quickly screw on the cap using proper technique to minimize contamination. Label the vial as NMN Soil Lysate A pt2, 8.24.18.

4. Retrieve a vial containing .5mL of arthrobacter and add it under non-aseptic conditions but with proper technique to the vial containing Soil Lysate A pt2 and quickly recap the vial. Rename the vial by adding “enriched” to its description and place the vial in the shaker.

5. Under a previously setup aseptic zone retrieve the vial of Soil Lysate A 8.22.18 and transfer its contents to a 15mL conical vial labeled Soil A Direct Isolation 8.24.18. Place this vial in the tray that is to be refrigerated by the TAs.

Observations)

The Soil A supernatant was much clearer after the second centrifuging compared to its original state, the supernatant was much more golden and translucent compared to the opaque and tan/cream colored supernatant before the second centrifuging. Soil Lysate A Enriched had about 10mL in it and Soil A Direct Isolation had approximately 5mL of lysate. There is a potential that contaminants could have gotten into Soil Lysate A Enriched when the arthrobacter was added under aseptic conditions.

Conclusions/Next Steps)

The next time I conduct soil washing greater care needs to be taken to make sure all critical steps are conducted in an aseptic manner to prevent contamination of the samples. Besides the potential that my sample was contaminated, I was able to create a Direct Isolation and Enriched Isolation from the filtration of the Soil A supernatant into lysate, which will be used in the next steps of bacteriophage isolation, plaque assays, and spot tests.