Soil Washing 8.22.18

Rationale)

To wash the soil sample I had gathered, Soil Sample A, in order to produce a lysate that can then be both enriched and left as a direct isolation, for later use in conducting a plaque assay and spot test in order to determine the presence of bacteriophage in Soil Sample A.

Procedure)

1. Set up an aseptic zone using CiDecon and 70% Ethyl Alcohol to clean the workspace and lighting an ethanol flame.

2. Retrieve LB broth and the sample of soil that was collected, Soil Sample A, under aseptic conditions and with proper aseptic procedure open the LB broth and add enough LB broth until the 50mL conical vial containing the soil sample is filled to 35mL line.

3. Close both the LB broth and the now partly filled soil sample vial under aseptic conditions and return the broth to the storage area.

4. Shake the vial containing the LB broth and sample consistently for about 16 minutes, after which mass the vial and record the mass on the vial in order for it to be centrifuged properly.

5. Centrifuge the vial at 3000g for 5 minutes.

6. Retrieve a .22µm filter and set it up under a fume hood, connect the vacuum to it and proceed to collect the centrifuged soil sample A vial and a pipette. Using the =pipette transfer the supernatant from Soil Sample A to the top of the filter apparatus.

7. Wait for the supernatant to go through the filter, however, due to excess particulate in the supernatant only about 5mL of supernatant are able to be filtered into lysate.

8. Without removing the vial containing the lysate from the filter apparatus, pour the unfiltered supernatant equally by weight into two 15mL conical vials and label as Soil A, using a scale make sure the mass of each is only .1g apart and adjust the volume of supernatant in each vial as necessary to achieve a deviation of .1g.

9. After the excess unfiltered supernatant is taken care of open the package containing the lid for the vial of lysate, once done proceed to remove the filter apparatus from the lysate containing vial and quickly place the lid onto the vial. Label vial as Soil Lysate A.

10. Place all three samples into the fridge for continuation on 8.24.18.

Observations)

There was too much particulate in the supernatant for it to be filtered as apparently indicated by it being fairly cloudy and filled with larger particles compared to the clear golden colored lysate that was produced, the vial containing Lysate was labeled: NMN 8.22.18 Soil Lysate A, the two vials containing the unfiltered supernatant were labeled NMN 8.22.18 Soil A.

Conclusion/Next Steps)

For future soil washing, less particulate needs to be in the supernatant that is to be filtered which could be achieved by a greater amount time in the centrifuge or less shaking of the vial once it has been centrifuged, the experiment will continue on 8.24.18 wherein it will be determined if the Lysate contains bacteriophage by conducting a Plaque Assay and Spot Test. Overall the experiment and procedures worked relatively well in producing the desired result