9.24.18 Obtaining and Washing New Soil Sample

Results from Friday, 9/21: As suspected, the plaque assay revealed that there was no phage, indicating that the small clearings were likely caused by competition among bacterial groups or other sources. Therefore, Soil Sample C is officially negative. The control plate also was contaminated yet again, and similarly to Wednesday’s (9/19) sample, it is believed that the cause stems from a contaminated top agar or LB broth.

Rationale: Since the results from the test on Friday showed contamination on the control plate and only an extremely small mark on the plate, it was concluded that a new sample would be needed to restart the process. The washing procedure was also fit into the lab session after obtaining a new soil sample (Soil Sample D) to be ready to perform a plaque assay and spot test on Wednesday.

Procedure:

1. Establish an aseptic zone.
2. Collected new sample of soil from tree at Teal Residence Hall. Recorded metadata in survey.
3. Added 2mL of soil to tube (“HMB Soil Sample D 9/24/18”)
4. Added 10mL of LB Broth to tube
5. Shook for 10 minutes
6. Centrifuged sample for 10 minutes
7. Used syringe and 22μL filter to filter supernatant of centrifuged sample. Placed into tube labeled “HMB Soil Sample D 9.24.18”.
8. Moved 100μL of filtered lysate to microcentrifuge tube for a direct isolate.
9. Added 0.5mL of Arthrobacter to “HMB Soil Sample D 9.24.18” tube to create enriched lysate.
10. Moved enriched lysate to shaker to be used on Wednesday 9/26.
11. Moved direct isolate to fridge to be used on Wednesday 9/26.

Observations:

• Control Plate still contaminated using the last LB Broth and Top Agar combination. Will be determined if it is the cause when next plaque assay is completed, as new Top Agar and LB Broth solutions have been created.
• Soil was very dark and already began settling out sand particles before centrifugation. Through this, it can be predicted that there will be a higher proportion of sand than normal.
• Mass of tube with soil and LB Broth was equal to 18.376g as measured before being placed in the centrifuge.

Next Steps:

• The enriched and direct lysates will be used for a plaque assay and spot test. These tests will show accurately whether or not phages are present in the soil obtained. Furthermore, it will be necessary to take extra care when using the new Top Agar and LB Broth to avoid contaminating the fresh samples, as this will have adverse effects on the control plates certainly and potentially the experimental plates as well.