10.1.2018- Spot Test and Plaque Assay Soil Sample D

Rationale: Lysate was obtained on Wednesday (9/26), but had yet to be tested from Soil Sample D. Therefore, it is necessary to determine whether or not a phage is present by using a spot test and plaque assay.

Procedure: Metadata results from Wednesday were obtained and recorded. 2mL Filter Sterilized Enriched Lysate (FSEL) created with syringe and 22µm filter. 10µL FSEL was added to 0.5mL Arthrobacter and was set aside for 15 minutes. 22.5µL CaCl2 was added to a control tube (“NMN HMB 10.1.18 Top Agar Control”) and experimental tube (“HMB Plaque Assay Soil D 10/1/18”). 2mL LB Broth added to both tubes. Arthrobacter with FSEL was added to experimental tube. 2.5mL Top Agar added to both tubes and poured onto plates. Plates were let sit for 48 hours. Obtain two new tubes (“HMB Spot Test Soil D 10/1/2108”) and (“HMB NMN 10.1.18 Top Agar Control-ST”). Add 2mL LB Broth to both tubes. 22.5µL CaCl2 was added to both tubes. 0.5mL Arthrobacter was added to the experimental tube. 2.5mL of Top Agar was added to both tubes, then the solutions were plated. Plates were labeled and let sit for 10 minutes. 10µL FSEL, Direct Isolate, and phage buffer were added to each respective section, then the plate was allowed to set for 15 minutes. Plate was incubated for 48 hours.

Observations:

• Metadata:
  • Soil Composition: 2.5mL Sand, 0.5mL Silt, and 0.5mL Clay were found in the sample. Results in 71.43% Sand, 14.29% Silt, and 14.29%
  • Percent Water: Sample’s mass when weigh boat was subtracted: 3.091g. Original mass of soil added: 4.126g-3.091g=1.035g/4.126g= 25.08% Water
• White particle was observed in direct isolate. It will be interesting to observe any possible effect that it has on the sample.
• Spots for spot test did not set as well as they normally have. This resulted in us not inverting our plates before overnight incubation.

Next Steps/Conclusion:

• Since the plates have been prepared, they will be able to be read and analyzed on Wednesday, October 3. If there are plaques, it will be possible to proceed with the plaque isolation process. However, if there are no plaques present, it will be necessary yet again to obtain a new soil sample, as there would not be any phages present in Soil Sample D.