Rationale:

The goal in the lab today was to analyze the results of the plaque assay performed on 10/01/18 and perform metadata experiments. If plaques are present, a serial dilution would be performed to get a high titer of phage. If the results were negative, the new soil sample will be cleaned and enriched for future plaque assays.

Results From 10/01/18:

• Plaque assay from 10/01/18 was negative, with contamination on the group control plate.
• The LB broth used for the plaque assay was contaminated again as well.

Materials:

• 10-mL LB broth
• DI water
• .22 micron syringe filter
• Conical Vials
• 0.5-mL Arthrobacter

Procedure for Enrichment:

• Established aseptic zone.
• Added soil up to 2-mL mark of conical vial and filled with LB broth to 12-mL mark.
• Vortex soil and broth mixture for approximately 15 minutes.
• Once vortex was completed, conical vial was weighed and DI water was added to balance out for centrifuge.
• Soil was centrifuged on table top centrifuge for 10 minutes.
• Soil was removed after 10 minutes and lysate was transferred to a new conical vial to separate from pellet.
• Filtered separated lysate through 0.22 micron syringe filter. Repeated until all lysate was filtered into a conical vial.
• After filtration, 0.5-mL of Arthrobacter was added to lysate to create an enrichment.
• Lysate was left to grow in shake incubator.

Procedure for pH and Soil Dispersion:

• Soil was added to the 4-mL line of a falcon tube and DI water was added to the 12-mL line.
• 3 drops of soil dispersion liquid was added to the soil and the tube was shaken for approximately 30 seconds.
• Soil was then put on a rack to settle for 48 hours.
• For pH testing, a small amount of soil was added to a vial and then filled with DI water.
• Soil was then shaken for 10 seconds and left to rest for 2 minutes.
• pH paper was inserted into the soil for 45 seconds and removed for examination.

Results/Observations:

• Lysate at the end of enrichment contained no particles, no apparent signs of error during procedure. Also LB broth used was clear and was kept in aseptic zone with caution to prevent any possible contamination.
• pH paper was removed and kept a yellow color relatively similar to the original just slightly dimmer.
• 
• Soil seemed to contain a lot of sand resting at the bottom right after the initial shaking. Could possibly even out after the settling for 48 hours.

Conclusions:

• From the metadata taken, the pH of the soil seems to be slightly acidic with a pH of around 5.5. This is the first soil sample taken that has had any pH below 7.0. Could possibly provide a more optimal environment for Arthobacter and phage, seeing as the previous basic samples have yielded no phage.

Next Steps:

• The next steps for this experiment are to perform plaque assays with the new enrichment to test for the presence of phage. Also additional metadata experiments need to be performed to gather water percentage and soil composition.