Title: Soils Metadata and Plaque Testing

Date: 8 October 2018

Rationale: In the previous lab session, the soil was washed, enriched and the lysate incubated. The metadata needs to be taken and the initial plaque tests need to be run.

Procedure:

– Aseptic zone created by washing lab bench with CiDecon and Ethanol, and an ethanol lamp was lit.

• Soil Metadata taken
  • % Water: 14.381g of dirt massed in weigh boat and set to evaporate under fume hood for 48 hours
  • % Sand, Silt, Clay: ~4mL dirt added to falcon tube, DI water added to 12 mL mark. 3 drops Soil Dispersion Liquid added, and shaken for 30 seconds. Tube placed in fume hood to disperse.
  • pH: small amount of soil placed in pH vial. DI water added to rest of vial. The tube was shaken for 10 seconds and a pH strip used to test the pH. The found pH was 6.0
• Incubated lysate retrieved from shake incubator and filtered through 0.2 micron filter to prepare a filtered lysate for a spot test and plaque assay.

The following recipe was used to make a spot test:

• 2 mL LB Broth
• 2.5 mL 2X Top Agar
• 22.5 microliters CaCl2
• 0.5 mL Arthrobacter

The mixture was plated and left to solidify for 15 minutes. 10 microliters of lysate was then spotted into a designated section of the plate along with a Phage Buffer negative control. The plate was then put unto the incubator for 48 hours.

The following recipe was used to make 1 plaque assay:

• 2 mL LB Broth
• 2.5 mL 2X Top Agar
• 22.5 microliters CaCl2

~4.5 mL added to 0.5 mL tube containing Arthrobacter + 10 microliters of filtered lysate. The plate was left to cool for 15 minutes and then incubated for 48 hours.

Conclusions: This soil sample constitutes the last attempt at finding a phage. The plaque assay and spot test can collectively indicate the presence of a phage that, if positive, will be picked in a following lab.