Date: August 29, 2018

Title: Plaque Assay for Phage Isolation

Rationale: Regardless of spot test result, a plaque assay is done to obtain another measure of phage presence.

Materials: Arthrobacter, LB Broth, 2X Top Agar, 1M CaCl₂, Enriched lysate, pipet, micropipette, plate, vials

Procedure: To create an aseptic zone, the following was done:

• Counter washed and wiped with CiDecon
• Counter sprayed with 70% EtOH and allowed to evaporate completely (to dehydrate and kill any bacteria on the counter and avoid contamination)
• Ethanol lamp lit to create rising heat and a current that protests samples from falling contamina

• mL Arthrobacter and 10 microliters of filtered enriched lysate (filtered at spot test)

In 50 mL tube the following was added (for 3 plaque assays and 1 group control):

• 8 mL LB
• 10 mL 2X Top Agar
• 90 microliters CaCl₂

~18 mL of solution

• Once the solution was finished, 4.5 mL was added to the small vial of Arthrobacter + lysate (enriched, filtered) and poured onto the plate. (NOTE: One vial was broken in the transition to the plate. The counter was re-sanitized, and another vial was prepared)

Results/Observations: The plate and the solution were yellowish and was put into the incubator to grow over the weekend. If the result is positive, I would expect to see growth on the plaque assay, and a negative result on the control. A negative result would constitute contamination on the control, or no growth on the plaque assay. Assuming a positive result, the next step would be to begin isolating the discovered phage. (See attached picture of resulting plaque assay plate before incubation)

Plaque Assay (Pre-incubation)