Date: Wednesday, August 22, 2018

Title: Soil Washing and Lysate Preparation

Rationale: By washing the collected soil and preparing two different kinds of lysate from a centrifuged supernatant, the process of isolating and discovering a phage will begin.

Materials Used: CiDecon, 70% EtOH, 50 mL test tubes, 15 mL test tubes, top filter, LB Broth, Arthrobacter culture, centrifuge

Preparation: 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 contamination.

Procedure: With the soil that was collected from an oak tree, the following was added to a test tube:

• LB Broth (poured to 35mL mark on tube) (Broth creates a favorable environment for bacteriophages to grow in)

Then, the tube was shaken for 15 minutes

Observation – The shaken tube was much lighter with the addition of LB Broth, turning the dark brown dirt to a lighter, coffee color uniform throughout the whole tube.

The tube was then inserted into a centrifuge and spun for 15 minutes to separate the supernatant and the solid soil.

After the tube was centrifuged for 15 minutes, the supernatant and the soil were separated clearly, with a dark solid at the bottom of the tube and a yellow supernatant filling the rest of the tube.

The newly acquired supernatant was then poured through a 22-micrometer top filter to filter out any bacteria (phages are just barely small enough to pass through.)

The supernatant was poured into the top of the filter and turned on via a vacuum to activate the filter.

The filtered supernatant, now lysate, was split into 2 different isolations

• A direct isolation, where pure lysate was poured into a 15 mL tube and refrigerated until Monday
• An enriched isolation, where lysate and 0.5 mL of Arthrobacter ATCC 21022 were combined and incubated until the following Monday

Conclusion/Observations: With two different isolations, different experiments and tests can be run to try and discover a phage. In the case of no growth, the process can be repeated until a phage is found. Within the enriched lysate, Arthrobacter, LB Broth, and the supernatant are all in one suspension, providing a favorable environment for growth and, hopefully, infection.