Rationale: After obtaining my lysate, I will be performing a spot test to see if there are any bacteriophage present in my lysate that will infect arthrobacter.

Procedure:

• First, we cleaned our lab area/station with CiDecon and 70% ethanol to create an aseptic area where we can work with our samples, minimizing the chance of contamination
• I then isolated my lysate via a 3 mL syringe and filtering; I ran the lysate through the filter and into a microcentrifugetube (Labled JY 8/27/18 FS lysate) [FS stands for filtered sterile]
  • The filter was 22μL in size
• My group then got 4 petri dishes; one singular dish for each member, as well as a dish for our team that we will use as our top agar control
  • Throughout this blog entry (and later entries), I will abbreviate top agar as TA
  • For my personal plate, I drew on the bottom and split the plate into three separate thirds and labeled them as “Negative Control, FS Lysate Soil A (Enriched), and FS Lysate Soil A (Enriched)
    • When I created my enriched lysate, I was only able to filter out 10 mL, so I wasn’t able to create an direct isolation
    • The plate was also labeled Spot Test Soil A
  • Our team plate was labeled “Team 2 8/27/18 Soil A TA Control”
• We then did calculations to see how much of the following materials we needed for our TA
  • For my personal TA, I need 0.5mL Arthrobacter, 4.5 mL LB Broth, 5mL 2x TA, and 45μL 1M CaCl2
  • For our teams TA, we needed 4.5mL LB Broth, 5mL 2x TA, and 42.8μL 1M CaCl2
• We used a blue pipette w/ a yellow sticker (The P200 pipette) [Min. 20μL and Max 200μL] to transfer 42.5μL of 1M CaCL2 to a 50 mL conical vial
  • This vial became our Team 2 TA vial
• I used the same pipette to transfer 45μL of 1M CaCL2 to a 50 mL conical vial
  • This vial became my personal TA vial, and was labeled accordingly with my initials, date and TA description
• We then used a cartwheel pipette with a 5mL tip to transfer 4.5 mL of LB Broth into both our team vial and my personal TA 50 mL vial
• I then took my vial to Lathan (Our TA) to have 0.5mL Arthrobacter added into my 50 mL TA vial
• We then added 5mL of 2X TA (Via a cartwheel pipette with a 10 mL tip) into both our team vial and my personal TA 50 m vial
• The contents of my vial was then immediately poured into my petri dish, and allowed to sit for 10 minutes for the TA to solidify
  • The contents of the team’s control TA vial was also poured into the team’s petri dish
• I then used a white pipette (The P10 pipette) [Min. 1μL and Max 10μL] to drop 10μL of my FS Lysate to both sections on my plate
• I also dropped 10μL of phage buffer into the Negative Control area of my petri dish
• All our dishes were placed into incubators at room temperature
• We cleaned and sprayed down our work area with CiDecon and 70% ethanol

Observations:

• In one of my thirds labeled “FS Lysate”, there were bubbles from when the TA was added
• Using the cartwheel pipette proved harder than anticipated, since it is based on how fast you release the liquid

Image of my plate after the drop of lysate was added

The circled areas are where we noticed bubbles in the TA

Next Steps:

• The next steps would be to see if the drop test turned out positive or negatively, and regardless of the outcome, proceed with a plaque assay, which will also give us information if there is bacteriophage in my lysate