Rational:

Today, we will be setting up and performing both a plaque assay and spot test for the enriched lysate of Soil B, as well as checking the results of the % water and % sand/silt/clay

Procedure:

• We first created an aseptic zone on our table using CiDecon and 70% ethanol, and lit an ethanol burner to create the aseptic zone
• I then massed my enriched lysate (21.59g) to find a partner for centrifusion. The enriched lysate was centrifuged at 3000 g’s for five minutes to pellet the arthrobacter
• While the lysate was being centrifuged, I checked the results of percent water and percent sand/silt/clay on my soil
• For percent water, I re-massed my soil+dish and got 11.72g, which meant the soil was 9.33g (Total dry weight – weight of dish)
  • I then took my dry soil weight and divided it by the wet soil weight (9.33/12.064), and got 77. I then subtracted 100 from this number to get 23% water
• For percent sand/silt/clay, I looked at my vial and determined where all the clay ended (Total 2 mL)
  • For sand, I determined it was 25% of the soil
  • For silt, I determined it was 50% of the soil
  • For clay, I determined it was 25% of the soil
• My table group decided that we will all be performing our spot test on one plate (1/4 per test), with the left over 1/4 being for our negative control (Phage buffer), as well as performing a plaque assay separately, while sharing one TA (Top agar) control plate
• For the spot test, we mixed together 2 mL LB Broth, 0.5 mL Arthrobacter, and 22.5 μL 1M CaCl2 into a 50 mL conical vial
  • The LB Broth was added with a cartwheel pipette with a 5mL tip, while the CaCl2 was added with the P200 pipette
• We then added in 2.5 mL 2xTA, and then immediately poured it onto our plate, and then let it solidify for 10 minutes
  • 2xTA added with the P200 pipette
• While waiting for the TA to solidify, we filtered our enriched and centrifuged lysate using a syringe and a 22μL filter
• We then spotted 10 μL of each of our enriched lysate on our separate sections of the dish, as well as spotted 10μL of phage buffer into our negative control 1/4
• We then moved the plate to an incubator
• For our plaque assay, we multiplied all our materials by four; we added 8 mL of LB Broth and 90 μL of CaCl2 into a 50 mL conical vial
• We then took our separate enriched lysates and added .5 μL arthrobacter into each one and let it sit for 10 minutes to infect
• We then added 10 mL 2xTA to our mixture, and immediately poured 5mL into our TA control dish (The TA will start solidifying after being added into the mixture)
• We then separately added 5mL of our TA mixture to each of our enriched lysate + arthrobacter mixture, and immediately added it to our plaque assay plates
• We let our plates sit for 15 minutes, and then moved the plates to incubation

Observations:

You can clearly see the separations of the dirt

My dried dirt, used to calculate % water

Our spot test dish; three of the quadrants are for the separate three members in our group, as well as 1/4 for our negative control

My Plaque Assay dish prior to anything added into it

My Plaque Assay dish with the procedure completed

• There was a significant color difference between the wet dirt and dry dirt
• The procedure for the spot test and plaque assay was performed more quickly than our first time

Next Steps:

Next time we come into lab, we will see if the spot test and plaque assay turn out negative or positive, and see if our controls worked