Rationale:

The goal of today’s lab was to perform both a plaque assay and spot test as well as examine the soil metadata from the lab prior.

Materials:

• LB Broth
• 2X Top Agar
• 2x 0.5 mL of Arthobacter
• Calcium Chloride
• Serological pipettes
• Micropipettes
• Balance

Procedure:

1. This procedure began with establishing an aseptic zone using CiDecon to wipe the table down, 70% Ethanol to spray on the table and let evaporate, and a burner to create a convection current for air flow.
2. I began with a spot test first, grabbing my enriched isolation labeled “GJA Enriched 2 09/10/18” and sent it off to centrifuge for 5 minutes at 3,000 g.
3. While that was occurring, I found my soil sample that was left to evaporate and weighed it to find the mass of the dry soil and calculate the percent of water in the soil. This is the data below along with the equation used:
   

   Empty Petri Dish (mi)	6.90 g
   Petri Dish and Wet Soil (mf)	11.60 g
   Mass of Wet Soil (mwet soil)	4.70 g
   Petri Dish and Dry Soil (mf2)	10.62 g
   Mass of Dry Soil (mdry soil)	3.72 g
   Mass of Water (g)	0.98 g
   %Water	20.85%

    

4. After calculating my percent water, I retrieved my enriched sample from the centrifuge and began to make the top agar by first adding 2.00-mL of LB Broth to a 50-mL conical tube labeled “GJA Top Agar 2 09/12/18”.
5. Next I used a 20-200 µL micropipette to add 22.50-µL of Calcium Chloride to my LB Broth as well.
6. Following this, I filtered approximately 1.5-mL of my enrichment through a 2 micron syringe filter and into a micro centrifuge tube and left that to sit while I transferred 0.5-mL of our Arthrobacter into my top agar solution.
7. Once the bacteria was added, I used a serological pipette to add 2.5 mL of the 1x Top Agar solution. Then I swirled my 50-mL conical vial gently to allow everything to mix and I quickly poured the top Agar onto my plate and left it to solidify for 15 minutes.
8. While my solution was solidifying, I examined my soil separation and poured off the supernatant so I could examine the sand, silt, clay composition of my soil.  Out of the 8-mL of soil present, 3.8-mL was sand, 2.5-ml was silt, and the remaining 1.7-mL was clay. This makes a percentage of 47.5% sand, 31.25% silt, and 21.25% clay
9. After calculating the soil composition, my group decided to perform a plaque assay as well with the remaining time we had available.
10. The group decided to make enough top agar for 3 plates plus a control. This meant we had 8-mL of LB Broth, 10-mL 2X TA, and 90-µL of Calcium Chloride.
11. We began with adding the 8-mL of LB Broth with a serological pipette into one 50-mL conical vial.
12. After adding the LB broth, the 90-µL of calcium chloride was added using a 10-100 µL micropipette.
13. Once the calcium chloride was added, I combined 0.5-mL of Arthobacter with 10-µL of my lysate and let that infect for approximately 15 minutes.
14. At the end of the 15 minutes, we added 10-mL of 2X Top Agar to the 50-mL conical vial through serological pipette. Immediately following this, I extracted 5-mL of the top agar solution, added it to a 15-mL conical vial with my arthrobacter and lysate solution, mixed it very quickly, and poured it into my plate to let it solidify for 15 minutes.
15. With the remaining 5-mL of top agar left, we plated into our control plate
16. Once the 15 minutes were up, the plates were immediately put into the incubator for 48 hours.

Data/Results

• 
• Water percentage of the soil was approximately 20.85%. This was moderately surprising because the soil when it was extracted was very wet, almost clay like.
• The top agar came out looking very similar to previous top agars, despite my lysate being not as clear as everyone else’s.
• The soil contains an extremely large amount of sand in comparison to silt and clay. This was unsurprising as our soil once dry was very crumbly and exhibited sand-like qualities.

Conclusions:

• Soil was roughly 1/5 water. This could be a factor of its location to a nearby water source. There was also a man-made fountain nearby, which may contribute to the amount of water in the soil.
• In addition to the water amount, the soil was a majority of sand present in the soil. This is most likely due to the dry location that the soil and tree is in.

Next Steps:

• The next steps would be to analyze the plaque assay and spot test for the presence of phage in the soil. If there is phage present in the soil, then we will begin to analyze the amount of phage present. If there is no phage present, then I have to redo another plaque assay and spot test or gather a new sample.