Rationale: Today’s goal was to correctly perform a spot test to determine the presence of phage in the soil samples that we had extracted in the week prior to this one. Hopefully, if done correctly with no contamination, spots will appear in our plaque after 48 hours of incubation.

Materials:

• Lysate
• PY Broth
• PY 2X TA
• 40% Dextrose
• 1M Calcium Chloride
• 0.5 mL of Arthrobacter host
• Agar plate
• Pipettes (Micropipettes and serological pipettes)

Procedure:

1. This procedure started with establishing an aseptic zone to decontaminate the work bench. The bench was sprayed and wiped down with CiDecon, then followed up with 70% Ethanol, which was spread on the table allowed to evaporate. Following this, I grabbed our burner to finish setting up the aseptic zone.
2. Next, I gathered the materials necessary to create the top agar that is needed for the spot test. I grabbed 4 plates (one for each group member and one for control), 2 serological pipette tips,a 50 mL conical tube, and I also set out four micro centrifuge tubes which will be used later in the lab. I then marked my plate with a sharpie to show where I would spot my direct isolation, my enriched isolation, and my phage buffer.
3. Next, I began to create the top agar, I measured out 4.5 milliliters of LB broth to add to the conical tube using a serological pipette. My pipette was having issues, and could not go past 4 milliliters of liquid, so I had to use the serological pipette for the 4 milliliters, then I had to use a micropipette to gather 500 micro-liters to add to my broth to equal 4.5 milliliters to add to my 50 mL conical tube.
4. After the LB broth was added, I needed to add calcium chloride to our top agar. Initially, the quantity of calcium chloride needed was unknown, but what was known was that we needed the molarity of calcium chloride to equate to 4.5 mM or millimolar. To solve for the volume needed, there is a simple equation that I used to get a final value of 45 microliters of calcium chloride
5. 
6. Following the calculation, the calcium chloride was added to our broth mixture using a 20-200 microliter pipette.
7. Instead of adding arthrobacter next, I filtered my enrichment from the previous lab through a 22 micron filter by syringe. I pulled out approximately 2 milliliters and pushed it gently through the filter into a micro-centrifuge tube.
8. After the filtration process, 0.5 ml of arthrobacter was added to the broth mixture.
9. Followed up immediately by pipetting 5.0 milliliters of PY 2x Top Agar with serological pipette and allowed the top agar to mix.
10. Then I very quickly plated my top agar and the group’s control plate, swirled the plate around, and waited 15 minutes for the plate to solidify.
11. After 15 minutes, I added 10 microliters of my direct, enriched, and phage buffer to their designated spots and put the tray to incubate for approximately 45 hours.
12. After 45 hours I removed the tray to analyze my results.

Observations/Results/Data:

• After examining the plate, it was concluded that there was no phage present in the lysate that I had extracted from my soil sample. The plate looked the same as it did when it was put in the incubator, no spotting whatsoever. This indicates a lack of phage as the arthobacter had not been cleared from the plate. There was no contamination as the agar looked exactly like the control.
• Plate had the same color, but had a liquid forming on the top. Unsure what exactly caused that. 

Interpretations/Conclusions:

• The results of the spot test indicate that there is no phage present in the soil sample that I collected the week before. This can be deduced by the lack of any clearing or any indication of bacterial death on the plate. If phage were to be present, there should be minor bacterial clearing in any part of the plate.

Next Steps:

• There may still be hope for the soil sample. The next step is to perform a plaque assay to view the results of that experiment, as plaque assays and spot test have the ability to yield different results. The plaque assay will be performed the next time I am in lab, and if the results are negative that means I have to collect more soil samples to test for phage.