Rationale: To perform a spot test to determine if there are any arthrobacter bacteriophages in the enriched lysate sample.

Scientific Research Question: Does the presence of arthrobacter bacteriophage appear more dominant in one oak tree species than others?

Description of Procedure:

1. The first step in this procedure was to clean the lab station. This was done with CiDecon and 70% Ethanol. An aseptic zone was created with a 100% Ethanol burner being lit.
2. First the enriched lyste was filtered using a sterile 0.22 uL filter to filter the added arthrobacter out of the sample. 2 mL were removed from the enriched lysate, and 1.5 mL was filtered into a 1.5 mL micro centrifuge tube, using a sterile syringe. The tube was labeled EF, which stands for filtered sterilized enriched lysate.
3. 4 petri dishes were then obtained, one for each person in Group 4 and one for the Top Agar control. My individual plate was labeled LIP 8-27-18 SP1 (Spot Test 1), and three sections were drawn onto the bottom of the plate, one for the enriched, direct, and negative control spot tests. The Control plate was labeled Group 4 8-27-18 Control TA.
4. Next, the top agar was made in a 50 mL tube. The tube was labeled LIP 8-27-18 TA1 (Top Agar 1). The below calculation was done to determine the amount of CaCl2 needed.
5. 
6. The below calculation was done to determine the amount of CaCl2 needed for the control plate.
7. 
8. 4.5 mL of LB Broth was added first in an aseptic zone with a sterile pipette, to both the experimental and control tubes.
9. Next, 0.5 mL of arthrobacter was added to the experimental tube, but not the control tube.
10. 45 uL of CaCl2 were added to the experimental tube, while 42.75 uL of CaCl2 were added to the control tube.
11. Lastly, the 2x TA was added to both the control and experimental top agar solutions and gently mixed with the micro pipette.
12. The Top Agar solutions were then poured onto the correct labeled petri dishes.
13. The plates were then allowed to sit for 10 minutes until the Top Agar solidified.
14. After the plates set, 10 uL of the enriched and direct lysates, as well as the phage buffer for the negative control, were spotted into their correct areas on the petri dishes. Nothing was added to the control TA.
15. The liquids were then allowed to absorb into the agar for 15 minutes.
16. The plates were then incubated until Wednesday. This process was complete at 4:35.
17. The  lab station was cleaned before leaving. This was done with CiDecon and 70% Ethanol. All used materials were properly disposed of.

Petri Dish Depiction: 

Observations/Results/Data:

Observations:

• There was not a lot of resistance when filtering the enriched lysate.
• The enriched lysate had a strong smell.
• The experiment was all done in an aseptic zone.
• The top agar took 10 minutes to solidify.
• When the enriched and direct solutions were spotted onto the petri dish, some of them rolled on the top agar before falling into place.

Results:

The experiment was completed (spot test). Group 4 completed 3 experimental plates and 1 control plate. The plated will be incubated in the lab until Wednesday 8-29-18.

Results on Wednesday 8-29-18: After 46 hours of growth, no plaques were found on the plate.

Interpretations/Conclusions/Next Steps:
The procedure was complete. It will now need to sit for approximately 48 hours, or until Wednesday, the next lab time. The procedure was slow, but went smoothly. The next step will be to check for plaques on Wednesday. If there are plaques, then a plaque assay will be done for both the enriched and direct lysates. If there are no plaques, then only a plaque assay will be done for the enriched lysate to check it once more.