9.19.18 Spot Test and Plaque Assay for Soil Sample C

Rationale: A Spot Test and Plaque Assay will be performed to analyze the composition of the lysates from Soil Sample C. The two tests will reveal if a phage is present through the presence or absence of a plaque on the bacterial lawn.

Procedure: (Metadata steps in italics)

1. Cleaned lab bench and lit burner for aseptic zone.
2. Obtained conical tubes and labeled “HMB Plaque Assay Soil C 9/19/18” and “Top Agar Plaque Assay Control 9/19/18”
3. Added 2mL of LB broth to both tubes
4. Added 25μL of CaCl2 to both tubes.
5. Used syringe and filter to create at least 10μL of Filter Sterilized Enriched Lysate. Placed in microcentrifuge tube labeled “HMB FSEL 9/19/18”.
6. It was observed that the LB Broth used in steps 3 and 4 was cloudy. This was thought to be due to contamination, so the control tube was kept and tested to confirm this thought while the other two tubes were remade (redid steps 3-4) with clear LB Broth.
7. Added 10μL of Filter Sterilized Enriched Lysate to 0.5mL Arthrobacter. Let sit for 15 minutes.
8. Obtained 3 new plates and labeled them “HMB Plaque Assay Soil C 9/19/18”, “Top Agar Contamination Control”, and “Top Agar Control 9/19/18”.
9. Added 0.5mL Arthrobacter with Filter Sterilized Enriched Lysate to conical tube labeled “HMB Plaque Assay Soil C 9/19/18”
10. 2.5mL of 2X Top Agar was added to all 3 tubes and was swished to mix.
11. Poured Top Agar solutions on to respective plates. Let sit for 15 minutes before incubating.
12. Obtained new 50mL conical tube labeled “TA Control for Spot Test HMB NMN 9/19/18” and “TA for Spot Test 9/19/18 HMB NMN”.
13. 2mL of LB Broth and 22.5μL of CaCl2 were added to Conical Tubes
14. 0.5mL of Arthrobacter added to experimental vial
15. 2.5mL of Top Agar was added to both Conical Tubes.
16. Conical Tubes were swished, then poured onto their respective plates.
17. At this point, it was observed that after letting the “HMB Plaque Assay Soil C 9/19/18” plate sit for 15 minutes that the Top Agar had broken and slid around the plate. Therefore, a new plate was created using steps 3-4, 7, 9-11 in that order. It was also given a new 15 minutes, then placed in incubator.
18. Weigh boat containing soil from Monday was reweighed and found to be 5.689g. 5.689g-2.43= 3.259g. 3.259g/4=0.815. 1-0.815= 0.185 –> 18.5% H2O.
19. Reexamined Soil Separation: 6mL of soil was separated. Measurements were found to be 2mL of sand (33.3%), 1mL of silt (16.7%), and 3mL of clay (50%). Soil was properly disposed of and tubes were cleaned.
20. pH tube and paper were obtained. Small amount of supernatant from soil separation sample was placed in bottom of pH tube and the rest of the tube was filled with Deionized Water. pH paper was placed in sample for 45seconds, then examined. Found to be a pH of 6.5 — more basic than previous sample.

Observations

• pH of Soil Sample C is more basic than Soil Sample B. It will be interesting to see if this has anything to do with the presence of a plaque as a possible secondary study to the overarching question currently posed.
• When Top Agar did not set, plate was shaken somewhat vigorously to attempt to prevent Top Agar from setting with bubbles present. This likely caused the plate to not set correctly, creating slipping of top agar. Important to note for next time.
• There was much more clay in Soil Sample C than found in previous samples.
• Cloudy LB Broth could be the result of contamination that has been previously observed throughout the lab.

Next Steps: The next component of the process is to reexamine the plates on Friday to determine whether or not there is a phage. If there is a phage, we will be able to move ahead in the process of picking a phage on Monday, but if not, I will obtain a new soil sample over the weekend.

Conclusions:

• Over-swirling Top Agar likely results in slipping and dividing of Top Agar.
• Cloudy LB Broth could have been the cause of contamination viewed on many of the plates.