9.21.18 Possible Phage Analysis

Results from Wednesday, 9/19: Plaque Assay experimental plate shows one clearing that could have been caused by phage. Both the control plate predicted to show contamination and the control plate expected to not show contamination showed contamination. Spot test showed a very small clearing that appeared similar to the one found on the plaque assay.

Rationale: Results from Wednesday, 9/19 showed that there was a very small clearing on the Plaque Assay test completed. Therefore, to determine whether or not a phage was the cause of the clearing, a new plaque assay was completed to test the composition of the clearing.

Procedure:

1. Aseptic zone established.
2. Plates were removed from incubator and analyzed.
3. 100μL of Phage Buffer was placed in microcentrifuge tube (“HMB PPL”)
4. Pipette tip was used to take up plaque from plate, added to phage buffer and mixed.
5. 2mL LB Broth added to control and experimental tubes
6. 22.5μL CaCl2 added to control and experimental tubes
7. 10μL of Phage Buffer and Possible phage solution added to Arthrobacter. Let sit for 10 minutes.
8. Added 2.5mL top agar was added to control tube and plated mixture after swishing about.
9. 0.5mL Arthro and possible phage solution added to experimental tube
10. Added 2.5mL 2X Top Agar to experimental tube. Swished. Plated overlay. Let sit for 15 minutes before incubating until Monday 9/24.

Observations:

• Both control plates that were used showed signs of contamination. One of the control plates was expected to show contamination, but the other should not have. This likely would have been from contaminated 2X Top Agar solution, which was not changed between the two samples.
• A small clearing was observed on the plaque assay. It appeared to be turbid, and its size was quite small. These factors made it uncertain whether or not a plaque was present because there was not an obvious presence.

Next Steps:

• The next step for this experiment is to observe the results on Monday and follow the next proper step in picking and diluting (if plaques are present) or obtain a new soil sample (if no plaques are present on the plaque assay).

Conclusions:

• Systematic contamination of control plates is likely occurring from repeated use of contaminated LB Broth and Top Agar. Many of the control plates completed in group 3 in the past weeks have been contaminated in addition to other incidents of contamination across the laboratory , so it is not unrealistic to conclude that it is likely that there is a common source.
• There likely will not be a plaque on the plate on Monday. While it is possible that a phage caused the small clearing or plaque, it is strange that there was not any other sign of phage. Furthermore, the contaminated control plate introduces a situation where bacterial competition could have caused the clearing. Therefore, it was necessary to test with another plaque assay to determine whether or not phage was present.