10.12.18 Plaque Assay

Rationale: Since the plates that contained a spot test and plaque assay from Soil Sample E displayed phenomena that resembled plaque, it was necessary to pick the areas and complete another plaque assay to confirm the presence of phage.

Procedure:

1. Aseptic zone was established.
2. Results from tests on Wednesday (10/10) were examined.
3. Plaque was picked and placed in 100µL phage buffer, then vortexed.
4. 10µL of phage buffer was added to 0.5mL arthrobacter. Let sit for 15 mins.
5. 2mL of LB Broth was added to tube.
6. 22.5µL CaCl2 was added to tube.
7. 2.5mL 2X Top Agar added to tube.
8. Plated on “HMB Plaque Assay 10/12/18”. Let sit for 10 minutes, then placed in incubator.
9. 2.0mL LB added to Control tube.
10. 22.5µL CaCl2 to Control tube.
11. 2.5mL 2X Top Agar added to control tube and plated overlay solution on plate (“HMB NMN 10.12.18 TAC PA”). Let sit for 10 minutes, then incubated.

Results from Wednesday, 10/10:

• As seen in the included pictures, there are clearings on the plaque assay and there is no growth on the control plate. However, on the spot test, there are no clearings found on any of the three sections. There is a strange pattern between the phage buffer and the enriched lysate section.

Observations:

• When creating the plate with the possible phage, there appeared to be a white substance in the overlay solution that was plated.
• Overlay solution set quickly and appeared to be smooth before placing in the incubator.
• The clearings on the plaque assay appeared to be clear, not turbid. If there a phage is confirmed, these clearings were caused by the lytic cycle rather than the lysogenic cycle.

Conclusions:

• Since there were plaques on the plate, it is likely that they were caused by bacteriophage, as there was no contamination on the control plate. A test will be run to confirm, but if the plaques observed on the initial plaque assay reappear, it would be a safe assumption that phage was present. These observations and predictions will be supported or rejected on Monday (10/15) after the confirmatory plaque assay is done.