Rationale)

To check my plaque assay and spot test from Wednesday, if the result is positive the plaque(s) will be picked and a lysate will be produced from the plaque and used to conduct a spot test, if the result is negative the plates will be disposed of another soil sample will be collected.

Results from Wednesday)

The plaque assay and spot test from Wednesday came back with a possible positive, as the plaque assay possibly contained a plaque which could have also been an air bubble.

Procedures)

1. Checked plates from Wednesday
2. Setup an aseptic zone
3. Labeled a micro test tube “NMN 9.21.18 PPL”
4. Filled tube with 100 microliters of phage buffer.
5. Picked the possible plaque in the plaque assay and swirled in the phage buffer to transfer, vortexed the tube to combine.
6. Labeled 50mL conical vial “NMN 9.21.18 Possible Plaque Assay Soil C” and other one “Top Agar Control for PA 9.21.18”
7. Added 2mL of LB broth to both vials
8. Added 22.5 microliters of 1M CaCl2 to both vials
9. Added 10 microliters of “PPL” to a .5mL vial of arthro, let infect for 15 minutes.
10. Collected two plates labeled one “9.21.18 Top Agar Control NMN, HMB” and the other “NMN 09.21.18 Possible Plaque Plaque Assay
11. Added 2.5 of 2xTop Agar to the control tube, swirled the tube and poured into the control plate. Let sit for 15 minutes then inverted the plate and incubated until Monday.
12. Added .5mL of infected arthro to the PA tube
13. Added 2.5mL of 2xTop Agar to the PA tube, swirl the tube and pour into the plaque assay plate. Let the plate sit for 15 minutes then invert and incubate until Monday.

Observations/Data)

I observed that the top agars contained thin filaments and the LB broth and top agar we used appeared to be from of contamination as based upon their high clarity. I also observed possible air bubbles in the plaque assay I performed which I subsequently marked with a sharpie to make sure misidentification did not occur.

Conclusions/Next Steps)

Based upon the possible positive result from my plaque assay on Wednesday I picked the supposed plaque and conducted another plaque assay using lysate produced from the plaque. The next step will be to check to see if the plaque assay comes back positive or negative, if positive purification will continue to occur by picking a plaque and performing another plaque assay, if the result is negative another soil sample will be collected and washed in the hopes of eventually isolating a plaque.