Rationale)

To conduct a spot test and a plaque assay in order to test, via the formation of plaques, for the presence of phage in lysate produced from Soil C and to finish collecting metadata on Soil C.

Procedures)

1. Setup an aseptic zone by wiping the work area with CiDecon and 70% ethanol, light an ethanol flame.
2. Labeled two 50mL conical vials “NMN 9.19.18 Top Agar for Plaque Assay” and “NMN, HMB 9.19.18 Top Agar for Plaque Assay Control” respectively.
3. Added 2mL of LB broth to both conical vials.
4. Added 22.5 microliters of 1M CaCl2 to both vials.
5. Retrieved “NMN 9.17.18 Soil C Lysate Enriched” and filtered enough lysate with .22 micron syringe filter to fill a micro test tube, labeled the tube “Soil C FLE NMN 9.19.18”.
6. The LB broth was found to be contaminated, the top agar control vial was kept to positively test for contamination of the LB broth and its lid was colored to denote its purpose, “NMN 9.19.18 Top Agar for Plaque Assay” was discarded, two new 50mL conical vials were then labeled the same as the discarded ones.
7. Added 10 microliters of FLE to a vial with .5mL of arthrobacter. Left to infect for 15 minutes.
8. Added 2mL of uncontaminated LB broth to both of the new 50mL conical vials.
9. Added 22.5 microliters of 1M CaCl2 to both of the vials.
10. Collected three plates with base agar, labeled them “NMN 9.19.18 Plaque Assay Soil C”, “Top Agar Contamination Control 9.19.18”, and “Top Agar Control 9.19.18”.
11. Added .5mL of infected arthrobacter to “NMN 9.19.18 Top Agar for Plaque Assay”.
12. Added 2.5mL of 2xTop Agar to all three vials.
13. Shook vials to combine components for 10 seconds and poured them into their respective plates, let them sit for 15 minutes then they were inverted and incubated for 48 hours.
14. Collected two 50mL conical vials, labeling one “TA Control for Spot Test 9.19.18 HMB NMN” and the other “TA for Spot Test 9.19.18 HMB NMN”.
15. Added 2mL of LB broth to both vials.
16. Added 22.5 microliters of 1M CaCl2 to both vials.
17. Added .5mL of arthrobacter to “TA for Spot Test 9.19.18 HMB NMN”.
18. Collected two plates labeling one “Top Agar Control Spot Test NMN HMB 9.19.18” and the other “9.19.18 HMB NMN Spot Test Soil C”, divide the latter plate into thirds labeling one NN, HB, and the other Control.
19. Add 2.5mL of 2xTop Agar to both vials, swirl to combine and pour into their respective plates. Shake the plates slightly and let sit for 15 minutes.
20. Retrieved the weigh dish labeled “NMN %H2O Soil C” and take the mass. The mass was 5.761, the mass of the water was thus found to be .599g, and the percent water of the soil was 14.975%.
21. Retrieve the falcon tube from 9.17.18 and find the milliliters of sand, silt, and clay. Sand was 2mL. Silt was .5mL. Clay was 2.5mL.
22. Added 10 microliters of each FLE and phage buffer to their respective sections of “9.19.18 HMB NMN Spot Test Soil C”, control and NN. Let sit for 12 minutes.
23. Retrieved a pH vial and added 2mL of water from the falcon tube, top off the vial with DI water, shake 10 seconds, then place a 1-inch strip of pH paper into the tube for 30 seconds. The pH was found to be 6.
24. Put the spot test plates in the incubator inverted, leave for 48 hours. Clean the workspace and leave.

Observations/Data)

We observed that our initial LB broth was cloudy, which indicates contamination, as our second container of LB broth was clear as LB broth is supposed to look. I also observed that there was 2mL of sand, .5mL of silt, and 2.5mL of clay in the falcon tube in which I dispersed the soil sample. Therefore Soil C was 40% sand, 10% silt, and 50% clay. The dried sample of Soil C was 5.761g with the tray and 3.401 without. Compared with the original 4 grams there was a total of .599 grams of water lost. Thus, the percent water of the Soil C was 14.975%. The soil pH was also observed to be 6.

Conclusions/Next-Steps)

From the data I gathered I can conclude that Soil C was a clay soil type, I can also determine the percent water was 14.975%, and the pH of the soil was slightly acidic at 6. Our next steps will include checking for the presence of plaques on both my plaque assay and spot test on Friday, which will determine if we can begin picking and purifying plaques or if we need to collect more soil. We will also check for the presence of bacterial growth in out top agar controls to determine if we need to reassess our process of conducting the procedures, as could occur with such things as contaminated LB broth which occurred when we conducted the procedures today.