Rationale)

Since the spot test from 9.12.18 was negative it is necessary to conduct a plaque assay in order to confirm that the lysate derived from Soil B was negative or deny it by having a positive result from the plaque assay.

Procedures)

1. Setup an aseptic zone by wiping down the work area with CiDecon and 70% ethanol, and lighting an ethanol flame.
2. Retrieve “Filtered Lysate Enriched 9.12.18″(FLE) and a vial of .5mL of Arthrobacter, under aseptic conditions add 10 microliters of FLE to the vial of arthro and let sit for 15 minutes.
3. Collect two 50mL conical vials, labeling one “Top Agar for Plaque Assay NMN 9.14.18” and the other “Control Top Agar for Plaque Assay HMB, NMN, 9.14.18”.
4. Retrieve LB broth and using a 10mL serological pipette add 2mL of LB broth to both the 50mL conical vial labeled “Control Top Agar for Plaque Assay HMB, NMN, 9.14.18” and the 50mL conical vial labeled “Top Agar for Plaque Assay NMN 9.14.18”, under aseptic conditions.
5. Retrieve the micro test tube of 1M CaCl2 from 9.12.18 and add 22.5 microliters of CaCl2 to both of the 50mL conical vials under aseptic conditions using a 10-100 microliter micropipette.
6. Collect two plates with base agar, labeling one “NMN, HMB Control Top Agar for Plaque Assay, 9.14.18” and the other plate “NMN Plaque Assay 9.14.18”.
7. Add the red-capped test tube containing both the Arthro and FLE to the 50mL conical vial labeled “NMN Plaque Assay 9.14.18” under aseptic conditions and swirl to combine.
8. Add 2.5mL of 2xTop Agar to both 50mL conical vials using a 10mL serological pipette under aseptic conditions.
9. Swirl both 50mL conical vials for 3 seconds to combine all ingredients, then proceed to pour their contents into their respectively labeled plates, shaking the plates slightly to distribute the top agar evenly. Let the plates solidify for 15 minutes.
10. After they have solidified invert the plates and place them in the incubator, letting them incubate over the weekend.
11. Clean the work area with CiDecon and 70% ethanol, and dispose of all used items into their appropriate receptacle.

Results From Spot Test)

The spot test I conducted came back negative, however, we did not create a top agar control thus this could have affected the results of the spot test.

Observations)

I observed that there were strands and particles that appeared when the top agar was mixed and when the top agar was poured into the plate.

Conclusions/Next Steps)

Due to the negative results of the spot test we had to conduct a plaque assay in order to positively confirm that the lysate from Soil B was negative in the presence of phages if it comes back negative or confirms the presence of phage in Soil B if the plaque assay comes back positive. The next step will be to check the plaque assay on Monday for plaques, wherein if present we will pick and begin isolating the plaques; if plaques are not present we will have to collect another soil sample.