Rationale:

To filter the enriched lysate using a syringe, collect the data to understand the soil’s % water and soil composition, and complete a plaque assay using the filtered enriched lysate.

Procedure:

The data for the soil composition and % water was first collected. To determine the % water, a scale was used to determine the initial and final masses which were used for calculations. For the soil composition, the different layers were observed and the volume of each layer was taken down for later use in calculations. After collecting all the data, the next step was to filter the enriched lysate. It was filtered using a syringe and 0.22 µm filter into a tube. In another tube, 1 mL of filtered enriched lysate and 0.5 Arthrobacter were combined and left alone for 10 minutes to infect. Next, 22.5 µL of CaCl2 and 2mL of LB broth were combined to form what was to become the top agar. After 10 minutes, 2.5 mL of 2X Top Agar was added to the Top Agar solution. The Arthrobacter and lysate were added to the top agar solution and poured onto the agar plate. The plate was left alone for 10 minutes for solidification. Then, the plates were placed in the incubator.

Results and Analysis:

% Water

(3.336/3.79)x100=88.0%

Soil Composition

Total: 4 mL

2 mL of sand

.7 mL of silt

1.3 mL of clay

(2/4)x100= 50% of sand

(.7/4)x100= 17.5% of silt

(1.3/4)x100= 32.5% of clay

Plaque Assay Plate

Conclusions and Future Plans:

First, data was collected from procedures completed previously (soil composition and % water) and used to perform calculations to determine the percentages. Then, the lysate was filtered using a syringe. Lysate and Arthrobacter were combined and left alone for 10 minutes. During that time, CaCl2 and LB broth were combined in a conical vial. After the 10 minutes, the Arthobacter and lysate were added to the CaCl2 and LB broth along with 2X Top Agar. The top agar was poured on a plate and left to solidify for 10 minutes then placed in the incubator.

Future Plans:

On Monday (9/23/18), the plates will be checked for plaques. If there is contamination of the control with no plaques on the plate, another plaque assay will be made. If there is no contamination along a negative plate, another soil sample will be collected in another area. If there is no contamination with a positive plate, the plaque will be picked and will be diluted using a phage buffer.