Rationale:

Today, the percent water, percent sand, percent silt, and percent clay will be calculated for later used to determine whether there is any correlation between the present of phage and the metadata. The spot test will be run only with the enrich lysate (since no isolated lysate was collected) will reveal whether or not the sample soil collected has any phages that target Arthrobacter specifically.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Weighed the mass of “KEA 9/10/18 Soil B enrich” conical vial.
3. Added a few drops of de-ionized (DI) water with disposable pipette to have a similar mass to a peer’s enriched lysate conical vial mass.
4. Centrifuged “KEA 9/10/18 Soil B enrich” conical vial at 3,000g for 5 minutes.
5. Finished collecting data for the percent water by weighing the mass of “KEA 9/10/18 Soil B” petri dish with the dry soil.
6. Finished collecting data for the percent sand, percent silt, and percent clay by analyzing and calculating from “KEA 9/10/18 Soil B” falcon tube.
7. Cleaned “KEA 9/10/18 Soil B” falcon tube with DI water.
8. Wiped a 0.22 μL tip with EtOH (70%) to prevent contamination.
9. Through aseptic technique (over an EtOH (100%) flame), used a 3 mL syringe with a 0.22 μL tip to filtered out “KEA 9/10/18 Soil B enrich” conical vial into a microcentrifuge tube.
   • Labeled the microcentrifuge tube as “KEA 9/12/18 enrich filtered lysate.”
10. Obtained two plates for a control TA, which was labeled “LEF KEA 9/12/18 Spot Test Control” and one for self, which was labeled “KEA 9//12/18 Spot Test.” Circles were also drawn to indicate where the enrich lysate and the phage buffer were placed.
11. Tripled the original recipe for performing a spot test.
12. Used a serological pipette with bulb with 10 mL tip to transfer 6.0 mL of LB Broth into a 50 mL conical vial.
    • Labeled this 50 mL conical vial “Team 2 9/12/18 Soil B TA.”
13. Transferred over with a serological pipette with bulb with 10 mL tip 7.5 mL 2X TA into “Team 2 9/12/18 Soil B TA” conical vial.
14. Used a P200 micropipette to add 67.5 μL CaCl₂into “Team 2 9/12/18 Soil B TA” conical vial.
15. Added 1.5 mL of Arthrobacter into “Team 2 9/12/18 Soil B TA” conical vial.
16. Used a serological pipette with bulb with 10 mL tip to transfer 5 mL of “Team 2 9/12/18 Soil B TA” mixture onto both “LEF KEA 9/12/18 Spot Test Control” and “KEA 9//12/18 Spot Test” plates and swirled to even out the TA.
17. Allowed 15 minutes for the TA to solidify.
18. Used a P10 micropipette to add 10 μL of phage buffer in the phage buffer circle on “KEA 9/12/18 Spot Test” plate.
19. Used a P10 micropipette to add 10 μL of the enriched lysate from the “KEA 9/12/18 enrich filtered lysate” microcentrifuge tube in the enrichedcircle on “KEA 9//12/18 Spot Test” plate.
20. Placed plates in incubator for the next 48 hours.
21. Cleaned lab counter with CiDecan and EtOH (70%).

Calculations and Metadata Soil Data:

Percent Water

Data Table

Equation for Percent Water

Percent Water = 9.65%

Percent Sand, Percent Silt, and Percent Clay

Data and Calculation Table

Percent Sand = 61.43%

Percent Silt = 17.14%

Percent Clay = 21.43%

Classifying Soil Texture

Based off the percent sand, percent silt, and percent clay, calculations using a soil texture triangle classifies the soil sample’s texture to be sandy clay loam. The point is plotted on the soil texture triangle below.

This image was retrieved from the Natural Resources Conservation Service website at https://www.nrcs.usda.gov/wps/portal/nrcs/detail/soils/survey/?cid=nrcs142p2_054167.

Calculations for the Amount of Materials Needed

Observations:

• “KEA 9/10/18 Soil B enrich” conical vial had a mass of 22.28 grams before DI water was added. After DI water was added, “KEA 9/10/18 Soil B enrich” conical vial had a mass of 22.32 grams.
• In the “KEA 9/10/18 Soil B” falcon tube, the levels of sand, silt, and clay were not straight across. The following photos illustrate how the levels tended to shift towards one side of the falcon tube than the other causing there not to be straight define lines of where exactly the silt starts and stops.

• As shown above, the sand had a light brown color. The silt had a dark gray tint. The clay had a dark cinnamon-brown color.

Next Steps:

Next time, check the spot test to see if there is any contamination, positive, or negative results. If contaminated, I will run another spot test. If not, I will run a plaque assay with the “KEA 9/10/18 Soil B enrich” lysate. The results from both the spot test and plaque assay will ultimately determine whether or not I have to collect more soil.