Rationale:

By conducting different procedures, different soil metadata, such as percent water, percent clay, percent silt, percent sand, and pH, is gathered for later use to see if there is any correlation in the data. By filtering and enriching the sample, the sample will be prepared for a spot test and plaque assay which will reveal the presence of Arthrobacter phages.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Started enrichment process by filling a 15 mL conical vial with 2 mL of the soil.
   • Labeled the conical vial as “KEA 9/7/18 Soil B.”

3. Through aseptic technique (over an EtOH (100%) flame), filled “KEA 9/7/18 Soil B” conical vial with LB Broth to the 12 mL mark using a serological pipette with a bulb with a 10 mL tip.
4. Shook “KEA 9/7/18 Soil B” conical vial for 15 minutes.
5. Weighed “KEA 9/7/18 Soil B” conical vial.
6. Centrifuged “KEA 9/7/18 Soil B” conical vial at 10,000g for 10 minutes.
7. Started the soil metadata procedures by filling a falcon tube with 10 mL of soil.
   • This falcon tube was labeled “KEA 9/10/18 Soil B.”

8. Filled the “KEA 9/10/18 Soil B” falcon tube with de-ionized (DI) water to the 30 mL mark.
9. Added three drops of texture dispersion liquid to “KEA 9/10/18 Soil B” falcon tube.
10. Used a disposable latex glove to cover the “KEA 9/10/18 Soil B” falcon tube while shaking it for 30 seconds.
11. The “KEA 9/10/18 Soil B” falcon tube was placed under a flume hood to sit for 48 hours.
12. Weighed a petri dish.
    • Labeled petri dish “KEA 9/10/18 Soil B.”
13. Placed and weighed approximately 3.5 grams of soil onto “KEA 9/10/18 Soil B” petri dish.
14. Set “KEA 9/10/18 Soil B” petri dish under flume hood for 48 hours.
15. To test the pH, added a small amount of soil to a small pH vial.
16. Filled this small pH vial with DI water to the top.
17. Closed thumb over top of small pH vial and shook for 10 seconds.
18. Allowed 2 minutes for the small pH vial to settle.
19. Placed a small piece of pH paper into the small pH vial and left it in for 45 seconds.
20. Recorded the pH and washed out small pH vial with water.
21. Back to the enrichment process, the supernatant was then pipetted into a 0.22 μm top vacuum filter to separate the lysate.
22. The lysate was collected in a 50 mL conical vial.
    • Labeled conical vial “KEA 9/10/18 Soil B enrich.”
    • Only collected a little less than 10 mL of lysate so all was used for the direct enrichment. None was saved for isolation.

23. Added 0.5 Arthrobacter to “KEA 9/10/18 Soil B enrich” conical vial.
24. Placed “KEA 9/10/18 Soil B enrich” conical vial in incubator at 28 ºC for 48 hours.
25. The lab counter was cleaned with CiDecan and EtOH (70%).

Observations/Data:

• With the addition of the LB Broth, the conical vial weighed 19.33 grams.
• The empty petri dish weighed 7.58 grams.
• The petri dish with soil weighed 11.00 grams.
• The pH paper revealed that the soil sample had a pH of 6.0 as shown in the picture below.

• As the falcon tube settled, the sand became apparent as shown in the picture below.

• The supernatant was a light shade of yellow. The pellet of Soil B was a lighter brown than the Soil A sample. The picture below shows the “KEA 9/7/18 Soil B” conical vial after it was centrifuged.

Next Steps:

On Wednesday, finish procedures and conduct calculations to obtain the soil metadata for the percent water, percent sand, percent silt, and percent clay. Also, the lysate in “KEA 9/10/18 Soil B enrich” conical vial will be used to conduct a spot test.