Rationale: To isolate bacteriopages through a spot test, and to collect more metadata for soil sample B.

Description of Procedures:

1. The workstation was cleaned using aseptic technique, and an aseptic zone was created.
2. The enriched lysate sample made on 9-10-18 was spun in the centrifuge for 5 minutes at 3000 x g. The mass before spinning was found to be 20.49g. The enriched sample was spun to form an arthro pellet, to help separate the arthrobacter from the lysate.
3. While spinning, the percent water of the soil was calculated. The mass of the dry soil with the weigh boat was found to be 6.896g. From this, the mass of the dry soil was found to be 4.54g. The original mass of the soil was 4.91g. Subtracting the original mass from the final calculates the mass lost to be 0.37g. Dividing the difference by the original mass and multiplying by 100 tells us that the percent water in the soil was 7.5%.
4. To set up the spot test, one plate was divided into four quadrants, one for each group member and one for the negative control. My quadrant was labeled LIP 9-12-18 ES (Enriched sample).
5. Next, approximately 1.5 ml of the enriched sample was filtered into a 15mL tube labeled LIP 9-12-18 Filtered EL (Enriched Lysate). A 0.22 ul syringe filter was used to filter the lysate.
6. To make the top agar, 2.0 ml of LB broth was pipetted into a 50 mL tube. 22.5 ul of CaCl2 was added to the LB broth, as well as 0.5 ml of arthrobacter. Next 2.5 ml of the top agar was added to the tube, and this solution was lightly mixed. It was then immediately poured on to the petri dish and allowed to sit for 10 minutes.
7. While the plate settled, the percent concentrations of sand, silt, and clay test was started. The vial was labeled LIP 9-12-18 % Soil C (Composition).
8. 10 ml of soil was added to the vial. The vial was then filled to the 30 ml mark with DI water.
9. After 10 minutes of sitting, the top agar was settled and the procedure for the spot test was completed. 5 ul of lysate was spotted into the correct quadrants for each group member. 5 ul of phage buffer was spotted into the negative control quadrant. This was then allowed to sit for another 10 minutes.
10. Three drops of dispersion liquid were added to the 50 ml vial containing the soil. The solution was shaken vigorously for 30 seconds. The tube was then put under the vent hood to allow it to sit until the next lab.
11. The workstation was cleaned and the materials were properly stored and disposed of.

Observations/Results/Data:

• Observations:
  • The TA had some bubbles when it was settled.
  • The soil clumped to the bottom of the vial of the percent composition tube while mixing.
• Results:
  • % H2O: 7.5%

Interpretations/Next Steps/Conclusion:
The procedure for the spot test was complete. In the next lab, the % composition of the soil will be determined. Also, the plate will then be checked for plaques.