Rationale: To attempt to isolate a phage by performing a plaque assay, after a negative spot test. This procedure will also finish calculating the soil concentrations of sand, silt, and clay.

Description of Procedures:

1. The work space was cleaned using aseptic technique and a 100% ethanol burner was lit to create an aseptic zone.
2. 10 ul of enriched lysate was added to 0.5 ml of arthrobacter. This was allowed to sit for 10 minutes.
3. Next, the top agar solution was made. One tube was used to make four 5 ml plates. 8 ml of LB broth and 90 ul of CaCl2 were added to the tube.
4. 2x Top Agar was then added to the tube and pipetted up and down to mix the top agar solution.
5. 4.5 ml of the solution was added to each of the groups arthrobacter and phage mixture, and immediately poured onto petri dishes. The remaining 4.5 ml was poured onto a control top agar plate. The experimental plate was labeled LIP 9-17-18 Plaque Assay. The control was labeled EAG, SS, LIP 9-17-18 Control Plaque Assay. The plates were then allowed to sit for 10 minutes.
6. While the plates were sitting, the soil metadata collection was finished. There were 7 ml of soil total, and it was found that 5.5 ml were sand, 0.5 ml were silt, and 1.0 ml was clay. The percent sand found was 78.57%. The percent silt was 7.14%. The percent clay was 14.29%.
7. After 10 minutes, the plates were inverted and placed in the incubator for 48 hours.
8. The work station was cleaned using aseptic technique and the materials were properly disposed of.

Observations/Results/Data:

• Observations:
  • There were small bubbles on the control plate, and one larger one on the edge.
  • There were 1-2 small bubbles on the control plate.
  • The soil had many layers of silt, all small, but different colors.
• Data:
  • % Sand: 78.57%
  • %Silt: 7.14%
  • %Clay: 14.29%

Interpretations/Next Steps/Conclusions:

The procedures were complete. The soil composition was found and the plaque assay, both the control and experimental, were completed. The next step will be to wait 48 hours to allow the bacteria to grow and plaques to form. If there are plaques, they will be picked. If not, then new soil will need to be found and the procedures will be started again.

Additional Questions:
1. Group 4’s Plaques: The group members in Justin’s group did not all collect soil from the same tree. The soil was from three different trees in the same area. This could account for the differing results. The soil Justin collected could have different phages from his group members, allowing him to have more and better defined plaques. However, the soil was most likely at least similar, as all three members of group 4 had plaques.

2. Lathan will need 4.01 ul to web his plate. (Work shown below).