Rationale

Today we will conduct a spot test to isolate bacteriophage and collect the remaining metadata on Soil Sample B.

Procedure

1. Mass the weigh boat labeled “EAG 9/10/18 %H2O Metadata” and calculate the percent water lost.
2. Use the aseptic technique to clean the area. With gloves on, spray CiDecon on the lab table and wipe dry. Then wipe 70% EtOH on the surface and let the excess evaporate.
3. Obtain your enriched sample created on 9/10/18 and mass it.
4. Spin the 50 mL conical vial labeled “EAG 9/10/18 Enriched Isolation” at 3000 g to pellet Arthro for 5 minutes.
5. Using a syringe and a 0.22 μm filter attached to the end of the syringe to filter 2 mLs of the supernatant produced into a micro test tube.
6. Label a petri dish by dividing the dish into 4 quadrants. Label one quadrant “EAG 9/12/18 Spot Test”, another quadrant “LIP 9/12/18 Spot Test”, another quadrant “SS 9/12/18 Spot Test”, and the last quadrant “9/12/18 Control.”
7. In a 50 mL conical vial labeled “9/12/18 EAG,SS, LIP Spot Test,” pipet 2 mLs of LB Broth in the vial using a 10 mL pipet and a suction device.
8. Micropipette 22.5 μL of CaCl2 into “9/12/18 EAG,SS, LIP Spot Test.”
9. Add 0.5 mLs of Arthrobacter into the 50 mL conical vial and quickly add 2.5 mLs of 2X Top Agar using a 5 mL pipet and a suction device. Lightly mix the tube and then pour into the petri dish.
10. Let the plate sit for 10 minutes.
11. Using a 5 μL micropipette, pipet EAG, SS, LIP, and phage buffer into four different quadrants in the plate.
12. Let the plate sit for 10 minutes and then place into the incubator.
13. Begin collecting the remaining metadata by determining the percent sand, silt, and clay of Soil Sample B. Obtain a 50 mL falcon tube and label “EAG 9/12/18 Sand/Silt/Clay Metadata”
14. Place 10 mLs of soil into the falcon tube and fill with DI water to the 30 mL line.
15. Add 3 drops of soil dispersion liquid and shake vigorously for 30 seconds. Let the falcon tube sit in the fume hood for 48 hours.

Observations

• Air bubbles occurred when pouring the Top Agar to the plate. Two air bubbles were present near the center of the control quadrant and one air bubble was present near the edge of quadrant “EAG 9/12/18 Spot Test.”
• When collecting the metadata on percent sand, silt, and clay of Soil Sample B, soil clumped up at the bottom of the falcon tube and the was not able to be mixed properly.
• The mass of the weigh boat is 7.192 g. The % water in the soil is 46.9%.

Conclusion and Next Steps

We will let the spot test and the sand/silt/clay tubes sit for 48 hours. The spot test results will be observed in hopes of plaque presence and the percent sand, silt, and clay will be determined.