Rationale

Wash the soil sample collected from section 3 of the Baylor campus quadrant map and produce a direct and enriched isolation from the lysate created.

Procedure

1. Clean the work area by using the aseptic technique. While wearing gloves, spray CiDecon onto the lab table and wipe dry. Spray 70% EtOH onto the table afterwards, wipe, and allow the excess to evaporate.
2. In the 50 mL conical tube labeled “EAG 8/22/18 Soil C” containing 15 mLs the soil sample collected, add approximately 20 mLs of LB broth or enough to fill the 35 mL line on the tube while working near an ethanol flame.
3. Shake the 50 mL conical tube vigorously for 15 minutes.
4. Measure the mass of the 50 mL conical tube by using an analytical balance.
5. Using a centrifuge, spin the 50 mL conical tube for 5 minutes on a 3000xg rotations/minute setting.
6. Filter the supernatant into another 50 mL conical tube labeled “EAG 8/22/18 Direct Lysate” by using a 0.22 μm top filter attached to a vacuum. (note: only 5 mLs of lysate was produced using the 0.22 μm top filter and an alternate method was then used to filter the remaining supernatant)
7. Place the remaining unfiltered supernatant from the top filter in two 15 mL tubes. Make sure that the mass of the two tubes are equal.
8. Spin the two 15 mL tubes in the centrifuge again with help of a TA for approximately 10 minutes.
9. While working near an ethanol flame and using a syringe, suction 2 mLs of supernatant from one of the two 15 mL tubes and then attach a 0.22 μm filter to the end of the syringe.
10. Manually push down on the syringe to filter the supernatant into the 50 mL conical tube renamed as “EAG 8/22/18 Enriched Isolation” until the volume on lysate in the tube reaches 10 mL.
11. Pour 5 mL of the lysate from the 50 mL conical tube labeled “EAG 8/22/18 Enriched Isolation” into a 15 mL tube labeled “EAG 8/22/18 Direct Lysate.”
12. In the 15 mL tube labeled “EAG 8/22/18 Direct Lysate,” pour in 0.5 mL of Arthrobacter. Give the tube to the TA’s.
13. Place both tubes “EAG 8/22/18 Direct Lysate” and “8/22/18 Enriched Isolation” into the fridge.
14. Clean the lab station using the aseptic technique (step 1).

Observations

After LB broth was added to the initial soil sample in the tube labeled “EAG 8/22/18 Soil C,” the tube had a mass of 53.51 grams. Despite being spun in the centrifuge for 5 minutes on the 3000xg setting, the supernatant that was produced in the 50 ml conical vial was still foggy and dark colored. After being put in the centrifuge a second time, the supernatant was significantly lighter in color and was easier to filter.

Conclusion and Next Steps

Since I was able to make a direct and enriched isolation, coming in on 8/24/18 was not needed. On 8/27/18, the enriched isolation will be used to make a spot test.