Results:

Both the “LEF KEA 9/12/18 Spot Test Control” and the “KEA 9/12/18 Spot Test” plates were contaminated. There was a yellow ochre liquid present in both plates. This might be because the plates were accidentally placed right side-up and were not inverted. The pictures below show these plates and the liquid.

Rationale:

Since the spot test was contaminated, I decided to run a plaque assay with the enriched soil B lysate. This will reveal whether or not there are bacteriophages in the soil sample that target Arthrobacter.

Procedure:

1. Cleaned the counter area with CiDecan and wiped it dry. Then, cleaned with EtOH (70%) and allowed it to evaporate.
2. Performed the following calculations to determine how much of the materials were required.

Calculations

3. Through aseptic technique (over an EtOH (100%) flame), used a cartwheel serological pipette with 10 mL tip to transfer 4 mL of LB Broth into 50 mL conical vial.
   • Labeled this 50 mL conical vial “KEA TA 9/14/18.”

4. Added 45 μL CaCl₂ with P200 micropipette into “KEA TA 9/14/18” conical vial.
5. Transferred 5 mL of 2X TA using a cartwheel serological pipette with 10 mL tip into “KEA TA 9/14/18” conical vial.
6. Used a P10 micropipette to add 10 μL of the enriched lysate from the “KEA 9/12/18 enriched filtered lysate” microcentrifuge tube into the test tube with 0.5 Arthrobacter.
7. Allowed 10 minutes for the mixture to set.
8. Used a cartwheel serological pipette with 5 mL tip to separate 4.5 mL of the Top Agar mixture into another 50 mL conical vial.
   • Labeled this 50 mL conical vial “KEA TA 9/14/18 control.”

9. Obtained two plates, one to run the enrich plaque assay on, which was labeled “KEA PA 9/14/18 enrich lysate” and the other plate served as a control, which was labeled “KEA PA 9/14/18 control.”
10. Used a cartwheel serological pipette with 5 mL tip to add and mix the remaining 0.5 mL of LB Broth into “KEA TA 9/14/18 control” conical vial.
11. Used the same cartwheel serological pipette with 5 mL tip to transfer the mixture onto “KEA TA 9/14/18 control” plate and swirled to even out the mixture.
12. Once the 10 minutes for the Arthrobacter and enrich lysate were up, poured this mixture directly into the “KEA TA 9/14/18” conical vial.
13. Used a cartwheel serological pipette with 5 mL tip to mix and transfer the mixture in “KEA TA 9/14/18” conical vial onto “KEA PA 9/14/18 enrich lysate” plate.
14. Allowed both plates to solidify.
15. Placed both plates inverted in incubator at 48ºC for the weekend.
16. Cleaned lab counter with CiDecan and EtOH (70%).

Observations:

• The mixtures in both “KEA TA 9/14/18” and “KEA TA 9/14/18 control” conical vial had started to solidify. The “KEA TA 9/14/18” conical vial mixture more than the “KEA TA 9/14/18 control” mixture since it had to wait 10 minutes before putting in the Arthrobacter with enriched lysate.
• The solidifying caused many air bubbles to form when mixing as shown below in the pictures.

Next Steps:

On Monday, I will examine both plates. If the plates are contaminated, I will run another plaque assay. If the plates are negative, I will find new soil. If the plates are positive, I will move on to do purification.