Rationale: After finding contaminated results in the previous lab, the purpose of this lab is to run another plaque assay.

Description of Procedures:

1. The work station was cleaned using aseptic technique and an ethanol burner was lit.
2. A pellet was seen in the bottom of the enriched lysate, so the lysate was spun again in the centrifuge for 5 minutes at 10,000 x g.
3. While spinning, enough top agar solution was made for four plates. 90 ul of CaCl2 and 8.4 ml of LB Broth were added to a tube.
4. The lysate was then filtered using a 0.22 ul syringe filter.
5. Next, 10 ul of lysate was added to 0.4 ml of arthrobacter and was allowed to sit for 10 minutes.
6. 2x TA was then added to the top agar solution and pipetted up and down to mix. 4.5 ml of the solution was poured into the arthrobacter and then poured directly onto a plate labeled LIP 9-26-18 Plaque Assay. 4.5 ml of the solution was poured directly onto a plate labeled Control LIP EAG SS 9-26-18.
7. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator.
8. The work station was cleaned using aseptic technique and the materials were properly stored and disposed of.

Observations/Results:

• Observations
  • The enriched solution had a pellet at the bottom and was a murky color, so it was re-spun and re-filtered.
  • There were a few bubbles towards the center of the experimental plate.

Interpretations/Next Steps/Conclusions:

The experiment was complete. The next step will be to check for plaques in the next lab. If there are plaques, they will be picked. If not, a spot test will be run.

Contaminated Plates: