Rationale

Today we will reconduct the plaque assay from 9/26/18 due to possible contamination in the Arthrobacter used by the class.

Procedure

• Established an aseptic zone.
• The enriched lysate produced on 9/19/18 had formed a pellet near the bottom of the vial, therefore the lysate was separated into two tubes of equal mass and was spun again for 10 minutes to reproduce a new enriched lysate. The supernatant formed after spinning was syringe filtered through a 22 µm filter.
• 8.4 mL of LB broth and 90 µL of 1M CaCl2 were added to a tube.
• 10 µL of the new enriched lysate was added to a vial containing 0.4 mL of Arthrobacter. It sat for 10 minutes.
• 2X TA was added to the first tube and 4.5 mL of the solution was added to the vial containing the new enriched lysate and 0.4 mL of Arthrobacter. The contents of the mixture were then poured onto a plate that sat for 10 minutes.

Observation

The cause of the pellets formed at the bottom of the enriched isolation from 9/19/18 is unknown. The proper technique was used, however the isolation was still unclear in color on 9/26/18. The TA poured onto the plate did not produce any air bubbles.

Conclusion

The results from the plaque assay will be observed again and a spot test will be conducted after.