Rationale:

The purpose of today’s lab was to continue the purification process with the phage particles gathered from the previous serial dilutions. If plaque assay results were positive, plaques would have been further picked to purify. If results were negative, plaque assays were to be rerun.

Results from Lab 10/17/18:

• As expected, top agar split and slid, giving no clear indication of phage presence. This was due to the irregular solidification of the top agar during plating the week previous.
• Plaque assays had to be rerun, as the results were inconclusive.
• 

Materials:

• LB Broth
• 2X TA
• Diluted Lysates
• Agar Plates
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2.  3 separate plaque assays were run with each dilution as well as one top agar control.
3. To begin, 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL added to a fourth top agar control vial.
4. Next, 22.5-μL of calcium chloride was added to the each of the conical vials.
5. 10-μL of each of the dilutions were combined with 3 separate 500-μL quantities of Arthrobacter and left to infect for appro.
6. After about 15 minutes, the diluted lysates were combined with their respective labeled LB Broth mixtures and 2.5-mL of 2X TA was added to the all 4 of the broths.
7. Swirled and plated top agars immediately and left to solidify.
8. Once solidified, they were flipped and added to the incubator until the next lab.

Results/Data:

• Top agar and LB Broth used were madd two days prior and contained no evidence of contamination. Also, top agar was not solidified before the top agar pouring process. This lead to a normal solidification of the top agar once plated and should not slide or split.

Conclusions:

• It can be concluded that this previous will yield accurate results in the phage dilution. Top agar did not seem to show any indication of splitting.

Next Steps:

• The next steps are to analyze the top agar results in approximately 48 hours. If the results are plaque positive, new plaques will be picked to get a high titer of phage and continue the purification process. If no plaques are present, then new diluted lysates will be made to rerun plaque assays.