Rationale: 

In today’s lab, the goal was to analyze the plaque assay results and continue with the purification process. This was the third round of plaque picking with no positive results yielded. If results from 10/24/18 were negative, aspects of the experiment would have to be modified.

Results from 10/24/18:

• Plaque assay results for all dilutions were negative, with no contamination on the top agar control.
• Phage particles could possibly be diluted out since original lysates were not very concentrated to begin with. Decided to increase the amount of lysate used in plaque assay procedure to give more phage particles in the dilution. Did not adjust any other part of the top agar solution and did not adjust any concentrations.
• 

  GJA 10^0 Dilution 10/24/18

  

  GJA 10^-1 Dilution 10/24/18

  

  GJA 10^-2 Dilution 10/24/18

  

  Top Agar Control 10/24/18

Materials:

• LB Broth
• 2X TA
• 50-µL of Diluted Lysates
• Agar Plates
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2. Gathered original lysate from 10/03/18 enrichment and filtered through 0.22 micron syringe filter
3.  3 separate plaque assays were run with each dilution as well as one top agar control and one plaque assay with the new filtered lysate.
4. To begin, 2.0-mL of LB broth was added into 4 separate conical vials and 2.5-mL added to a fifth top agar control vial.
5. Next, 22.5-μL of calcium chloride was added to the each of the conical vials.
6. 50-μL of each of the diluted lysates were combined with 4 separate 500-μL quantities of Arthrobacter and left to infect for approximately 15 minutes.
7. After about 15 minutes, the infected diluted lysates were combined with their respective labeled LB Broth mixtures and 2.5-mL of 2X TA was added to the all 5 of the conical vials.
8. Swirled and plated top agars immediately and left to solidify.
9. Once solidified, they were flipped and added to the incubator until the next lab.

Results/Data:

• Plaque assay procedure went well with no complications. The increase of lysate by 40-µL is almost negligible for the final concentration of the 1M calcium chloride and 2X TA, so those values were not adjusted. The original enriched lysate was a dark red color, seemingly contaminated from a airborne bacterium that had been found contaminating other lysates as well. Also, for the 10^-2 dilution a different top agar had to be used since there was no more left to use.

Conclusions:

• The increasing of the lysate should be able to increase the phage population to combat the serial dilution. Original phage concentration could be weak due to the small and few plaques present from the plaque assays run with the original enriched lysate. There is a possibility of the 10^-2 dilution being contaminated or not being similar to the rest of the plaque assays run as it was made with a totally different top agar.

Next Steps:

The next steps for this experiment are to check the plaque assay results in the next lab. If the dilutions are positive for plaques, the purification process will be continued by picking a plaque from one of the dilutions to further purify. If no plaques are present, then plaque assays will be rerun with an even higher lysate volume.