Rationale: Due to the aggressiveness of my phage I will have to dilute my lysate all the way to 10^-6. Also since there is not much arthrobacter available I will have to conduct a spot. test of each of the dilutions rather than a plaque assay for each.

Procedure:

1. Took 10µL of lysate and placed in a microcentrifuge tube labeled 10^0. Added 90µL of phage buffer.
2. Took 10µL of 10^0 sample and placed into tube labaled 10^-1 and added 90µL of phage buffer.
3. Repeated up until 10^-6.
4. Made top agar by mixing 8mL of LB Broth, 90µL of CaCl2, and 10mL of 2X TA.
5. Divided plate into 6 sections.
6. Added 4.5mL of solution to control plate.
7. Added 4.5mL of top agar to 0.5mL of arthro then plated.
8. Let plate sit for 15 minutes then. spotted dilutions 10^-1 to 10^-6.
9. Placed in incubator for 24 hours. Note: accidentally didn’t leave plate inverted.

Observations:

The spot’s were large and weren’t good plaque’s to pick from until 10^-6. The calculated titer according to the spot test is 2.1*10^9. Also a lot of spots had arthrobacter start to regrow in the center of the plaques.

Conclusions and Next Steps: My unpurified lysate has a high titer. I need to purify from here so I picked a plaque and I’m going to run a plaque assay with it and then go directly to trying to web a plate. I may also try to web a plate with the 10^-6 dilution.