11.14.18 Dilutions and Spot Test for Titer

Rationale: Since plates overnight had lysed all of the arthrobacter on the plate, phage buffer was added to the plates to flood them to collect bacteriophage that was present on the plate. Today, it was necessary to process this by creating dilutions and running a spot test to determine titer, as it is necessary to discover whether or not a high titer lysate has been obtained.

Procedure:

1. Aseptic zone was established
2. Filter column was used to filter the phage buffer from the plate into a tube labeled “HMB from CEW 10^0 lysate 11/14”
3. Dilutions from 10^0 to 10^-9 were created by adding 90µL of phage buffer to 9 different tubes and adding 10µL of the previous dilution into the tube (10µL 10^0 into 10^-1, 10µL 10^-1 into 10^-2, etc)
4. 2mL of LB Broth was added to a tube containing 0.5mL Arthrobacter
5. 22.5µL of CaCl2 was added to the tube with Arthrobacter
6. 2.5mL 2X Top Agar was added to the tube with arthrobacter, then poured over a plate with 9 distinct sections labeled after briefly swishing
7. Plate was let sit for 10 minutes
8. 10µL of each dilution was added to plate on the respective section in the form of a drop
9. Plate was let sit for another 10 minutes, then placed in the incubator overnight.

Observations:

• 2X Top Agar used was not at the optimal temperature, which led to some slippage of the overlay. Many of the 2X Top Agars in the incubator were contaminated however, so options were limited.
• Drops seemed to absorb better than they have in previous spot tests, which could be a result of the problematic top agar overlay.
• Overlays that are likely to slip seem to be darker yellow in color and have more grainy surfaces.

Results:

• Plaque Assays from Monday (11/12) resulted in completely lysed plates through the 10^-3 dilution. This is possibly indicative of a high titer presence, which is the end goal of the experiment. Hopefully, as results are seen on Thursday (11/15) or Friday (11/16), it will be possible to view plates that confirm the presence of a high titer.
• 

Next Steps and Conclusions:
If the spot test is functional despite the slightly slipped Top Agar, it will be possible to view the results that show the presence of a high titer or not. If the overlay is not functional, it will become necessary to retry the overlay with a new combination of fresher, uncontaminated Top Agar and LB Broth. This will allow for definite conclusions to be made about the titer of the lysate.