11.5.2018 Plaque Assay for High Titer Lysate

Rationale: Since the plaque assays from Friday (11/2) were visualized and calculated to have a titer of 6.8×10^7, it was found to be necessary to use lysate from Friday to create multiple plates of 10^-2 dilution to be flooded to obtain a lysate of higher concentration.

Procedure:

1. Established an aseptic zone.
2. Added 10μL of 10^-2 lysate to 5 different 0.5mL Arthrobacter. Let sit for 10 minutes.
3. Obtained 5 plates and labeled “CEW 11/5”.
4. Added 10mL LB Broth to conical tube.
5. Added 112.5µL CaCl2 to conical tube. Swished to mix.
6. Added 2mL of solution from conical tube to each tube with 0.5mL arthrobacter and lysate.
7. Added 2.5 mL 2X Top Agar to each tube. Swished and poured onto 5 separate plates.
8. Added 2mL LB Broth, 22.5µL CaCl2, and 2.5mL Top Agar to conical tube
9. Poured all tubes onto 6 separate plates (1 plate was top agar control and was labeled accordingly).
10. Let sit for 10 minutes before placing them in the incubator.

Results:

• The lysate obtained had a concentration of 6.8×10^7 pfu/mL. This was a medium titer. The results of the plates created on Friday (11/2) are shown below.
• There was very slight contamination found on the top agar control.
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Observations:

• Two of the five plates had top agar overlays that did not set correctly, which led to the overlay slipping on the plate. This was likely caused by small variations in the 2mL additions from the conical tube to the individual tubes with Arthrobacter. The other three plates were set correctly and should be able to be used as normal.
• Lysate was three days old when it was used. After usage, it was made known by Dr. Adair that it may not have the same effectiveness due to the degradation of phage. This may impact the results that are able to be seen on the plate.

Conclusions/Next Steps:

• Since the lysate did not have a concentration of 10^8 or more, it was not able to be used as a high titer lysate. However, it is getting closer to that value, and with the plaque assays done today it will likely be possible to flood the plates to obtain a lysate with a higher titer. Thus, it will be necessary on Wednesday to flood the functional plates and use the new lysate obtained to create new dilutions that would theoretically contain phage at a higher concentration than previously obtained.