Rationale:

To create a gridded multi-spot test in order to determine the titer of the lysate produced from the flooding of the plates from 11.12.18.

Procedures:

1. Established an aseptic zone.
2. Filtered the lysate from the four plates flooded by Dr. Adair using a .22µ vacuum filter to produce 10^0 lysate.
3. Diluted 10^0 to create dilutions down to 10^-9 using the procedure of 90µL phage buffer and 10µL of the next highest dilution.
4. In a vial of .5mL of arthro, added 2mL of LB broth and 22.5µL of CaCl2.
5. Added 2.5mL of 2xtop agar and plated on a previously grid labeled plate with 10 boxes for the 10 different dilutions, let solidify 15 minutes.
6. Spotted 5µL of all dilutions onto the gridded plate in their respective boxes.
7. Created a top agar control using 2mL LB broth, 22.5µL of CaCl2, and 2.5mL of 2xtop agar, plated it.
8. Let both plates solidify for 15 minutes then incubated.

Observations:

The plates produced on Monday were all completely lysed as of Tuesday, Dr. Adair proceeded to flood them in order to obtain the phage before it died off, this is what was filtered today. The top agar appeared to be solidifying more quickly than in previous plaque assays and spot tests.

Conclusions/Next Steps:

The next step will be to check the spot test on Friday and from there determine the titer of the lysate that was produced and used to create the spot test. From there it can be determined if the lysate is a high enough titer to be used for TEM and DNA extraction and analysis.