11.19.18 TEM Preparation and Redo of Titer Calculation

Rationale: Since the titer calculation plate from the last lab did not display results due to a slipped top agar, it was found to be necessary to redo this calculation to ensure a high titer lysate was present. However, it was found to be possible to continue with the procedure of TEM as each other individual that used Claire’s base lysate had found a high titer lysate from the last round of amplification. Therefore, since we all did similar procedures with similar results, it was concluded that there was a possibility that I too had a high titer lysate.

Procedure:

1. Aseptic zone established
2. 100µL of lysate was added to a microcentrifuge tube
3. Parafilm was placed on petri dish
4. 15µL of lysate was added to the parafilm at the center
5. 15µL of water was added to the parafilm to the right of the previous drop two times
6. Uranyl Acetate was added to the parafilm at the rightmost point.
7. Copper mesh was placed on the lysate for 5 minutes
8. Mesh was transferred to first water drop for 2.5 minutes
9. Step 8 was repeated with second water drop
10. Mesh was transferred to uranyl acetate for 1 minute, then dried with filter paper. Placed in B8.
11. 2mL of LB Broth was added to a tube with 0.5mL of arthrobacter and an empty conical tube
12. 22.5µL of CaCl2 was added to both tubes
13. 2.5mL of 2X Top Agar was added to both tubes, then the solutions were swished and plated on respective tubes.
14. Original lysate was used to create dilutions from 10^0 through 10^-9. These lysates were added to labeled sections on the experimental plate in 5µL drops
15. Plates were incubated overnight.
16. Station was cleaned

Results:

• Plate prepared on Wednesday (11/14) that was intended to show the titer of the lysate had issues with the consistency of the top agar. This was predicted to be due to the temperature of the agar when it was used. The plates created today seemed to confirm that the top agar had been allowed to cool for too long, which likely caused the problems with Wednesday’s plate. The same problem was not encountered today.
• Lysate titer will be reported as the plate created in this lab session is examined this week.

Observations:

• Top Agar and LB Broth was very clear and was made fresh. This will help reduce the chances of contamination.
• Drops did not settle into plate for spot test. Therefore, it was found to be necessary to place the plate in the incubator right side up with instructions to avoid flipping or moving the plate to give the drops time to settle.
• With the TEM preparation, there seemed to be confusion regarding shiny-side up and shiny-side down with the copper mesh. Any adverse results could have been caused by this confusion.

Conclusions:

• Since the top agar worked today after being taken fresh from the incubator rather than from the table, it can be concluded that the temperature caused the problems that were seen in the previous top agar. The TEM will allow for bacteriophage visualization, and the plate run today will confirm whether or not a high titer lysate has been used for the TEM.