November 10

Flooding a Plate, Serial Dilutions, and Webbing a Plate (11/9/18)

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Results:

No contamination appeared on the plates, and just as expected no plates were webbed as shown below.

Rationale:

The “KEA 11/7 PA 100150 µL” plate will be flooded and filtered out. Plaque assays will be performed using 400 µL, 450 µL, and 500 µL of the “KEA 11/5 FS lysate 100” and also 15 µL and 10 µL of 100, 10 µL of 10-1, 10 µL of 10-2of the new lysate with a goal to receive a webbed plate.

Procedure:

  1. Once an aseptic zone was established, 4 mL of phage buffer was poured onto “KEA 11/7 PA 100150 µL” plate.
  2. The plate was shaken on an incubator for one hour.
  3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/9 FS lysate 100.”
  4. 10 µL of “KEA 11/9 FS lysate 100” was added with 90 µL of phage buffer to create 10-1dilution which was vortexed.
  5. 10 µL of “KEA 11/9 10-1” was added with 90 µL of phage buffer to create 10-2dilution which was vortexed.
  6. 400 µL, 450 µL, 500 µL of “KEA 11/5 FS lysate 100” and 15 µL of 100, 10 µL of 100, 10 µL of 10-1, and 10 µL of 10-2of the new lysate were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
  7. 16 mL of LB Broth, 180 µL of CaCl2, and 20 mL of 2X TA were combined into a conical vial.
  8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
  9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “KEA 11/9 Control.”
  10. These plates were placed in the incubator at room temperature.

Observations:

  • The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl2needed for 8 plates.

Original Recipe

 X8

2 mL LB Broth

 16 mL LB Broth
2.5 mL 2X TA

20 mL 2X TA
22.5 μL CaCl2

180 μL CaCl2
  • Air bubbles formed when pouring the plates.

Next Steps:

The best-webbed plate from the plaque assays performed today will be flooded to collect more lysate with a goal to reach a high titer. Also, with the lysate created today, the titer will be calculated. The goal is to have a high titer by Wednesday, the last day to view phages under the transmission electron microscope.


Posted November 10, 2018 by Kathryn Adkins in category Kathryn Adkins

About the Author

Kathryn Adkins is currently a freshman attending Baylor University majoring in neuroscience with a minor in biochemistry.  She hopes to one day earn an M.D./Ph.D. and become a pediatric oncologist and cancer researcher. Kathryn volunteers at Cook Children’s Hospital in Fort Worth and is actively involved in AMSA (American Medical Student Association) and BURST (Baylor University Research in Science and Technology).

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