Rationale:

The purpose of today’s lab was to check on the plates that were set in the incubator the day prior to observe them before any bacteria was able to recolonize. If the plates were lysed or highly webbed, they were to be flooded immediately to gather the lysate while phage was still present.

Results 11/12/18:

• Plates had almost completely lysed with very few indicators of any bacterial lawn surviving. This is good as it indicates high phage presence and it was decided to flood each plate and combine lysates to calculate titer in the next lab.
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  20 µL Plaque Assay 11/12/18

  

  15 µL Plaque Assay 11/12/18

  

  10 µL Plaque Assay 11/12/18

Materials:

• 24 mL of Phage Buffer
• Vacuum Top Filter
• Serological Pipette

Procedure:

1. Began by establishing an aseptic zone.
2. Added 8 mL of phage buffer to each of the three plates.
3. Once added, plates were set on the shaker to gather phage particles for approximately an hour and a half.
4. After time had passed, plates were removed from the shaker and a vacuum top filter was assembled under the fume hood of the lab.
5. The lysates from each plate that had been gathered from the flooding process was then poured and filtered through the 0.22 micron filter into a new conical vial.
6. Then the lysate gathered from 11/07/18 flood procedure was transferred to the new conical vial, yielding a total 25 mL of flooded lysate.
7. Lysate was then labeled and stored in the fridge for next lab.

Results/Data:

• Although 24 mL of Phage buffer was added to the plates, only 20 mL of lysate was recovered from the flooding process, the remaining 5 mL of lysate came from the 11/07/18 lysate that was combined with the other 3 floods. Lysates were flooded for a little over an hour and a half, allowing plenty of phage particles to be picked up from the lysed plate. This should increase the titer of the lysate and the large volume of lysate allows for DNA extraction to be possible once a high enough titer has been acquired.

Conclusions:

Once factor that could have contributed to the loss of about 4 mL of potential lysate is reabsorption of the phage buffer by the top agar. The reason why plates were flooded so quickly was to avoid any Arthrobacter regrowing and recolonizing as phage begins to die out due to the lysed plate. There should be still be phage present on the lysed plate, just unable to reproduce due to the lack of hosts available.

Next Steps:

The next steps for this experiment are to spot titer out the new combined lysate to determine a new titer after 3 separate floods.