Rationale:

The purpose of today’s lab was to move towards obtaining a high titer of lysate for TEM and DNA extraction. Currently the lysate is both at a low volume and a low titer so flooding and spot titering is necessary to move forward with the experiment.

Results:

• The plate made with the lysate gathered from flooding last week had completely lysed, the plate with the 60 µL of diluted lysate had split. After closer examination it was determined that the plate had completely lysed, leaving the phage to die off, allowing for the Arthrobacter to recolonize and regrow onto the plate.
• It was decided to run another plaque assay with 3 different volumes of lysate instead of the 60 µL used last time to hopefully web a plate well enough to flood.
• 

  PA Made with 60 µL 11/07/18

  

  Flood TA Control 11/07/18

  

  60 µL TA Control 11/07/18

  

  FLood PA Regrowth 11/07/18

Materials

• LB Broth
• 2X TA
• Calcium Chloride
• Flooded Lysate
• Agar Plates

Procedure:

1. Established an aseptic zone
2. Ran 3 different plaque assay procedures with 3 different volumes of the flooded lysate: 10 µL, 15 µL, and 20 µL of lysate.
3. To run a plaque assay, 2.0 mL of LB broth was added to 3 separate conical vials as well as 2.5 mL of LB broth into a 4th vial for the top agar control.
4. After LB broth, 22.5 µL of calcium chloride was added to all 4 of the conical vials.
5. Then, 3 separate 0.5 mL of Arthrobacter was combined with the different volumes of lysate and left to infect for 15 minutes.
6. Once the 15 minutes were up, the infected Arthrobacter was combined with the LB Broth and 2.5 mL was added to each of the conical vials.
7. After combining, the top agar was then plated immediately into their respective plates and left to solidify for about 15 minutes.
8. Once the 15 minutes were up, the plates were then flipped and stored in the incubator.

Results and Data:

The experiment went with no errors and none of the components appeared to have any visual signs of contamination during the experiment. The top agar did not have any bubbles during plating, as a new procedure involving the serological pipette was used to plate the top agar.

Conclusions:

The difference of lysates included into the top agar should reduce the presence of phage particles in the top agar, increasing the probability of having a properly webbed plate. If the plates do lyse again, they will need to be immediately flooded to capture the phage particles present before the phage dies off and Arthrobacter regrows and recolonizes.

Next Steps:

The next steps for this experiment are to flood the plates as soon as possible to obtain as many phage particles from the plate as possible. The lysates gathered will then all be combined into one conical vial to gather a high volume of lysate to calculate a titer from. The goal is to continue moving towards a high titer of lysate.