By Taylore King
Much has changed since my blog post concerning the plethora of phage plaques that erupted from my first attempt at direct plating.
I now have an isolated phage specimen.
After seven rounds of T-streaking, contaminated plates, and the burning question of whether or not the plaques represented one or two phage morphologies, my efforts were successful and I prepared my Medium Titer Lysate dilution series. I plated dilutions ranging from 10-1 to 10-7. After the plates were incubated for 24 hours, allowing phage to lyse the M. smeg bacteria, I found that the 10-2 dilution was the ideal dilution that would create the perfect web plate.
With this information, I began to prepare my High Titer Lysate (HTL). I diluted six solutions to 10-2 in order to form six web plates. After the plates were incubated for 24 hours, I flooded each plate with 8 mL of Phage Buffer (PB) and allowed the set of six plates to sit for an hour, “extracting” phage. I then collected the PB/phage mixture from all of the plates, pipetted it into a conical tube, and filtered the solution. This filtered solution was my final High Titer Lysate.
From my HTL, I transferred 1 mL (1000 μL) of PB/phage into a microcentrifuge tube using a micropipette and centrifuged the tube for an hour to form a small pellet of phage. I then removed 950 μL of the supernatant liquid and added 100 μL of new PB to the microcentrifuge tube and allowed it to sit for another hour dissolving the phage pellet.
With this final solution, I walked to a neighboring building with our Teaching Assistant and another student to the imaging lab that contained two electron microscopes. Here, I watched as a biologist prepared my phage solution for Electron Microscopy and began to image it using the thirty-year old electron microscope.
As he adjusted the resolution of the microscope, a phage was born. I cannot put my excitement into words as I saw the image of my phage form on the computer screen. All 198 nm of tail and 75 nm of capsid were worth the frustration of contamination and endless T-streaking.
I had isolated a phage.