By Laura Hinsch
The scene of this tale opens upon a brightly lit laboratory on a warm September day. Fresh pressed blue lab jacket and smudge free goggles in hand, I am ready to take on the fascinating challenge of hunting for phages. At this point, let it be noted that I had not taken a class where I got to work in any sort of lab for two years. Of all the equipment that was placed in front of me on those lab tables, that first day, only half of it had I ever seen or used before in my life. Unprepared for the challenge of the day, I embarked on the preliminary task of direct plating. The frustration I felt that first day was immense. It took me hours to complete the task asked of me. Aseptic technique was a concept I struggled to master and I got more bubbles then phage on my plates. I was certain after that first lab that I was going to be a failure. However, I liked the content of the lab enough to try again. I had to get better the second time, right? On the second day, I discovered that direct plating was a failure, so I went ahead and performed an enrichment of my sample. I was told that this would most likely yield results, but I am stubborn and was convinced that it was my poor lab technique that had destroyed my direct plating sample. With that, I decided to attempt direct plating once more with a different sample. Despite my desperate attempts to ensure good technique, the direct plating was still a failure. In contrast, the enrichment sample worked and my agar plates were growing a beautiful array of phages. The most dilute plate was covered in a variety of phage morphologies. My plates were bursting with so many morphologies and phages that I was lucky to have any identifiable phage morphology at all.
Jump forward several weeks and I have a nice morphology working in my favor. It’s small, 1-2 mm in diameter and lytic. I have completed three rounds of streaking with consistent morphologies and I’m ready to generate my MTL (medium titer lysate), which means I am more or less ready to purify my phage. I touch a micropipette tip to one of my morphologies, complete the dilution, and let it incubate. When I return several days later, to my horror my phages seem to have disappeared! My 100 dilution and my 10-1 dilution plates are almost entirely lysed, as expected, however my 10-2 plates and lower were not showing the consistent reduction in amount of phages that they should. The phages went from too many to count, to between twenty and fifty, to none at all! I struggled to understand how I could go from too many phages and morphologies to barely enough. Luckily, after another round of dilutions and the generation of and MTL (with the help of some math), my phages once again reappeared in the form of a beautiful webbed plate.
Overall, in this lab I have gathered a fantastic toolbox of lab techniques as well as, a greater appreciation for the ever existing and propagating bacteriophages that inhabit our environment and surroundings.