By Dillan Villavisanis
Digging up dirt around campus and hoarding it into plastic bags on the first day of college probably isn’t the best way to meet new people, as many of my other classmates were doing. But this was the first step in a yearlong journey in a class dedicated to identifying and characterizing a novel bacteriophage. Besides, it’s much easier to like phages than people (just kidding . . . kinda).
We started by utilizing a direct plating technique, in which the sample was filtered and immediately plated with the bacteria Mycobacterium smegmatis. These bacteria were plated with the potential phage mixture, in hopes of identifying plaques. Plaques indicate that phages have infected the bacteria, and the phage can be analyzed and further proliferated. I was forewarned that the chances of identifying a phage using this technique were smaller than the phages themselves, so I wasn’t disheartened when my plates failed to contain plaques.
We moved onto an enrichment culture technique, in which the soil sample was mixed with a variety of other solutions and incubated to promote the development of phages. While many of my classmates found success in this method, I only identified a single plaque, which turned out to be too small to proliferate. As a result, I obtained a new soil sample outside the Undergraduate Teaching Labs and began the enrichment culture process again.
Fortunately, this sample was rich with phage and provided many plaques. Now that I had identified a phage, I needed to isolate a single morphology. This is to say all the plaques should be of the same physical appearance, in diameter, shape, and color. This is important, as the goal is to isolate a single type of bacteriophage.
First, a dilution series was established to create a gradient of several concentrations of phages across several different plates. This would provide the optimal opportunity to select a single plaque to be streaked. After the dilution series, a streaking technique was used in which a single plaque was selected and streaked onto a new plate. This would create a plate of new phages, which could then be streaked from again. In doing this several times, the streaking patterns could be analyzed.
After streaking over six different times across a multitude of plates, a single morphology was identified. My particular morphology is that of many small plaques, that contain a slightly darker ring around them.
Currently in the lab, I am working to establish a Medium Titer Lysate, or MTL. This MTL will allow me to calculate the phage titer and to create an HTL. Phage Hunting has been equally fun as educational. I am excited to continue my work in the lab and see where the phage hunting leads me.