Phage Phuns aren’t even that Phunny

My apartment is next to a hiking trail marked by two large boulders. This trail leads into Yang Ming Mountain National Park and connects to a complex network of trails that weaves throughout a dormant volcano. As I hike this trail, streams of sunlight penetrate the thick canopy and gently light up the thin layer of fog that permeated the entire forest. Growing up within the borders an of national park, I am no stranger to nature. In fact, my passion for science was instilled by the rainforest that surrounded me. Prior to the semester-long phage hunting class, I was enrolled in a decade long class that involved hunting for the various insects that lived in the trees and under the rocks of the forest. Digging in the soil, I would find myself returning home with the usual earthworm or, when I’m lucky, a grub that would eventually metamorphose in a stag beetle.

While I thought that my usual outdoor excursions would be cut-short by my move to Hopkins, I was proven wrong before I even started the first day of class. Opening up my email, I saw a message from Dr. Fisher detailing a sample collection process for potential phages in soil. We were to venture out in search of the most interesting soil samples we could find in hopes of uncovering new and exciting phages. The similarities to what I’ve been doing were uncanny.

To those unfamiliar to it, nature can seem quiet and lifeless. However, years of exploration have open up my eyes to a lively and exotic world. In almost every puddle, tree branch, or clump of soil, one can find some sort of organism living in its niche environment. What I didn’t realize before was that this under-appreciated world was larger than I could have ever imagined.

To identify or even “see” the myriad of biological entities that roam the micro-environments of the soil involves a little more than just careful observation. Phages are, if anything, extremely small and plentiful, making it hard for a single individual to be characterized. To start, the soil sample I collected was shaken up with an enrichment broth solution and filtered for a direct isolation solution. This solution was then plated with Mycobacterium Smegmatis. Ideally, a reaction between the phages and the Smegmatis would cause a plaque to form. This, however, did not occur in the first four weeks of class. For weeks, I would come back to class only to find a contaminated, disrupted, or perfectly Smegmatis covered plate.

After four weeks of futile attempt after futile attempt, I came to the conclusion that two variables had to be changed. First, I started using a new soil sample. The sample I had first collected did not yield any results, leading me to question that quality of the sample. I decided to collect new samples directly from the grounds right outside the undergraduate teach lab. Second, I also learned to be more efficient and effective with my plating procedure. I discovered that the time it takes for the agar to set can be longer than expected. Aseptic technique is also a very crucial step to execute correctly in order to yield non-contaminated plates.

Armed with the knowledge and experience, I set out round two with high hopes. With the new soil sample, I filtered and did a serial dilution. Then, I directly pipetted 30 microliters of filtrates onto marked spots of my different plates. After 48 hours, I observed that there were plaques forming on the marked spots! Finally, after four weeks of work I finally got to see my phages show themselves for the first time.

With the plaques, I can now finally start the streaking process to isolate a single phage. Unlike my bug collecting adventures as a child, the process of finding a viable plaque was a long but very rewarding one. The journey has only begun, however, as generations of streaking is required to isolate a single phage sample.

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2 Responses to Phage Phuns aren’t even that Phunny

  1. barronchiu says:

    Were you able to find a way to “extract” pure colonies from infected plates? I was able to do this from a tip by Jake. You can take rogue uninfected colonies to eventually achieve a completely uninfected plaque.

  2. vickyytwang says:

    “This, however, did not occur in the first four weeks of class. For weeks, I would come back to class only to find a contaminated, disrupted, or perfectly Smegmatis covered plate.”

    As someone who also had trouble with attempting to purify a phage, I understand all too well your struggles with plaque detection and identification. It was very comforting to know that another phage hunter was in the same boat as me (for at least a month of class)!

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