A Phage That Refuses To Be Isolated

By Justin Yeh

On my first day of Phage Hunting, I recall walking into a lab that was saturated with expensive equipment, bright lightning, and scientific knowledge. Awed by what I saw, I was humbled to be able to take part in a freshman research class at Johns Hopkins. Despite the hardships I have faced so far in class and the cold mornings that I have to go through to fight my bed sheets off my body, I don’t mind getting up a bit earlier to go to my favorite class in college.

As the name of the course conspicuously states, the objective of Phage Hunting is to isolate a phage from a soil sample, purify it, take a picture of the phage, and to hopefully sequence it. But this can only start by obtaining a soil sample, which I got from a soil patch near a tree by the tennis courts behind AMR I (yes, a very specific location). After placing a spoonful of my sample in a tube and flooding it with Phage Buffer, I extracted the supernatant, combined it with M. smegmatis – the bacteria that the phage will infect – and plated the live solution. This process was the direct plating of a soil sample without enrichment (which we will get into later).

So the next meeting of PH, I discovered that I had agitated my Top Agar and could neither see any phages – if there were any in the first place – nor a healthy bacterial lawn. I was frustrated, yet incredibly charmed by the delicate nature of biology based labs. But soon enough, the frustration evaporated.

On that day, the class took a field trip to a Community Garden near JHU to obtain a second soil sample, which will be mixed with an enrichment solution to encourage phage propagation. Needless to say, the enriched sample will have a higher chance of containing phage than an un-enriched sample, like the first sample I took. Scrambling back to the lab with bags of soil, the enrichment process began. And just as the enrichment process had promised, I observed phages on my plate in the next meeting.

The next step was to isolate the phages by morphology. To start off, a process called streaking is performed. It is not the hardest procedure, but it can take several trials, costing many plates, Top Agar, and wooden sticks. Long story short, I spent several weeks trying to obtain plates with uniform morphology. Had I went to a school without adequate funding, I would have run the institution beyond bankrupt within a course of two weeks. Anyway, my enrichment plates yielded two different morphologies: large cloudy plaques and small, clear plaques. And I streaked my posterior end away for those two and a half weeks in an attempt to homogenize the plaques. The large plaques were corrupt from time to time and often decided to have small plaques piggy back on the plate. But on a side note, the positive side to the streaking weeks was that I was the first one in the FFC lunch line for two weeks.

So, I was able to isolate the large plaques better than I could the small ones – and therefore the large plaque producing phage were chosen to be used in the titer assay, and medium titer assay, the step which I am currently on. And yes, I admit, I am kind of behind schedule; but I am happier than ever that I have homogenized my phage population. So therefore it is not too hard to figure out why I have titled my blog posting “A Phage That Refuses To Be Isolated.”

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