From Puddle to Picture

by Alexandre Mason-Sharma

I first learned about phages in my seventh grade biology class, and ever since I’ve been fascinated by their almost alien appearance and traits, I even used them in a poem for an English class when I ran out of rhymes. There is something slightly unsettling about such a perfectly geometric, biological machine infecting live organisms in the billions, unobserved by even the strongest conventional microscopes.

Obviously then,  I was thrilled to hear about the JHU Phage Hunting Class, I even woke up at the ungodly hour of 6AM in the summer to be sure I would get a place during registration. Over the course of this class, I’ve learned a lot about lab technique that I am sure will help me far into the future, and done some amazing things in the process.

The year began with a confusing trek through some very deserted parts of a forest river I had no idea existed in the middle of Baltimore. Along with my friend Bettina, and a very unhelpful iPhone, we finally discovered the river our professors had told us would be the perfect place to find a good phage sample. Our enthusiasm waned somewhat however when we saw the Maryland State Health Department warnings about the water we were trying to reach… After some debate over whether phages from a horribly polluted river would be more or less awesome than the garden variety, we decided it would be in our own best interest to find a different spot. As a result, we settled on a rather unpleasant smelling puddle of rainwater and a shady patch of soil under a tree. Scooping up earth with butter knives, the samples were transferred to tubes and placed overnight in my suite’s mini fridge, much to my roommate’s surprise.

From then on, it was lab work, and the long hard process of isolating and purifying any potential phages in the sample began. It was at this point that my second sample from the tree was discarded, 22 micron filters apparently having some difficulty in dealing with thick slimy mud.

My first sample AMS1 filtered fine however, and was enriched and plated with M. smegmatis. After incubation and a week’s time, a tiny putative plaque was observed. This clear pinprick was alone on the plate and my initial expectations were low. I feared I would have to submit to ‘adopting’ the abundant progeny of some luckier phage hunter. Nonetheless, I streaked this dot and plated it. I also began an enrichment and dilution of my sample, centrifuging and diluting from  and then plating to observe.

My results were inspiring!

My 0, -1, and -2 dilutions all showed both clear and turbid plaques across the plates

These two morphologies were each approximately 3mm in diameter. To separate the two, my -1 plate was streaked onto two lines, one for the turbid plaque TPA and one for the clear CPB.

The lab protocol required 3 successive streaks to guarantee a single phage per plate, and it was at this point that my good luck began to run out. My first streak suffered E. coli contamination and was abandoned. The second attempt at streak 1 succeeded but unfortunately streak 2 was not incubated initially then after re-incubation suffered contamination, with no plaques observed on TPA. As a result, the streak 1 plates were flooded with phage buffer, the solution syringed out after 15 minutes and filtered. Streaking then began again. However, there were some strange properties of the new streaks. CPB plaques were smaller than typical and TPA showed only 1 putative plaque. On the second streak, normal morphology returned to CPB, however all TPA plates were clear. To cut a long story short, CPB streaked out fine for three runs whilst TPA disappeared and was discarded as an option.

As a result, by early October, I was left with a single phage CPB, ready to analyze. A plaque from my third streak of CPB was used to make 4 serial dilutions of 10:1 and plated, giving an initial titer of  pfu/ml

CPB was then re-designated AMS24 and a lysate harvested. This lysate was determined to have a titer of  pfu/ml. After several failed platings however, the lysate was discovered to have dropped to 7300 pfu/ml! A new lysate therefore had to be made from past plates.

After so many setbacks, the end of the semester was looming, and it was time for my 10 plate infection, or rather my 8 plate infection, 2 plate’s worth ending up on my jeans. Needless to say, top agar does not make for pleasant laundry. Moreover, the agar did not harden and so a second 10 plate was needed. Luckily this one worked and after flooding a high-titer lysate was harvested. My platings for titer calculations all yielded web plates. Good news for my titer, but bad news for the maths behind it. Diluting further and plating out gave a titer of   pfu/ml, more than enough to move onto the most exciting stage, electron microscopy.

Preparing my sample, we left the lab with a tub of ice and microcentrifuge tubes and walked across to another Dunning Hall. Preparation was a lot more interesting than I thought; nothing makes science exciting like liquid Nitrogen and Uranium compounds…

I had never seen an electron microscope before, and they’re certainly a lot more complicated than I imagined. Nonetheless, I was surprised by how easy a sample was to observe. Sitting in the darkened room, green lights reflecting off lenses and the steaming fog of Nitrogen coolant, with the hissing sound of airlocks I held my breath as a grainy black and white image crackled into view. Then I saw them, my phages, a semester’s work in a single image. They were a pretty average size and length, but the contrast from the stain was beautiful. There were also a surprising number of burst capsids present.

Sadly, there won’t be time for me to isolate my phage’s DNA, but I’m not complaining. I have some incredible electron micrographs of a virus I discovered in a puddle, and that’s an awful lot more than most college freshmen can say!

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