Dirt, Contamination, and a Lot of Mulch

I have to be honest. Phage Hunting was definitely the class I was most excited about before I arrived on campus, and it still is.

Our first assignment was to collect dirt. Procrastination is bad, don’t get me wrong, but I just could not get myself to go start digging up dirt in front of everyone. However, when I decided to conquer my fears of getting weird stares from people who do not know about phage hunting, almost everywhere I looked, there was mulch. There was some sand as well, but I was looking for dirt. After trekking around campus and analyzing the ground wherever I went, I finally found some dirt in the Bufano Gardens! And guess what? There was only a couple of people around. So, I found a nice area underneath a tree and happily brought my finally collected sample to class.

The first lab was definitely stressful since everything was new to me. I could definitely feel the heat from the Bunsen burner as I slowly learned how to plate and tried to avoid causing bubbles to form! And, I learned to wait 25 minutes for my top agar to set because disfigured plates are just not good.

I did not get any visible plaques on my direct plating, but I was hopeful for the enrichment dilutions! Sadly, when I removed the tape that held my stack of plates together and slowly placed each plate on the counter one by one, I saw nothing. No observable plaques in sight. One of my plates was even completely blank even though I know that I put top agar on it the lab before. I was pretty confounded and disappointed. All that meticulous work and no phages!

However, life goes on! I guess this could be an answer to one of those infamous interview questions about how I recovered from a failure. Well, I got up from my fall and went out to get more dirt on my quest for phages!

A couple of other students also did not get any phages, and we were told that there was an abundance of phages near the FFC (a dining hall). So, we headed outside, and I felt no shame as I collected dirt for science. Again, mulch is everywhere on this campus, and we finally found a spot near the side of the FFC, just dug down underneath the mulch, and collected the sought after dirt. We returned to lab, and I prepared my enrichment for Sample 2.

Two labs later, I got my stack of taped plates and started slowly placing them on the table as suspension built up.


I looked at the first plate, and I don’t think anyone has ever been happier to see bacterial contamination. I knew it didn’t look like plaques, but I was so thrilled that I didn’t get blank plates again.

As I went further down the stack, wait for it, I got phages!!!! It was pretty exciting, and it definitely made my day!

I had to take pictures, and I took at least 3 pictures of each phage-laden plate, afraid that one of the precious pictures of my viruses would get accidentally deleted. Now that I think of it, pictures of plates make up the vast majority of the pictures on my phone at the moment. As I was creating my presentation, I felt like I spent forever looking at the plates and cropping the picture to perfection.

As the lab continues, I definitely feel awesome as I prepare plates like a pro (at least I feel like a pro). The movements have almost become like a second nature to me. Of course, today I prepared my MTL, so some new techniques are coming my way. But, I am excited to see where my phages will take me!

Happy Phage Hunting!

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What in the World is a Micropipettor?

My first day of phage hunting can only be described as a blanket of confusion wrapping a cluster of bad decisions while clear panic rained down from above. We had recently come back from digging dirt out of the “Phage Garden”, a lovely plot of land nestled almost secretly away, producing homely vegetables as well as, more importantly, phages. I had been pretty up to speed at that point, clasping my little tube of dirt, and was excitedly anticipating what was to come. Phage hunting! When registering for classes, this was the one I had been the most excited for. As a computer science major, most of my registered classes were lectures, sitting in a room while typing away on a laptop. Not that coding didn’t excite me, but the prospect of getting a hands-on experience in a laboratory that involved getting up and moving around was something different.

What I didn’t expect was to immediately be thrown into it. Sure we were given a briefing and a handout, but after an hour or so we put on our lab coats and got to work. I distinctly remember mimicking other students without really understanding what in the world I was supposed to be doing. You see, prior to showing up 15 minutes early for this class, I had never step foot in a laboratory. I had seen a Bunsen burner perhaps twice in my life, and the most complicated experiment I had ever done was probably poke a dead mink that stunk up the single hallway of my high school for weeks (art schools in Oregon don’t exactly offer the most high-quality science facilities).

Somehow though, with the help of both my classmates and student teachers, I managed to at least complete direct plating. I did end up having to do the process twice; the first time, I didn’t know the difference between a pipette and a micropipettor, and ended up plating a little over 1 ml of soil filtrate instead of 50 ul. But at the end of the two and a half hour session, I felt a sense of exhilaration. Sure I may have spilled some agar and used six more syringe filters than necessary, but at least I didn’t set anything on fire.

I relaxed more in the coming labs. After multiple rounds of trial and error on the first day, I knew more or less what the handout was trying to tell me to do. Despite the fact that my plates from direct plating failed to produce any phages, I was reassured by the fact that these results were common, and went on to make my enrichment sample. By the time I got to making 10-fold dilutions, I not only knew what a micropipettor was, but also how to use it. This time, the results were favorable. Plaques appeared! I internally gave myself a high five, proclaimed science to be the coolest thing ever, and proceeded to the process of streaking.

Now here I am, on to my fourth round of streaking. I have currently narrowed it down to what may be two different morphologies of phages: one small but with a clear edge, the other slightly larger with fuzzy edges. Perhaps they are the same type and I am just over-analyzing it, but either way I’m excited to see the results. Despite a bumpy start, this class is by far my favorite. Failure only promotes growth, and I am confident that I can tackle whatever challenges are thrown my way. Besides, science is just so darn fun.

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I never thought I’d say that I was excited to see a plate full of bacteria. But on the fateful afternoon of September 8, I discovered that my plate was not only full of bacteria, but full of bacteria being eaten by viruses- and I was absolutely elated! Now you probably think I’m crazy, so let me backtrack and explain myself a bit…

Just a few short months ago, I was in the process of registering for classes. Phage Hunting was something that had caught my eye, but as a social science major, I figured I shouldn’t prioritize it. And so precisely at 4 AM in California, I clicked ‘Register’ and with maxed out credits, left Phage off the list. Almost immediately, however, I realized how much I regretted my decision- I really wanted to take Phage. Unfortunately, by then it was too late. Phage had filled immediately, so I placed myself in position 7 on the wait-list for an 18 person class, and well, waited.

The weeks that followed were a whirlwind of meeting my future classmates on Facebook, eating too much In-N-Out, and saying goodbye to my high school friends. By the time I arrived on campus at JHU, I had all but forgotten about Phage. That is, until I received an email on the second day of classes saying that I had gotten off the wait-list. In the next 24 hours, I was on ISIS for far longer than I should have been, trying to deal with scheduling conflicts and trying to decide which class to drop. The one thing I knew for sure was that I was not going to let this opportunity get away again.

So that’s how I ended up here: In the Phage Hunting lab, holding my plated 10^-3 enrichment dilution sample, looking fondly down at the plaques my new phages had made in the field of M. smegmatis bacteria. I hope you’ve realized by now that by plate full of bacteria, I mean an agar plate- not a plate full of rotting food! I was discouraged when my direct plating yielded no results, but after opening the incubator to discover such distinct and beautiful plaques, I think my heart may have actually skipped a beat- and I knew that this lab was the place for me. Even now  T-streaking over and over and over doesn’t get mundane because I know that the next time I’m in the lab, I’ll get to see what new patterns my phages have made, or if there was another strain of phage hiding between the ones I’d been nurturing.

Currently, I’m waiting to see the results from my fourth T-streaks of three different strains of phage. One strain has very small, very clear plaques, and I’m fairly certain it contains a pure phage. The other two strains have larger, fuzzier plaques, but there is slight variation in morphology on both plates. I haven’t experienced much hardship in terms of contamination or otherwise, and I hope I never do. But no matter what comes my way, this lab has been a phenomenal experience so far, and I’m confident that any challenges will only push me to learn and experience more. And so regardless of my major, I know that taking this lab course has been quite possibly the best decision I’ve made so far as a Hopkins student. And who knows- maybe my major will end up changing to accommodate my love for being in the lab. Happy hunting!

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“Phalaphel” Emerges

By Suborna Panja

After 4 months of nonstop plating and re-plating, my phage “Phalaphel” has emerged! While the idea of discovering my own phage seems exciting, there has been an incredible amount of frustration. To all those future phage hunters out there:

1. Don’t be afraid of discouraging results. It happens to all of us.  For example after multiple rounds of streaking to isolate a pure phage, I ended up discovering I was unsuccessful when I plated my MTL. As a result, I had to do the entire streaking process over again.

2. You will become very familiar with contamination. Pretty much everything we work with has to be sterile. Even the smallest bit of exposure to non-sterile items can contaminate your plate.

Once I got past the endless amount of streaking and repetition, all the hard work finally paid off with a successful MTL and then later HTL. The HTL is the key to determining tons of characteristics about the phage. So once I got to this part, most of the repetition was over.

Nothing is more rewarding than using the HTL to see my DNA concentration and then finally my phage! I admit, because of the constant amount of errors I have run in to, I was partially convinced that I would have no phage. I thought that perhaps I screwed up at the last step and had nothing. But to my excitement, my phage was clearly there!
Phages look nothing like I thought they would look like. Perhaps I had this preconceived notion that bacteria looks like blobs of DNA enveloped in some sort of plasma membrane. Well, in fact, that’s not the case with phages. Phages have round heads with tails coming out of them. The size of the head and length of the tail varies per phage; my phage happens to have a 50 nm head, and 230 nm tail. The first time I saw my phage, its round head reminded me of a falafel, hence, I named my phage “Phalaphel.”

Well, the end is not near, it is here. It’s been a long and unexpected journey filled with many scientific obstacles, but persistence is the key to success. Phage hunting has definitely been one of the most challenging projects I’ve ever participated in. The idea of not only characterizing something that has never been discovered before, but also characterizing something that you can’t even see seems a bit impossible. But you’ll be surprised what you’ll find from a simple sample of dirt.

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Tears upon T–Just Kidding; There is no Crying in Phage Lab

By Michael Shang

It’s been an eventful month and a half. When we last left off, a new morphology had cropped up in my MTL dilutions (despite the previous step having a seemingly pure population and after it had already been subjected to around 8-ish streakings), and I was determined to find out what caused it. Weeks later, much information has been gleaned . . . but none has helped me move forward. Alas, after a semester of lab, and after coming in every day, Monday through Thursday, to squint at my plates and curse the Phage Gods once again, little progress has been made.

And yet, the information I’ve found is invaluable — the phages that make the new big plaques take two days to incubate, as I found out from coming in every day (e.g., I would plate the big ones on Monday, come in on Tuesday and notice that they made tiny pinpricks on the plate, and then come in on Wednesday and see their size explode to the usual “big” size). And, perhaps most important, I’ve found that yes, the two different morphologies are indicative of two different phages, rather than one phage behaving differently.

That information came, funnily enough, from EM–electron microscopy–which is supposed to be one of the last steps we take in the process of phage hunting. Instead of doing EM on an HTL, I ended up doing it on my MTL.  And the results are simultaneously encouraging and discouraging. Because the tails are 20 nm apart in length (190 nm vs 210 nm), the two plaques are definitely indicative of two different phages. The good news is that it confirms my suspicions; the bad news is that it means I have two phages. Another interesting thing: even though there seems to be so much more of the “small” phage, when I went to do EM, the big one was easily visible on the screen; finding “smalls” was actually quite hard to do (it didn’t seem like there were many of them).

Alas, after sticking with my small inverted-bullseye-morphology-phages for an entire semester, I’m forced to conclude that I must switch to the big ones. The decision was not an easy one, and I almost shed a tear — kidding, kidding (I exhausted my reserve of those a long time ago).

Even if I don’t get graded for it, though, I still want to figure out what’s going on. Even though I’ve had a myriad of woes, I think this is the coolest thing about science: when stuff doesn’t go your way and weird stuff pops up, you could be on the brink of discovering something. Anyway, let’s hope that, next semester, I’ll have more luck.

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The End.

By Meg Chen

“Please collect a few spoonfuls of dirt in a ziplock bag and bring the sample with you to the first day of class.

It’s been a long, yet quick journey.  The path from a random soil sample to a beautiful, unique phage feels so unreal.  But now that I come to think of it, it must have taken much longer than three months for the first researcher(s) to experiment with different procedures for isolating plaques and develop the most effective one.  For example, who came up with the idea of streaking a plaque?

One of the most important things I learned along the way was lab technique.  Early on, I would stay past 5 every day, checking my work at each step.  Yet I would still end up with contamination.  I figured that since there was no way to keep every single phage out of the microcentrifuge tubes, I resorted to methods to eliminate outside phages, such as cleaning my tools with CiDecon before putting my phage solution in and working closer to the flame.  One day, I actually worked dangerously close to the flame and burned a hole through my gloves (though the fire went out quickly and did not get to my skin).  Even though I successfully eliminated outside phages from my tubes and plates, I realized that I would have to find ways to minimize contamination instead.  For example, instead of reaching into the container to pick out microcentrifuge tubes, I learned from Dr. Fisher that I was supposed to dump them out and close them quickly.

I slowly grew accustomed to the method of sterile technique through trial and error, and by the third week, I was out of the lab by 4 most of the time.  I still remained skeptical that my plaques all came from phages in my enrichment plating and not from the surrounding air, and I saved 2 plates for each round of streaking and dilution series so that I could go back and re-streak if I run into contamination another time.  After putting top agar on my negative control plate, I would shut the lid quickly, sometimes shattering the outside of the petri dishes.  I always took one more tube to pick up the top agar with than I actually needed in case I accidentally touched the tip of one tube to a foreign surface.  When our whole class experienced contamination due to the old smeg, my plates with top agar and phage buffer alone did not have any contamination even though I had been slightly less careful.  This was when I realized that I had been too paranoid all along, and that the chance of my phage coming from the air is lower than what I originally expected.

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Webbed Plate of Lies

By Megha Telur

I can honestly say that Phage Hunting has been my most enjoyable class this semester. Everything about the class just clicked; research, lab work, and biology. I aspire to one day be able to do genetics research at Hopkins, so being in a UTL biology lab was an absolutely amazing feeling. It seems strange to many but I love being in the lab (I also love long airplane flights!) it’s where I feel comfortable and strangely enough, at peace. The wonderful huge open windows added to the charm of my second home, the UTL.

As much as being in a Johns Hopkins lab thrilled me the first few weeks of phage hunting were overwhelming. I found that I was extremely dependent on my lab manual and the TAs to get through basic procedures. With time the process became more and more intuitive and I was able to make judgement calls on my own. I knew when I had isolated my phage,  I picked plaques to streak, and I knew when additional plating or diluting needed to be done. Nonetheless, no matter how much progress I had made, nothing could prepare for the ultimate test of patience: the webbed plate of lies.

I calculated and diluted several times to no avail. My web plates never came out right!!! Many times there was hardly any lysis much less complete lysis of the bacterial lawn on the agar plate. After weeks of no success (I plated web plates at least 4 times) and discussions with Dr. Fisher and Dr. Schildbach , I finally flooded my partially webbed plates for an HTL. It was determined the initial concentration of my phage was so high (I couldn’t successfully calculate an initial titer) that the risk in flooding partially webbed plates was minimal.

Thankfully, the risk paid off in the end. Not only was I able to quantify enough DNA, but I was able to complete the procedures on time. This experience was a quick but effective lesson in independence and strategic decision making. I realized that the manual doesn’t have all the answers and deviating from the standard procedure is sometimes necessary. If there is substantial evidence and solid reasoning creating your own path may actually lead to the best results-especially if it means avoiding a dreadful webbed plate of lies.

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