“This is what it must feel like to watch an ultrasound when you’re pregnant.”

Phage hunting wasn’t the first time I patiently waited for a micro-centrifuge to stop buzzing or held my breath while opening a sealed flask containing bacteria, nor was it the first time I contaminated way too many plates. However, it was the first time I took something so minuscule, we needed a microscope the size of a room just to see it, from a soil sample and studied it. I remember going for a run to Wyman Park to collect soil (you know, when I was still intent on fighting the freshman fifteen). At the time, I didn’t know much about bacteriophages or how we were supposed to isolate them from a clump of dirt.

In my experience, research is tedious. You start with a procedure. You mess it up, i.e I would forget to turn on the Bunsen burner, contaminating my plates; I would forget to refrigerate my HTL and end up lucky that it’s stable at room temperature; and sometimes, I would even forget to add M. Smeg so nothing would grow on my plates. But finally, you get it right. The excitement is unreal and you wonder why growing bacteria makes you so happy. And then you repeat the procedure again. And again. And maybe one more time after that.

By the end of the semester, we had dedicated so much time to our phages that they were like our temporary children (without the crying and diapers). We got excited to learn more and more about them through our experiments. Perhaps the greatest part of the course was finally being about to see what our tiny phages looked like via the electron microscopy images. The experience was almost like my classmate described it: “This is what it must feel like to watch an ultrasound when you’re pregnant.”

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What is Success without Failure?

On the first day of phage hunting, upon entering the lab for the first time, I was expecting to see the materials already set out for me, and a lab notebook with instructions neatly lying in the middle of my personal lab bench. Instead, all I see is a Bunsen burner which having always scared me to death, did nothing to alleviate my anxiety.

If I were to describe phage hunting in two words back then, it would have been Unexpected Freedom. On that first day, we were given a quick outline of what we were supposed to do, and then proceeded to be let loose in the lab like a pack of clueless college freshman.

The shock only lasted a few more classes, though, thankfully, and by the fifth day or so I had learned how to navigate around the lab, carry out the various protocols with ease, never flip my plates to prevent the agar from shifting, and bond with the Bunsen burner to make the most out of my aseptic technique. All my blunders and contamination problems in the beginning of the semester diminished and it was smooth riding from there. I had found my flow, and felt empowered and unstoppable in my white lab coat.

The following days can be summed up by the following:

Enrichment? Check…1st streak? Check…2nd streak? Check…3rd streak? Check…4th streak, Check…MTL? Check…Web Plate? Che- Aww crap…

And that’s when I hit the first of my biggest walls. The 6 web plates that I made to flood and collect as my HTL (Highly concentrated stock of phages) came out poor in that there should have been a gazillion plaques on each plate, but they only had about 15. In the end, it took me two more tries to finally obtain my HTL.

But the pride in my HTL evaporated when I realized that there was more work to be done in isolating its DNA. It took me five tries of tedious washes with isopropanol and centrifuging to obtain a high enough concentration, and that was the class right before final presentations, another stressful time.

While I thought my problems ended after giving my final presentation, immediately after I learned that my DNA was not refrigerated. So, as of now, my struggle in Phage Hunting continues as I prepare myself for another round of DNA prep and, considering that I have ran out of HTL, need to work desperately to make this last one count.

So what are my thoughts on Phage Hunting three months into it? Despite all the work and challenges, Absolute Delight. Working in the lab and being empowered with a Bunsen burner and a pipette relieves the stress from school and studying all the time. But most of all, after reflecting on my achievements, seeing how far I’ve come, and finally seeing my phage via electron microscopy, I am certain that I will never regret taking Phage Hunting.

 

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Going Through the Motions

Bunsen burner on, 90 μl of phage buffer plus 10 μl of phage. Repeat, repeat, repeat. 10 μl of mixture into 0.5 mL of smeg. Repeat, repeat, repeat. Twiddle my thumbs for 20 minutes and annoy Kushi, my bench partner, with sarcastic humor. 4.5 mLs of Top Agar into the smeg, suck it up, expel, suck it up, expel, mixing it well. Carefully open a labeled plate and dispel the top agar into the plate, carefully re-suck up the bubbles. Repeat, repeat, repeat. Wait 20 minutes for plates to dry while checking my email. Carefully stack plates, tape them, scrawl “SRS” across the tape and open the smelly incubator. Place on the M/W shelf. Wash hands, go home, and take a nap. Come back next class, and retrieve plates. Oh look plaques! It must have worked. Time to repeat the process.

The past couple of months of Phage Hunting has somewhat turned into a blurry concoction of repeating similar lab tasks in hopes of getting similar results that indicate that something is working. Sometimes it’s difficult to lose sight of what that something is exactly. Almost as if by magic results just appear and it’s easy to not fully think about what they mean. By going through the motions it’s easy to miss the significance of what this class is really showing us. The plaques aren’t just little clearings in bacteria, well okay I guess they are, but they’re where a phage decided to set up shop, to live and eat and reproduce. And these plaques aren’t simply just indication of something working, they’re indication of purification of a phage that may ultimately lead to being able to add my phage to a database. And this database may one day lead to the development of new medical therapies to treat disease, or may help an epidemiologist understand the biodiversity in the phage world. Wow. That’s a lot bigger than just some clear spots on a fuzzy bacterial lawn, but that’s what we, as phage hunters, must keep our eyes on.

It honestly feels horrible when you get out your plates, and they look weird. And they smell weird. And they have e.coli, and you want to scream because now you’re behind 3 days and very confused what went wrong. But you just have to keep going through the motions, while keeping your eyes on the bigger picture. Yes I’m getting VERY sick of plating dilutions, but seeing my phage on the database will make it worth it. Knowing that I have made a contribution to a larger scientific community will make the trials and tribulations of this class worth it in the end. You start to feel a sense of ownership with your phage, almost like a puppy, and when it behaves, you feel proud and when it doesn’t, you want to scold it. But in the end it’s best to just keep your cool, go through the motions once again, and remember what the end goal is.

Tomorrow in lab I will take my HTL to do electron microscopy. Suddenly this mythical lab technique only read about in high school biology class has a purpose, and a very important purpose to my phage hunt. By using this insanely amazing technique, I will hopefully obtain a picture of my puppy, errrr excuse me, I mean phage.

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Do I dare?

As an 18 year old freshman born and raised in Los Angeles, attending Johns Hopkins University was a dream come true.  Traveling across the nation to one of the world’s leading research institutions, and I, this wide eyed freshman had the opportunity to discover my very own…dun dun dun…bacteriophage!  True, I recalled the very fundamental background regarding bacteriophages from AP Biology but that material was minimal.  Little did I know that I would soon embark on an entrancing scientific quest.

My eyes grew wider from the first moment I stepped into the Undergraduate Teaching Labs.  From the wows echoing in the room as Dr. Fisher picked up a marker and wrote on the erasable wall, to all of us first year students receiving our very own white lab coats, fascination beamed from cheek to cheek.

And on that very first day, a lab full of frail freshmen attempted to begin a collegiate research project.  Let’s be real now, no one truly knew what they were doing on that first day.  This is blatantly obvious from any picture dated from the first two weeks of lab.  But what was key and a major learning objective of phage hunting as a whole was that we as freshmen were educated as young scientists.  We truly learned!  Not the type of learning you do when you have an exam two days from the point in time you begin studying, but the type of learning  where curiosity and wonder drive understanding.  Being the eager phage hunter that I was, the most challenging part of the entire semester project was killing off two of my three children (now wait a minute let me explain).

I became very dedicated to phage hunting.  I began to love every streak, restreak, and re-restreak I did.  As a result, when I was fairly confident that I had three unique and morphologically distinct bacteriophages and the time came when I had to select just one phage to pursue the rest of the project with, you could only imagine my hesitation.  How do you pick which child lives on?  Though this may seem dramatic, I believe the reason why I felt so attached to my phages was because picking one phage means losing two: that’s two genomes that could potentially be sequenced, two phages that could possibly hold the key to phage therapies.  With science, unearthing new technologies is inevitable and some may say scary, but the possibility of not discovering when given the opportunity to do so is even scarier.  For that reason, killing off phages B and C (sending them to the refrigerator) and keeping phage A was categorically the most daring part of phage hunting thus far.  Yet, in doing so, I have learned so much.

Whether actually knowing how to use a Micropipettor to mastering the screwing and unscrewing of caps off of sterile tubes using just one hand, phage hunting taught me not only useful lab techniques but also how to persevere amidst failure.  Failure was ubiquitous throughout the semester.  Often times, failure occurred over and over again at the same point along the research project.  However, through teamwork and persistence, the science prevailed.

Phage hunting has been an integral part of my first semester at Johns Hopkins.  It has added to my love of intellectual inquiry and has led me to want to learn in a more comprehensive manner and for that, I am grateful.

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Finding a Phage

Back in April, I came to Hopkins to visit for SOHOP. I’d been accepted, but I still had to make the decision of whether I would move halfway across the country to come to Johns Hopkins, or stay in my home state (was it really that hard of a choice?). So I woke up at 2 a.m. my time to fly out here. Although most of that trip passed by in a caffeine-fueled haze, a few things stood out. Among them was the brightly lit UTL and the number of times my hosts insisted I should sign up for Phage Hunting. And by some luck, I got into the course.

I neglected to collect a soil sample before coming to lab, and I was glad I did. My first day of class featured a field trip to the Phage Gardens. I scooped up a soil sample from under some tomatoes, and we returned to the lab to start working on the samples.

As we started working with the sample, a few things came as a shock. Unlike the C labs of the past, our course was not defined by strict time periods. We could come and go as we pleased, and worked at the pace guided by our progress rather than set deadlines. Furthermore, to keep materials sterile, we had to throw out many things after one use. As I pipetted liquids from one test tube to another, I couldn’t help but watch the pile of waste pile up. Nonetheless, I couldn’t help but be drawn in by the excitement of isolating my phage.

My initial attempts at direct plating yielded putative plaques that turned out to be nothing more than air bubbles. My enrichment sample and dilutions, however, yielded several plaques. All were large, but some were totally clear, some were hazy, and some looked like a bullseye. Next, I started streaking the samples (A, B, and C ) in an attempt to dilute them out. This entailed using several sterile sticks or micropipettes to draw a series of intersecting lines on an agar plate. Although I started the process with three plaques, some disruption in the agar and aggressive phages hindered this process.

Ultimately, I discarded plaques A and C and chose to stick with Plaque B. On my third, and hopefully last streak, however, I found the plate totally clear. This meant the phages had killed all the smeg bacteria (M. Smegmatus is the bacteria used in lab), but it also made it impossible to distinguish each plaque from each other. I redid the streak from the previous plate as well as taking a streak from the totally clear plate. To my surprise, the next streaks revealed small clear plaques rather than the large ones I had seen previously. Unfortunately, this meant that I have to redo the streaking until I receive three consecutive identical plaques. Hopefully, my next round of plaques will isolate soon so that I can start creating stock phages.

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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.

DRUM ROLL PLEASE….

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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