Two and a Half Hours in the Life of a Phage Hunter

By Sophia Chen

Walking into class every day is a surprise. I never know what to expect when I walk in, often just resorting to wondering what strange thing has grown on my plates today. This thought often runs on replay in my head as I hang up my backpack and grab a pair of gloves, never stopping even when I go back to my area to put on my lab coat and disinfect my station. After having dragged out the moment long enough, I usually go to grab my plates – a process that involves a lot of picking up other people’s random plates and being convinced mine are not there before finally finding them (one time I actually thought my plates had been lost, only to find that they had been placed to the side in a place I had never thought to look). Taking my stack of plates back to the lab desk, I peel off the neon green tape keeping them together and hold the first one up to the light, hoping for good results.

So far, I have been very lucky. Most times my initial apprehension has turned to delight as I spot my precious phages growing on a lawn of otherwise spotless, contamination-free M. smegmatis. Other times, frustration reigns supreme when I see the evil yellow taint of invading bacteria crawling across the plate. At these moments I curse the phage gods and head back to waste more plates and top agar, resigning myself to starting all over again. These experiences accurately describe the roller coaster of emotions that is phage hunting. In two and a half hours of phage lab, I go through more emotions than in five hours of lecture, a fresh change of pace and exactly why I like phage hunting so much.

These emotions appeared even before the first day of class, when I received the email about collecting dirt samples. My first reaction was (among many others) confusion and not a little bit of apprehension. As I read over the instructions, I seriously reconsidered my class choices, wondering what in the world I had signed up for. Deciding to take a leap of faith, I went to a remote corner of campus to collect my sample, hoping that no one would remark upon the strange girl digging a hole in the ground with her plastic spoon. But there was no need to have worried at all because by the end class the next day, all my doubts had disappeared. After about 30 minutes of introduction that first day, we jumped straight into lab and have never looked back since.

That first class involved us direct plating our soil samples (a fail) and creating our enrichment culture. From there, we plated our phages and started the long process of isolation. It took a while but after about five billion rounds of streaking and ten billion wasted plates, I finally isolated my chosen strain of phage. Creating omy MTL next was a little trickier (I had some problems with contamination) but eventually even that came through and soon I will have my HTL.

Throughout phage lab I have been learning so much. I have gone from wondering how to do aseptic techniques with only two hands, to accepting my limitations and being tolerably good at it (although I still wish for more than two hands). I have gone from almost burning myself multiple times on the invisible Bunsen burner flame each class, to now only having a couple of close calls each week. And I have gone from confused lab notebook writer, to only semi-confused lab notebook writer, a change that required a lot of scribbles and resulted in quite a messy-looking notebook. I can’t wait until the Phage Olympics and the sequencing of phage genes begin but until then, may the odds be ever in your (phage’s) favor!

Posted in From the Phage Hunters

All About That Phage

By Dillan Villavisanis

Digging up dirt around campus and hoarding it into plastic bags on the first day of college probably isn’t the best way to meet new people, as many of my other classmates were doing. But this was the first step in a yearlong journey in a class dedicated to identifying and characterizing a novel bacteriophage. Besides, it’s much easier to like phages than people (just kidding . . . kinda).

We started by utilizing a direct plating technique, in which the sample was filtered and immediately plated with the bacteria Mycobacterium smegmatis. These bacteria were plated with the potential phage mixture, in hopes of identifying plaques. Plaques indicate that phages have infected the bacteria, and the phage can be analyzed and further proliferated. I was forewarned that the chances of identifying a phage using this technique were smaller than the phages themselves, so I wasn’t disheartened when my plates failed to contain plaques.

We moved onto an enrichment culture technique, in which the soil sample was mixed with a variety of other solutions and incubated to promote the development of phages. While many of my classmates found success in this method, I only identified a single plaque, which turned out to be too small to proliferate. As a result, I obtained a new soil sample outside the Undergraduate Teaching Labs and began the enrichment culture process again.

Fortunately, this sample was rich with phage and provided many plaques. Now that I had identified a phage, I needed to isolate a single morphology. This is to say all the plaques should be of the same physical appearance, in diameter, shape, and color. This is important, as the goal is to isolate a single type of bacteriophage.

First, a dilution series was established to create a gradient of several concentrations of phages across several different plates. This would provide the optimal opportunity to select a single plaque to be streaked. After the dilution series, a streaking technique was used in which a single plaque was selected and streaked onto a new plate. This would create a plate of new phages, which could then be streaked from again. In doing this several times, the streaking patterns could be analyzed.

After streaking over six different times across a multitude of plates, a single morphology was identified. My particular morphology is that of many small plaques, that contain a slightly darker ring around them.

Currently in the lab, I am working to establish a Medium Titer Lysate, or MTL. This MTL will allow me to calculate the phage titer and to create an HTL. Phage Hunting has been equally fun as educational. I am excited to continue my work in the lab and see where the phage hunting leads me.

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Hello, World!

By Chang Ha

Back in April, during the second week of SOHOP (Spring Open House and Overnight Program), I visited the Homewood Campus in hopes of learning something new about Johns Hopkins and its college life. After all the pre-scheduled events , I was finally introduced to my host who was willing to spread his wisdom to us confused souls from his hands-on, “real” college experience.

Phage Hunting

That was the phrase that I remembered most vividly from his recommended list of classes. From the whole list of suggested classes, the word “Phage” and “Hunting” stood out the most for various reasons.  (And one class NOT to take: with professor *CENSORED*.)

Phage Hunting

“That’s the name of the class?” or “That doesn’t sound too official” or “That actually sounds quite interesting” came to my mind as I was thinking about what kind of class it could be.

Here I am now, writing this blog post. I am usually very indecisive, but I can say this one thing with full confidence and determination.

This is the best class that I have ever taken in my life. Hands down.

At the first day of class, I was provided with a brand new lab coat. Wearing that white, professional-looking garment made me not only motivated, but also very excited. I found myself feeling motivated to do work – which was unthinkable.

Wow, I am actually in a college lab.

Then, we were introduced to the pipettes, goggles, all the other sterile equipment which were at our disposal. Then I watched the professor demonstrate how to use the pipette with an aseptic technique. Then she discarded the pipette.

She discarded the pipette

The simple act of throwing a piece of equipment into a trash bin came to a shock. I then realized that this is college laboratory, not an hour-long, boring, and grumble-inducing high school chem lab anymore. We cared that much about the actual result than simply imitating a lab experience.

This was my first impression from the first few days of phage hunting which left me with confidence that I will like this course throughout the whole semester.

I then began my journey to hunt for phages.

I collected my soil sample just outside of my dorm in hopes of finding plaques. I made sure that the soil was not too dry or too damp (for some reason I believed that that would change the result of the experiment). I then brought the sample to the lab and did “Direct Plating” and went through the “Enrichment” process.

For direct plating, we flooded our soil sample with Phage Buffer solution and plated it directly onto the agar dish.

Sadly, no plaque.

Well, it was hard for me to confirm that, because I initially had absolutely no idea of what I was looking at. For me, (at that time) the “good plates” were supposed to contain several holes, and the “bad plates” were not supposed to contain holes. Mine had no holes.

Instead, my plate was contaminated. The content of the plate was cracked, smelled nothing like the M. smeg (bacteria that we were using to grow phages) but instead smelled something closer to E. coli (or the professor said so). I then realized that my errors were actually displayed in front of our eyes. I failed to follow the aseptic procedure correctly, and this was the result.

It was as if the agar plate was yelling:

You Done Messed Up Aaron : You Done Messed Up - by Anonymous

Still clueless, I moved onto the Enrichment process, where I had to filter the soil sample and dilute it into 100, 10-1, 10-2, 10-3,and 10-4 dilutions with the phage buffer. Remembering my error from the previous experiment, I made sure to take extra care in following the standard phage lab procedure and not contaminate my dish with the ‘evil’ contaminants again.

Then I found plaques.

It was one of those few moments where I was genuinely proud of what I have accomplished. I found my plates in pristine conditions with no cracks, and with a number of small blank regions which were nothing close to the regular bubbles that sometimes formed on the plate.

I then moved on to streaking, where I would try to isolate my dish full of beautiful plaques with a series of streaking in order to purify and collect a single type of phage only. After four or five different delightful (yes, delightful) series of streaking, I came to a conclusion that I finally got a single, pure phage.

As of now, I am currently doing a titer assay, a process in which the number of phage in a volume is calculated.  Although the process is slow, I am happy to see that my babies (phages are babies) are growing and thriving well under my care.

Overall, the class of phage hunting shed new light on me in terms of how college and real life is different from what I have experienced so far. I am learning rapidly, and am learning a lot of valuable lessons in an effective manner.

It’s hard to believe that I am getting all these valuable experiences in a class that I may not be in, had it not been for my host from the SOHOP event. For that, I thank you very much Hunter.

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Phage at the End of the Tunnel?

By Ashley Yoo

I’m not going to lie . . . I was scared coming into the lab the first day. I didn’t want to ruin my sample or the materials on the very first day, and the fact that the Bunsen burner was always on but never visible had me on edge. It’s been 4-5 weeks since then, and I’ve become comfortable with the lab that (hopefully) my phages will be able to call home for the time being.

The first couple days of enrichment, direct plating, and lots and lots of M. smeg were a blur. That is until I ended up with a thick, yellow contamination on my streaked plates that left me worried sick. I retraced my steps, and after consulting with the T.A.s and Doctor Fisher, I decided to filter my sample and streak again. The next time I walked into the lab and observed the plate . . . the contamination was still there! What I prayed wouldn’t happen happened. To keep the story short, I was pretty confused and a little distraught. This time, rather than retracing the steps in my head, Dr. Fisher watched as I showed her how I would go about filtering my sample. From there, I discovered that I was making a careless mistake that contaminated the microcentrifuge tubes: I touched the lid. Though it may seem like a minute detail, it made the world of a difference. I filtered my sample again, and streaked a new plate, to come into the lab next time to find a perfect gradation of plaques on my plate. Coming from a rough patch, this made me want to jump for joy (but my sanity kept me glued to the ground). I went straight to work, more confident in my work this time.

Since then, I’ve begun to differentiate between two main phages: one that has plaques with hard edges and one with soft edges. My heart skips a beat every now and then fearing the resurfacing of the horrible yellow goo. However, as I begin to accumulate plates free of contamination and progressive dilutions, I’m beginning to build my confidence in the lab. My phages may not be 100% pure, but it’s getting there, and I’ve been thoroughly impressed with my progress.

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A Study in Phage: The Musings of a Novice

By Taylor Veracka

I’ll admit it: my first day of phage hunting was rocky at best. I was a nervous wreck, what with it being my first college class ever and all. Even though I knew what the class was about from attending SOHOP in the spring (Dr. Schildbach’s expo was what made me want to take the class in the first place), I was still unsure of what to expect. Upon entering the lab, I was immediately intimidated by the clean, austere beauty that was laid out before me. Yes, I truly thought that the lab was beautiful. I still do! It was unlike any other learning space that I had encountered, and the fact that I was admitted into its confines, that I was actually going to be allowed to utilize this space so as to further my education, was borderline incomprehensible.

Diffident in my abilities, my hands shook as I tried my best to pipette using sterile technique. I found it incredibly difficult, and didn’t really believe the TA’s when they said eventually it would become second nature. I finished that first day almost an hour after the class was supposed to end, mentally drained, nervous about the next class, but proud of the work I had done.

To my surprise, by the next class I had sterile technique almost down to a T. My direct plating didn’t yield any plaques, but that was okay. I performed the enrichment culture swiftly and efficiently, and actually finished early (a stark contrast from the previous class). I left that day feeling incredibly confident and excited about phage hunting: I knew without a doubt that I was meant to work in a lab. It made me feel more confident in my post-college ideas and dreams.

The actual experiments only added to the amazing experience that I’ve had just far: the first time I saw phages on my enrichment culture dilution plates, I was absolutely thrilled. My 100 dilution plate was rife with plaques (though, as I discovered later, I did not yield as many as much of my peers, some of whom needed to dilute their samples past the 10-4 plates).

The streaking that followed was immensely fun: although I was a bit heavy handed with the streaking stick the first time, eventually I developed a passable technique. Seeing all the different types of morphologies eventually dwindle down to a single strain was very gratifying. My chosen morphology became a relatively large bull’s-eye shape. These plaques had distinct, dark circles on the inside, and a lighter, slightly-fuzzy outer ring. Class after class I streaked from my plates and felt such relief each time the morphologies matched. (I did have a scare once, because upon first glance, it appeared that my plaques had taken on a new morphology—still bull’s-eye shaped, but smaller—but it turned out that there were just so many of them that they were so squished together and appeared smaller because they were right on top of each other.)

It was almost sad when I moved on to make my titer, because I enjoyed streaking so much! But the change of pace is good—I was getting complacent. Currently I’m waiting to see how my dilutions from my MTL came out, and I’m so excited to see them. That’s the best part of this class: no matter what, I always look forward to phage hunting, and I always enjoy it. The first day was clearly just a fluke, and I feel as if I’ve made such strides as far as my knowledge of research. Signing up for phage hunting was definitely the best decision that I’ve made in college thus far. It is so rewarding, and I feel so proud about the work I’ve done.

Posted in From the Phage Hunters

Turning a new p(h)age in this chapter of my life

By Jihae Snyder

“What classes are you taking?” is definitely one of the most frequently asked questions I have been bombarded with basically every day since I arrived at Hopkins 56 days ago.

With the urge to roll my eyes, I reply mechanically, “Chemistry, Chem Lab, Calc, Italian, Shakespeare and His Goddess, and Phage Hunting,” almost as easily as I reply to the other common questions of, “How do you pronounce your name again?” and “OH! You’re from California?? Do you know my friend so-and-so?”

I then begin to count.

You see, I have discovered that a way to avoid getting annoyed at the inevitable next question, “What is phage hunting?” is to count the seconds between my answer and their question . . . sort of like counting the number of “Mississippis” until thunder claps after lightning strikes.

It was a rocky first couple of weeks trying to figure out how to give an explanation, but I think I’m slowly starting to figure it out.

I heard about Phage Hunting when I went to a biology presentation at the admitted students’ SOHOP trip in April of this year where Professor Joel Schildbach was presenting. He mentioned a couple of interesting things of which I only remember vaguely something about penguins and a fascinating class called “Phage Hunting.”

Needless to say, I thought that if I decided to come to this school, I would definitely take the class. I did and I did.

It is hard to imagine that after these weeks of being in the lab, it all started with a couple spoonfuls of crusty dirt from the small lawn lot outside of McCoy.

When I went into class that first day, I was totally lost. Class went over by an hour and I was terrified of what I had gotten myself into.

However, the next Tuesday came and went, and then Thursday, and then Tuesday, and then Thursday, and somehow Tuesdays and Thursdays have become my favorite days in the school week.

As insanely nerdy as this may sound, and I’m sure it does sound pretty nerdy, I actually am excited to get to lab each class and check if my previous class’s attempts were successful. I enjoy being meticulous in my pipetting. I find vortexing fun. I love holding my plates up to the light at the beginning of each class with a small rush of anticipation and excitement to see if my results came out how I intended them to.

I feel like I’ve learned so much in this class, like how to do direct plating (although let’s be honest, it doesn’t seem very effective), how to create an enrichment culture from which to isolate phages, and how to streak (a billion and one times).

I’ve been either extremely lucky, or I’ve been doing something right because somehow I ended up being one of a few people ahead of the rest of the class. This is scary because I have to constantly ask one of the TA’s or professors for help, and I second guess everything I do. Thus far, it has worked out okay.

Recently, having successfully identified and isolated a single phage morphology, I was able to create a Medium Titer Lysate from which I calculated the number of plaque forming units needed to create a High Titer Lysate. From this, I plated several amounts from my 10-4 dilution of the MTL to create the perfect web-pattern plate.

Today, I took my finally attained web-pattern plate to determine the accurate amount of MTL dilution to create plate six plates of phage-infected M. smegmatis bacteria.

If all goes well, on Tuesday, I should be able to finally name my phage, something I have been looking forward to since I heard about this class way back in April.  I’m thinking about Formics, after the highly intelligent ant-like alien species from one of my favorite book series, or perhaps, or perhaps something majestic, like Nicolas Phage. I’m so torn.

Whatever I end up going with, and where this labventure takes me, I’m looking forward to the rest of this class.

May the odds be ever in your favor, and happy hunting!

Posted in From the Phage Hunters

A Love for Discovery

By Maria Moncaliano

We are hunters. What do we hunt? PHAGES.

Hunting for phages has been an experience . . . and it has only been three weeks.

I came to college excited to do research. So I signed up for a nifty research class that not only included a project, but actual lessons on how to properly conduct research. Little did I know, I would learn so much more than how to use a pipette.

A lab in college is nothing like a lab in high school. I am thankful for that every day. Twice a week, I am in charge of my research project. I am taking care of my phages as they grow on my plate. It has not been smooth sailing by any means . . . and I have needed more than a little help (a perfectionist by nature, I ask a lot of questions).

But that’s what I have professors and upperclassmen for. To learn from their experiences. And this bunch happens to be very smart and very helpful (thank you).

So let me take you on my mini-journey as I undertake my first research project. It all started with a pile of dirt.

MM blog1

There is nothing strange about a Johns Hopkins student kneeling on the floor, under a tree, in the center of the quad, with a spoon and ziplock bag scooping up dirt in the middle of the afternoon.

Nothing Strange.

Really though, I had no idea what I was expected to do with a pile of dirt, but after 15 minutes of class that first Wednesday, I understood that it was much more than that.

There are tiny viruses in this world that can kill bacteria. There are so many variations that the student who scooped up dirt from Bloomberg probably got different phages than I did.

And I am going to discover a phage of my own.

Sure, it seems easy now. In the moment…it was a tad overwhelming.

That’s what college is for right? To get through those overwhelming experiences and emerge bigger, better, and badder than before. Yes? I think so.

So I put my dirt in a flask, added some nutrients, put in some smeg (bacteria for the phages to kill), and waited for phages.

And phages came.

Not at first…

My first plate was a fail…my negative control was contaminated, unfortunately, but it’s ok!

The first lesson I learned about research is that when we fail, we do not give up…and that is a lesson that applies everywhere.

So I re-did the plates and, to my great joy, I got phages!

MM blog2

You can imagine how happy I was when I saw those clear little spots on my petri dish, proclaiming their presence to the world.

As I examined the morphology, I was reminded of why I wanted to be a phage hunter in the first place.

Because I love science. And I love learning. And observing. And, above all, discovering.

I look forward to discovering a lot this year. About myself, about life, and about phages.

So here I am, a Blue Jay with a petri dish, looking forward to the day she can create a research project of her own.

Until then, happy hunting!

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