What Doesn’t Kill Y̶o̶u̶ Smeg Makes You Stronger

One of the first things Dr. Fisher told us on the first day of Phage Hunting was that this class would not be a typical one. She warned us that we would have to get used to having people at multiple different stages, and come to accept the fact that not everything follows a perfect timeline or goes according to plan.

How many times have you heard “you learn from your mistakes,” or, “what doesn’t kill you makes you stronger”? These phrases just seem like mindless clichés that people say when they can’t think of anything else. But let me tell you, this class has more than proven to me that failure is not a reason quit.

Going through direct isolation, plaque assays, serial dilutions, and enriched isolation, all of which resulted in absolutely no plaques, was discouraging to say the least. Especially because I was told multiple times that it’s nearly impossible not to get results from an enriched isolation, due to the fact that the 7H9 amplifies the phage in the sample. Despite this, I am a very determined person, and I wasn’t ready to adopt someone else’s plaques, so I decided that I would try an enriched isolation with one last new soil sample; this would be my fourth sample.

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This is pretty much what all my plates came back looking like up to this point: wonderfully plaque-less lawns of smeg.

For this last attempt, I told myself I would follow the procedure to the T, and take my time making sure I was doing each step correctly. But right from the get-go, something had to stray from protocol. We didn’t have enough 7H9 in the lab, so each student got only 10ml of enrichment broth instead of the suggested 20ml. Since enriched isolation hadn’t worked for me when I had the correct amount of it, I was not confident that it would work this time with half the amount. Despite this, I was determined to keep a positive attitude, and carry this last effort to the end.

After I’d finished filtering my sample, I handed off the conical tube containing the filtrate, thinking it was going into the incubator. The next lab period I went to get my sample from the incubator, but I could not find it. It turned out that my filtrate had actually been in the fridge for the last 48 hours. So, at this point, it pretty much felt like my experience in this class was a real-life embodiment of Murphy’s Law, which states that “anything that can go wrong, will go wrong.” What’s interesting is that the plaque I ended up choosing actually came from this “fridge” sample.

I decided to do a spot test right away with the filtered enrichment culture I’d obtained from two separate filtration procedures to make sure there was phage in this sample that was capable of infecting smeg as a host bacteria. The next lab period, when I checked the results of the spot test I could not believe my eyes. I’d gotten so used to seeing full lawns of smeg that it took me by surprise when I actually saw the circular clearings I’d been waiting for for weeks.

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Phinally!

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Plaques on plaques on plaques

I’ve only been in Phage Hunting for a matter of weeks, but I’ve already become familiar with a variety of research techniques (of which I knew absolutely nothing about before taking this class), I’ve learned more about the world of bacteriophages, and I’ve come to understand more about the nature of research. But, perhaps the most important thing I’ve learned from this course (besides how to do aseptic technique like a boss) is that achieving success after rounds of failure is sometimes more fulfilling than success that comes without any obstacles.

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Success is not a straight path

Throughout the semester of Phage Hunting this has become my motto that I have sticked to the entire time. Since the beginning, I have failed to even obtain evidence for the presence of phages. I was stuck like this for the first two months of the course. Because I have continued to try to find phages, I have now obtained data from my restriction enzyme electrophoresis and an image of my phage from an electron microscope. I’m now in the process of archiving my discoveries, something I thought I would never be able to do in the first few weeks of phage hunting.

Phage hunting has taught me that the only way to succeed is to learn from failures and mistakes. In every plaque assay that I created I would always end up with a nice bacterial lawn with no evidence whatsoever of the presence of phages. I would end up with this same result for two months straight. Day in and day out I would follow the same procedures for isolation to end up getting nothing again. I even had to collect four different soil sample from different places across campus At one point a slight glimmer of hope appeared in which a small clear spot was observed on one of my plates. However, that hope was soon dashed as when I streaked the clear spot to test if it was a plaque, it simply turned out to be another bubble. At this point I was completely disappointed as I’ve spent a month and a half wasting two and a half hours every Monday and Wednesday to end up with no results. At that point I was ready to drop the course as I felt as though I would not end up with any data to create a presentation or write a final term paper.

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However, through some divine intervention, also known as Wendy, I was finally able to obtain a phage that I could isolate and finally begin the process of getting to know my phage. I had been looking forward to doing this since the beginning of the year. As I began to isolate my phage I felt more and more like I was becoming a scientist. I finally felt that the time I spent each day was actually worth it as now I was finally producing results that I could measure and observe. Since the day I finally obtained my phage and I going through the entire project with no other obstructions. I had isolated my phage through streaking and calculated the phages concentration. I then got to finally see my phage when I viewed it under the electron microscope. At that moment I realized that despite all of my failures I was still able to find my own phage and characterize it. This made me believe that success is almost certain, it only takes a certain amount of patience, determination, and stamina to finally reach it.

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Success is not simply a straight path that you can follow easily. It is in fact a series of paths that mostly end in deadens from which you must blackout from and start a different path that will again most likely end up in a dead end. However one of those paths will finally be the correct one and success will finally be found Many of today’s discoveries could never have happened if the person simply gave up their work. For instance Einstein was not simply able to conjure up his well known equation E=mc^2 in a matter of a single day with no mistakes. He had to spend many months experimenting and deriving equations that simply were not correct. If Einstein had decided to give up the first time he failed he would have never been able to calculate any of his commonly known and frequently used equations as well I would never have obtained a picture of a phage that I was finally able to find and isolate.

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Practice Makes Perfect

I expected the first day of phage hunting to be a syllabus day, much like the first day of every other class had been. And it was–at first. The first hour or so of class was spent going over the syllabus, but after that, we were thrown into the lab to start actual work. In high school, all labs started with the teacher going over what were supposed to be doing, step by step, making sure that everyone understood what was going on and what the procedure was (partially for the sake of learning, but mostly so we didn’t burn the school to the ground). In phage lab, however, we read our manuals and follow the procedures independently. And because everyone is at different points in their experiments at different times, often we’re doing the work completely independently. Thus, on that first day, we were thrown into the lab with more independence than I’d ever had in a lab before.

On that first day, all we did was the direct isolation procedure. This involved flooding a tube containing my environmental sample (dirt that I had collected from near the polluted river) with enrichment broth. After we did that, we let it shake for an hour in the incubator, and then had to filter it. Filtering it was a real struggle–I felt like I needed two more hands to move everything around and open/close things at the right times! Thankfully, I had my lab partner to provide those two more hands.

The next day, we spent the whole class plating one experimental and one control plate. At the time, it was almost overwhelming, and I felt like I needed even more hands than I

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This petri dish took about an hour of work to prepare (and it’s not even that great–look at all the top agar shifting!)

needed when just doing direct isolation! How was I supposed to be able to open a pipette, open the top agar, suck up top agar, close the top agar, open the smeg tube, dispense the top agar into the smeg, mix the smeg and the top agar together, suck up all the top agar, close the smeg tube, open my petri dish, dispense the top agar and smeg, swirl it around, close the petri dish, and move it all away from the flame while only having two hands? And how was I expected to do all of this without the top agar solidifying in the time it takes to do all that? And how was I supposed to do all of this using aseptic technique under the flame from my bunsen burner?

 


At first, the answer to most of those questions was to have my lab partner open and close everything for me. Then, I did the same for her when she plated her direct isolation, but I was worried about how we were going to manage plating while also both having to do whatever procedure our experiments called for on a given day.

But, as with any scientific procedure, it’s much easier after the first time you do it. If the records I’ve been keeping in my lab notebook are correct, I’ve now plated 79 petri dishes with top agar and smeg. If I plated all of those at the same rate I did the first one, I would have been in the lab for hours and hours every time I did a serial dilution. And while I’ve never actually timed myself, I can probably plate a petri dish with top agar and smeg in 30 seconds–thank goodness for that. Practice truly does make perfect, and I can navigate through the lab and procedures with more confidence and much more efficiency now than I could on the first day of lab.

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Third Time Isn’t Always the Charm

Why is my lysate so much more yellow than everyone else’s?

Pretending to not panic, I casually checked out all of the other lysates in the rack. They were mostly clear, some with a faint yellow. Yet, there mine was, a distinctively darker yellow.

As I retrieved my lysate and went back to my bench, I noticed that I did not have any leftover phage buffer from last class. In order to collect the lysate, I flooded my webbed plate with phage buffer, allowed it to sit, and then collected it into a separate conical tube. At least, that was what I was supposed to have done. In place of where I thought I would have my leftover phage buffer was a tube of yellow enrichment broth, and my heart sank.

I had flooded my webbed plate with enrichment broth rather than phage buffer.

Since I already threw away my round of plates that were from serial dilutions, I had to perform serial dilutions and plaque assay again.

The number of times I have messed up in this lab is more than I would like to admit, and it seems as if I had a talent for making mistakes at every opportunity. However, as the end of November loomed upon me, I realized this was no laughing matter. I was running out of time, and just when I thought I had my lysate and could progress along, I had to mess up.

There were no alternatives – with all my materials gathered once again, I performed serial dilutions and plaque assay, followed by flooding the webbed plate. I ensured to add all the right materials, and finally, I got my lysate. I thought the worst was over, since I only had to obtain my EM images and extract DNA next. I found comfort in the fact that I will only be getting results from here, and ensured to be extra careful in extracting my phage DNA in preparation for gel electrophoresis.

Some time between the first and second round, I handed over some of my lysate to (hopefully) obtain EM images. Four other classmates and I went at the same time, and I was the last to get my image since I spent the first part of class obtaining yet another DNA sample. As each person got their EM images successfully, my apprehension of not getting anything concrete only amplified exponentially. My heart raced as the professor proceeded to examine mine, and the encompassing darkness only contributed to the tension.

“Look, another Myroviridae!”

Words could not explain how relieved I was to know that I did get something. I was, once again, hopeful for the future of my phage, only to be disappointed by my lack of DNA data from 3 rounds of gel electrophoresis.

To briefly summarize my dismay:
– 1st time: no band
– 2nd time: no band
– 3rd time (even after the TA’s tried DNA precipitation over Thanksgiving break): no band

Fortunately, there were many others in the same boat as me, and Dr. Fisher told us that the purpose of getting DNA was really only for whoever will be sequencing his or her phage. While I was slightly disappointed initially, looking at my EM images reminded me of how far I have come in both contributing to this SEA-PHAGES project and in cultivating my scientific self.

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The EM image of Phomnie (Phomnie is the name of my phage)

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Phage Hunting: Big Wins, Big Losses

Being a phage hunter for two months now, I’ve come to realize that success does not come without failures, trials, and mistakes. That’s exactly what I felt as I searched hours on end for plaques in hopes of someday yielding a phage.

Luckily, I was blessed with a good sample right from the get-go. A few days before class started, I learned that I had to go out and find a soil sample somewhere and bring it to class. I was thinking about all of the places I had been since I had moved into Baltimore a couple of weeks prior; I was thinking anywhere from the famed park behind campus to Camden Yards, where I had gone to my first Orioles game just a few days prior. Of course, it seemed like a long shot going all the way to a baseball game just to collect a soil sample, so I had to resort to a closer location – a MUCH closer one. In hopes of being different, I went out as far as I felt safe walking. At the time, I didn’t know how to get to the park behind campus, so I instead decided to just pace around until I wound up in front of the Wyman Park Building, just across the street from Mason Hall on the other side of campus.

My first few days in phage hunting were guided by wishful thinking and being hopeful for the best results. Doing my first streak, I was looking for every spot possible on my plate that looked like it could have been a plaque; over the course of time, I learned that some of the spots I was streaking from came from cracked agar, and wasn’t anything special to my results. After only a second try with the same sample I started with, I yielded what appeared to be plaques… lots and lots of them. I was excited, thrilled at the possibility of (eventually) finding a phage.

That one soil sample I found in front of the Wyman Park Building took me through a journey of exploration and discovery, all mostly occurring on my lab bench in UTL G72. I spent weeks streaking, trying to find good results, failing, making silly mistakes, and questioning my progress in the class. If it weren’t for the people around me, from those dreading at their lack of evidence to those making phage puns all class, I feel like this journey would not be possible. While hunting for phages may seem to be dreadful at first, it actually felt like a beautiful struggle. Although I was making mistakes, I was learning more about myself as a scientist than I ever have in any high school lab. The feeling of independence and the desire to obtain solid results is what motivated me to keep trying throughout the class. Now that I have some EM images of my phages does not mean that I have succeeded completely just yet. I still have some room to make mistakes, challenge myself, and discover more about the scientist within me. It’s a long journey, but it’s totally worth it. That Halloween afternoon when I saw actual images of the phages I had been attempting to isolate for so long, I knew that my hard work paid off.

What advice can I give to any future scientist? To put it bluntly, you will have times when you will fail. However, failing does not automatically mean complete and total failure. It is what you make of your failure that determines how well you will be able to execute the rest of your trials. I learned this from countless days of streaking incorrectly, doing serial dilutions, and having several missed attempts at a successful DNA prep. Don’t think that it’s the end of the world just because your lysate didn’t yield a high titer (in fact, I actually had a high titer at first – 3.47 x 10^-9 pfu – but I somehow left my lysate out over the weekend, and it may have decreased my titer significantly). Don’t let contamination hold you down either; my plates were contaminated and I was still able to streak from areas without contamination with successful results. As long as you go back, acknowledge what you did wrong, and work to fix your mistakes, you’re on your way to becoming an ideal scientist.

I’ve taken so many things and learned so many lessons from this class. It’s not just about phage discovery, but scientific discovery as well. It’s exciting, it’s challenging, and can even be tiring at times… but it’s totally worth it.

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There Are Plenty of Other Phages in Baltimore

“There are plenty of other fish in the sea,” applies to many situations, sharing optimism with someone feeling lost and hopeless. We know it to be true: there have to be other fish out there, since you have a whole sea at your disposal. I wish someone had told me this after my first failed dirt sample. Encouraging words like, “there are plenty of other phages in Baltimore,” would have eased my initial fears of perpetual phageless-ness. There had to be other phages, since I had the whole city of Baltimore (and technically the whole world) at my disposal.

My dirt sample, which I excitedly collected on the first day of classes, was taken from the garden behind the Center for Social Concern. I immediately started the direct isolation procedure, trying to extract coveted bacteriophages from the dirt. After plating my liquid from direct isolation… Nothing. No observable plaques to show that I had any phage concentration. How? In a world with a bacteriophage population of 10^31, how did I manage to find the one patch of dirt that had nothing?

I still don’t know. So many different things could have gone wrong with that one dirt sample, but instead of sitting around and hoping for plaques to magically appear, I went out and got another one. This time, I searched close to my second home, the Undergraduate Teaching Labs, and took a sample from right outside the door I walk in every Monday and Wednesday afternoon.

This was my second sample, and it slowly but surely turned into a success. Nothing compares to the feeling of seeing your first plaque, knowing that you found this phage, your phage, and continuing to isolating it. 1 Enriched Isolation, 5 serial dilutions, 12 streaks, and what seemed like a million plates later* – I had isolated my bacteriophage. Sometimes, when you’re searching for something, it’ll be right in front of you and you won’t even know it. I was looking for bacteriophages, and I found mine steps from the building where I started my search.

After isolation came visualization. We had spent most of the semester talking about phages, working with phages, praying for phages – but I had never seen one up close. Sure, there were intriguing pictures from phage-related research online, but it wasn’t the same. Viewing the electron microscopic image of my phage was surreal. I had gotten just what I wanted: my very own bacteriophage.

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The EM image of my wonderful phage

I registered for Phage Hunting over the summer, thinking I would go to lab twice a week, follow a few procedures, and continue on with my day unaffected. I was wrong. Phage Hunting is a class of discovery, independence, and genuine enjoyment. I look forward to my two labs every week, more so than any other class. I walk in and immediately feel like a scientist, not like a student absentmindedly following someone else’s calculated steps. Every new finding yields celebration and excitement, and every failed result offers opportunity to improve and analyze. I believe I am a better scientist, student, and thinker for having taken Phage Hunting. In a world full of bacteriophages, I isolated my own. In a university full of classes, I chose Phage Hunting; I would do it again in a heartbeat.

*Disclaimer: Numbers slightly exaggerated. I only used 999,974 plates.

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What phage hunting has taught me about life

–Wendy Xie

“This is too cool! It looks exactly like the phage picture in my middle school biology textbook!” This was the first reaction after I saw my phages under the electronic microscopy with my own eyes. With all the efforts made in 8 weeks, I finally got to see the tiny little creatures that I separated from the soil independently.

 

During the whole process of separating the plaques and purifying the phage, it is true there were a lot of difficulties that I have gone through: contamination happened every now and then and it was hard to get isolated plaques from direct streaking and so on. The most disappointing of all is that, sometimes even though you have done everything correctly according to the protocol, still you don’t always get the result you have expected.

 

I picked four plaques of different morphologies and carried them all through the whole purification process until the third generation, but unfortunately among four of them, two third-generation plates were not completely pure and there were plaques of inconsistent morphologies on them. For the other two third-generation plates, I collected lysates from them but it turned out that the concentration was not high enough so constantly I wasn’t able to have enough DNA when running the gel.

 

I got quite upset when my third trial of the gel electrophoresis still didn’t turn out very well, but after calming down and thinking about it, I started to realize what all these “failures” have brought me. There’s some similarity between life as a researcher itself and the small, beginner-level phage hunting project we are doing right now: It is true that sometimes even though you have done everything right and followed exactly what the protocol said, things just does not turn out in the way you want to be. It is the same in research, or even in life itself. There are always too many factors that we have no control over which influence the results, but still it is worth working hard and wishing for the best. Similarly in life, there are too many things we can’t control but failures in life do not make it not worth trying. More importantly, most times it doesn’t matter that much to get the successful results–what really matters is to learn from the experience. From the phage hunting, no matter whether I will be able to successfully analyze the DNA features of my phage in the end, I have learned a lot about basic lab skills in molecular and cellular biology, have built a lot of friendships with all my peers in lab and have had such a fun time exploring the micro-world looking for tiny little microbes which we barely care about in daily life, and I truly appreciate all of my gains from it. I never realize how much I do love all the hands-on researches, even the “annoying” bench work, until I actually have made a lot effort looking for the phage.

 

Phage hunting is truly my favorite class of this semester. It really leads me to do some real science and discover something new rather than seating in the lecture hall learning things that have been discovered previously. It is true that scientific researches can be tedious, but one professor from JHU once I talked to told me that, “despite the fact that, among all the experiments you conduct, a great portion of them just do not turn out so well, but still it feels so proud of yourself to think that you may be the only person in the world who is doing research in this field and discovering something nobody has found before”. Even not sure if I will be a researcher in the future right now, but phage hunting is definitely a good experience and start-point.

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