The Hunt for Phage

By Evelyn Chiu

I had no idea what I signed up for when I decided to add phage hunting to schedule while browsing through courses back in July. Probably something along the lines of looking at a microscope, furiously prodding at a pile of samples to try to find some phages.

The first assignment that we got for this class was to dig for some dirt. Having taken AP Environmental Science back in high school, digging for soil samples is nothing new to me. Obtaining dirt without a shovel, though, was a new experience. I imagine I was quite a sight, squatting in the sculpture garden furiously smashing at the ground with the blue plastic silverware they gave us on move in day.

Our goal in this class, basically, is to try to isolate a pure phage sample from the soil we got. At first it sounded easy, especially when I was reading over the lab manuals. Filtration, pipetting, plating . . no problem, I’ve done all those before in previous research experience. Except, as I soon found out, what we’re aiming for isn’t all that easy.

What we all want

Some things that we have done these weeks include direct plating, enrichment, and streaking. Lots, and lots, of streaking. Like almost everyone else, my direct plating was a failure and yielded no results. My enrichment culture, however, gave me two plates that I could work with. I then proceeded on with my task to isolate a pure phage by streaking, streaking, and streaking again. Except, I ran into quite a lot of problems. First, it was contaminated top agar. Then, it was cracked top agar. Then contaminated streaking sticks. And of course, the dreaded incidents where there are no plaques present, except for one . . . that always turns out to be an air bubble.

Everytime

After 5 attempts at streaking, I have finally come up with what appears to be a good line of plates that is likely to produce three generations of single phage populations. They’re roughly medium size, with hazy edges and a bull’s eye morphology.

The family tree

Hopefully, my next couple of streak tests will be successful and I will be able to get an isolated and pure population soon. This class has been fun so far, and I am quite excited to see which phage I will end up with, as well as which line our class will ultimately choose.

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Looking for Phages

By Fion Shiao

On the day of convocation, my friends and I went to a walking trail near school where I decided to obtain my dirt sample. I chose a spot full of vegetation thinking I’M GOING TO GET A LOT OF PHAGES! Being extremely-self conscious, I sat there as I used a spoon to transfer dirt into my plastic bag.

On the first day of lab, I was very excited about getting my own lab coat, my own lab bench and my own glasses. Following the SEA Lab Manual’s instructions, I extracted phage from my soil sample by adding PB buffer and filtering it. I then did direct plating by infecting my phage sample with M. smegmatis and plating them on agar plates. At the same time, I also prepared enrichment culture.

The next lab day, I checked my direct plating for results, and I sadly had no phages. Fortunately, there was still hope in my enrichment culture. I diluted my enrichment culture into different concentrations and plated them. Checking my results the next class, some of my diluted plates had phages. I then picked four distinct plaques with different morphologies and streaked them on plates before adding M.smegmatis and Top Agar with the aim of isolating phages. As an extremely creative person, I of course named my plaques A, B, C and D.

I thence began my endless journey of streaking. In this ongoing process of streaking, I unfortunately had to abandon A and B. After looking at my third streaking, my type C and D plaques still had different morphologies, so from C and D, CA , CB , DA and DB were created. After streaking for four times, one of the TAs pointed out how my plaques are all too concentrated. As it turns out, having too many phages is not always a great thing. He advised me to try a method, which worked for another student with the same problem. Following the method, I used a stick to dab on the plaque I wanted to isolate and swirled the stick in 100 μL of Phage Buffer before streaking. Last class, I checked my plates, and the dilution technique worked!! However, my plaques’ size still varied so much that I streaked a big plaque and a small plaque from each plate to test whether the sizes vary because of the phage’s morphology or because the different sized plaques come from different types of phages. With a bad habit of not throwing out my old plates, my stack of plates has been growing higher each class while I await for my plates to yield plaques with identical morphologies.

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The Streaking Continues…

By Catherine Yin

It’s been three weeks since this class has started and I’ve already learned so much. I know, this sounds so cliche, but it’s true. Even the first day of class overwhelmed me as we got to work with lab equipment I’d never handled before. And the first time I saw my plaques? I swear, my eyes started getting misty and my maternal instinct kicked in.

So far it’s been an eventful journey to find my one true phage. Direct plating didn’t yield any results, sadly, but my enrichment cultures didn’t let me down and that’s when the streaking began. Today was my fifth time streaking and I’m glad to see my progress from generation to generation. There were some rough times in between (really hazy phages that I could barely see and contamination on my agar plate) but they’ve been isolating at a nice pace. Although all this streaking can get repetitive after a while, I feel excited and giddy about finally getting to meet my phage!

I didn’t really know what to expect when I signed up for a class called Phage Hunting but I’m glad I did. I hope you guys are all basking in this journey of finding your own phages. And now it all comes down to which phage will ultimately be chosen? *Cue suspenseful music*

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A Day in the Life of a Phage Hunter

By Katie Link

It’s Tuesday, September 16th, and the mid-morning sun shines through my cheap dorm curtains and gently awakes me from my long, restful slumber. Most days, my alarm clock unwillingly forces me out of bed and into hours upon hours of lectures, homework, and awkward encounters with fellow freshmen whose names I have forgotten.

Tuesdays and Thursdays, however, are different, and it’s all because of a class called Project Lab: Phage Hunting. Not only does the class start at the lovely hour of 1 pm, leaving the mornings to work (or more likely sleep in), but it also already has become one of my most enjoyable classes this semester.

Direct Plating of Enrichment Sample: Success!

Direct Plating of Enrichment Sample: Success!

I decided to enroll in Phage Hunting way back in July for two primary reasons: one, the 2-credit course fit perfectly into my schedule, and two, it appealed to the part of me that hopes to explore the world of scientific research. Though my prior research experience is limited, one of the reasons I decided to come to Hopkins was the vast number of research opportunities available to undergraduates. When I saw that there was a course that allowed me to start to work in a lab my first day of freshman year, suddenly my prospective schedule filled with huge intro-level science courses looked a little more exciting.

Flash forward to today, 1:15 pm. With my lab coat, glasses, and gloves on and lab bench sterilized, I’m ready to take on the world (or at least a few bacteriophages). These past few weeks have been all about taking a hunk of dirt and enticing a select few phages to become isolated on their own separate plates. Spoiler alert: it’s a lot harder (and also more exciting!) than it sounds.

The motto of a new Phage Hunter

Although the direct plating of my sample did not produce any plaques, the plating of my enrichment sample has been going very smoothly. I feel like a proud mother, as my phages seem very well-behaved. When the plates from my enrichment dilution first showed beautiful plaques of multiple kinds of phage, I was so overjoyed that I emailed my mom back home pictures of my plates. Today will be my fourth day selecting phage plaques from the previous class period and streaking them on agar plates, and I’m excited to see what last week’s plates look like.

1:25 pm: After evaluating my plates from last time, at least two of my plates might have isolated phage! Could this possibly be the end of streaking (and a seemingly endless number of agar plates)? This excitement is short-lived as a TA has informed me that I’ll most likely need to replicate that result another two to three times. It’s also clear to me that some of my earlier plates have plaques of different diameters, indicating different types of phage. It’s back to plating.

Another 20 minutes or so pass, and the rhythm of the lab has consumed me. It has only been three weeks and I already feel comfortable using the equipment independently. The excitement of working with such “grown-up” equipment, however, has certainly not worn off. It’s definitely a step up from the equipment used in my high school science courses. Just a few weeks ago, my hands were shaking as I began to use the pipetting equipment for the first time. Now, after nearly a month as an official Phage Hunter, I am confidently working on auto drive as I streak my plates and then “feed” my baby phages with a hearty serving of M. smegmatis (and top agar). Yum.

With my plates finished, I finish up writing my observations and procedure in my black, hard-cover, official-looking lab notebook. This is another area in the realm of research in which my self-confidence has increased substantially. Even though I was self-conscious about my laboratory writing skills in the beginning (and my drawing skills are not up-to-par when drawing diagrams), these last few weeks of practice have certainly honed these skills.

My work here is finished, and after sterilizing the bench one last time, it’s time to leave the lab for the day. While it’s tough leaving my little phages behind, I’m hoping my luck as a Phage Hunter continues and today’s plates will show isolated plaques again. No matter what happens though, I know I’ll always be looking forward to my next day as a Johns Hopkins Phage Hunter.

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Phages!

By Heather Han

When I was looking for potential courses during summer, I knew that the Phage Hunting class would be the right choice for me. For how could it not? Throughout high school, my favorite class had been AP Biology, not because of the interesting lectures as much as because of the numerous labs that I had been granted the opportunity of doing. The ability to perform labs in which we did experiments from modifying E. coli plasmids to breeding and learning how to identify the sex of fruit flies (Drosophila melanogaster) fascinated me.

Yet, upon learning about the first assignment, I began to have some doubts about the class. Digging dirt? Why would I ever want to do that? But the first day of class absolved some of my doubts. After spending only half an hour in the classroom going over the syllabus, we moved to the lab and immediately began to prepare the enrichment culture. I was pleasantly surprised. I did not expect to do as much as I did the first day of class (even though we did have to stay after class for a while). But what surprised me the most was that I got my own lab coat! My own PERSONAL lab coat, a lab coat only for me! Having my own coat made everything seem that much more official.

Although direct plating did not initially yield any phages, my enrichment culture soon did. We prepared our enrichment culture by transferring about 2 teaspoons of soil sample to an Erlenmeyer flask, then adding 40 mL of sterile water, 5 mL of sterile 10C7H9/glycerol broth, 5 mL of AD supplement, and 0.5 mL of 100 mM of CaCl2 to it. On the second day of class, we once again transferred our sample, except that this time it was from the flask to a 50-mL conical tube. The TAs then proceeded to collect our tubes so that they could spin the entire class’s sample at the same time at 3000 rpm for 10 minutes. I then filter-sterilized my enrichment sample and transferred 1 mL of it into a microcentrifuge tube. Since directly plating the enrichment sample would most likely produce a plate covered in plaques if phages were present, we were instructed to dilute our sample before plating. In the end, we plated 6 plates, each containing 0.5 mL of M. smegmatis, 4.5 mL of top agar, along with either 50 µL of phage buffer (negative control plate), 50 µL of 100, 10-1, 10-2, 10-3, or 10-4 dilutions of the sample. We let the plates sit undisturbed for at least 20 minutes before the TAs went around to collect them and place them in a 37 degree Celsius incubator for 24 hours.

By the time of the third class, I had phages! While my 100 plate was completely covered with plaques – the entire plate appeared to be transparent, my 10-4 plate contained plaques that were more spread apart. In the end, I chose 2 plaques of different morphology (one of which was big and cloudy, the other small and transparent) from my 10-4 plate and streaked them onto 4 new plates – 2 of each kind – as well as a negative control plate.

We had to repeat the streaking process again on the fourth day of class in order to better isolate our phages. When I began the process of choosing plaques of phages to plate again, I noticed that there appeared to be plaques of two different morphologies on one of the plates. This means that I might have 3 different types of phages rather than the initial 2 that I thought I had. But I guess I won’t find out till the next class! Seems like there will always be new information for me to learn from Phage Hunting!

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Good company makes for good conversation: the 2014 SEA-PHAGES Symposium

Just about a week ago, I had the wonderful privilege of attending the 6th annual SEA-PHAGES Symposium. Situated on the gorgeous Janelia Farm Research Campus in Virginia, the symposium lasted for a whole weekend and hosted more than 240 students and faculty from over 75 colleges around the nation.

I was pleasantly surprised that everyone who attended was as excited about bacteriophages and research as I was. During dinner, Laura (one of my peers from Dr. Schildbach’s section) and I chatted with Carnegie Melon phage hunters about different ways to perform genomic sequencing. We couldn’t stay and talk for long, as we were quickly swept into a whirlwind of presentations and talks. Students, faculty, and even Dr. Graham Hatfull himself (a celebrity in the phage hunting world – he founded the national Phage Hunting program) gave captivating presentations about their own experiments and experiences with research. The guest speaker this year was Dr. Martin Chalfie, a Nobel laureate who shared the 2008 Nobel Prize in Chemistry with two other scientists for discovering and developing the green fluorescent protein (GFP). His presentation was not a lecture on the wondrous studies and experiments he had performed (which I am sure there are many), but rather he weaved a fascinating story about his path of scientific discovery and its implications on society as well as his own life. He did briefly discuss the logistics of his experiment and how it led to the ultimate discovery that GFP can be used to identify cells expressing specific proteins – his clarity of presentation was astounding to me, as even I could easily understand what a man of his caliber was teaching. However, the thing that struck me the most was his description about starting out as a young scientist, not unlike the ones sitting in front of him. He made it sound like research was easy to pursue as long as you were curious and determined enough. This opened up my eyes to a plethora of possibilities because, to be honest, I was never really quite certain that I would be able to find solace in the intimidating field of biological research.

However, my job at the symposium was not to just simply listen to and soak in all of these wonderful presentations – I also had the opportunity to present a poster about the experiments performed by my class during the school year. There were two sessions in order to accommodate the multitude of posters that were being presented. I was scheduled for the second session, so I used my free time during the first to talk to a few presenters and see what other schools were doing. Some schools decided to pursue independent projects in the wet lab, like we did, while others focused more on bioinformatics or applications of phage therapy. One of my favorite posters included a presentation from North Carolina State University, where the students there were working on finding a phage that could target Paenibacillus larvae, a type of bacteria that can infect and kill honeybee larvae, as a means of rescuing the dwindling honeybee population. It was so fascinating and inspiring to see that the research we are doing has an immediate application to the real world.

symposium2

The second poster session, during which I presented, sailed by smoothly enough, probably because our class had a poster presentation right before the semester ended, which left me feeling confident that I was relatively well prepared for any questions headed my way. I remember that one of the researchers asked me what the most interesting thing I learned throughout the course was. Looking back on it, he might have been asking me for an interesting fact about phages or even a question that our experiments left unanswered, but before I could reflect on his intent, I blurted out that I learned how hard research was. All the experiments we performed had procedures and guidelines and even expected results; however, along the way, plates got contaminated, HTL ran out, and many of my experiments failed. Despite not everything working out as expected, the most important thing I learned in class was that a contaminated plate is not the end of the world- you’re going to stumble a few times before you end up with the results that you want.

I am so thankful to have had the opportunity to participate in the Phage Hunting Symposium as well as the class itself – from dealing with contamination to actually getting to see my phage under the electron microscope to meeting people equally as excited about science as I am, I have been able to learn something from every single one of the ups and downs I’ve experienced in this class. Thank you to Dr. Fisher, Katie, and all the Mon/Wed Phage Hunters for making this class one of my most rewarding freshman year experiences!

sympos1

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Ode to Scientific Method

By Aviana Duca

Over my spring break, I was helping my little brother with his middle school science fair project, titled Ode to Snot. His project was about the effects that nasal mucus has on odor perception. For those that didn’t hear my brother’s hypothesis and abstract at the dinner table for a week straight, who knew that the much-neglected nasal mucus has such an important role in transporting odorants to receptors in the nose? He designed his experiment to have a control group, a test population, and a well thought out procedure to determine the accuracy of his hypothesis. I was able to shed some light on the scientific process and help him analyze and interpret data. Working with him allowed me to realize how far I have come as a scientist and this only excites me as I begin to work on my own experiment.

Working with my brother helped me to realize that no matter what age you are, what your experiment is, or what you are trying to prove, the scientific thought process is universal. The scientific method is a logical thought process that is used by scientists to acquire and interpret data. Experiments involve observation and two different kinds of reasoning strategies, inductive and deductive reasoning. Inductive reasoning is when broad generalizations are made from specific observations. This is helpful when designing an experiment and coming up with what to test. Deductive reasoning is when a scientist starts out with a general statement and preforms an experiment to reach a conclusion. The scientific method uses deduction to support or disprove the hypotheses.

What makes the scientific method so attractive is that it can be applied to both complicated problems and everyday occurrences. It is a step-by-step process that leads to a conclusion. While my brother’s experiment and my own are at two different levels, the steps we follow are fundamental to our success. Working with my brother helped me to organize ideas for my project and the direction that I want to take. I look forward to getting back into the lab and moving to more hands-on experimentation. I also look forward to see what results and data will be obtained and what discoveries will be made. Watching him go through the process of testing a hypothesis reconfirmed my love for science.

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