Gone Streaking!

By Ernest Ekunseitan

Phage Hunting this semester has been a roller coaster. It’s had its ups, downs, and everything in between. The most tedious part of phage hunting to this point was the constant streaking. By streaking, I don’t mean running through lab naked, but rather using sterile sticks to spread out the phages on agar plate (aka the less fun streaking).

Before we could start streaking, we had to go through the process of first direct plating (which yielded no plaques), enriching our samples, and then dilution where we finally caught our first glimpses of plaques. We could now begin our process of streaking.

Streaking is actually a fairly simple process. You begin with a sterile stick and touch this stick to a nicely separated and distinct plaque. You then use this stick to draw a horizontal line on the plate. You grab a new stick and draw a short vertical line down the center of the horizontal line. The last step is to grab another sterile stick and draw a “squiggly” line back and forth for towards the bottom. The entire process is simply meant to spread out the phage from an area of high concentration to low concentration in order to get distinct and separated plaques.

StreakPlate

We needed to streak until we obtained one plaque morphology and then streak 3 more times to prove that we only had one morphology. After proving that I only had one morphology, I was then able to go on to create my web plate, MTL, and HTL ( the real fun stuff). All that said Phage Hunting has definitely been my most interesting class this semester, much more fun than Intro Chem Lab, and I’m anxious to see how my phage turns out.

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The Trials and Tribulations of Phage Lab

By Myles Wood

Whenever I first walked into the lab, I didn’t know what to expect. Having no prior lab experience, my expectations were low, and my esteem lower. (Having an awesome lab partner is key, I would soon learn.) While reading my notebook, I remember that that everything seemed like a foreign language. It just didn’t make sense. I clearly remember getting to lab and worrying about every small little step. Many parts of the process soon became plagued with errors. As the lab progressed that day I remembered Russ say, “Don’t worry, man,” and I tried to believe him. This was the start of an epic journey filled with failure and triumph.

I have made my fair share of mistakes, and then some. However, it is from these extrapolations that I have obtained with an intimate understanding of the process and the biological underpinnings that constitute our efforts. I can say that I have repeated almost every step, and I can tell you with absolute certainty that this determination is what enables one to grow in phage lab. I have streaked plaques more than 2×1028 times, diluted phage concentrations for weeks, and attempted to calculate a “web plate” an even more shameful amount of times. It is from these trials that we grow and appreciate our efforts.

As of my progress, the journey has been long and the learning curve steep, but it has always been fun nonetheless. I began with a handful of dirt, and from this sample I have been able to derive a distinct bacteriophage. It is from this distinct phage I was able to make my first MTL (Medium Titer Lysate) and, more recently, I have was able to make a HTL (High Titer Lysate). With this precious concentration, I can begin to sequence the genome and use the electron microscope to further characterize my phage. When that day comes, I will fall to my knees and weep. But until then, the journey continues.

Posted in From the Phage Hunters

The Ten Commandments of Phage Hunting

By Miguel Tusa

Mistakes are a part of life in the lab, but at what point must we draw the line and say, “Alright, you really screwed up this time.” The answer to this might surprise you, because indeed there are more back-up solutions and quick fixes than one could ever imagine. Trust me, I’ve tried them all. While maybe it’s time for me to just clean up my act and be a little more careful, there is something to be said for those life lessons you learn through your second, third, fourth, and maybe even fifth time going through the procedure. With this said, I’ve taken the liberty of shifting away from telling an epic narrative of how my phage came to be. Instead, I offer a little bit of wisdom for those of us who just can’t seem to get it right.

I. Thou shalt have no other gods before the negative control.

 No matter how pointless you think this is, just do it. There’s nothing worse than having something potentially ground-breaking, but you just can’t decide if it’s actually just a speck of pesky M. smegmatis.

II. Never be afraid to ask questions, none of us know what were doing.

 As cliché as this phrase is, I couldn’t emphasize its importance more: not knowing something doesn’t make you an idiot; it just means you’re about to learn something new. Don’t pretend you know exactly what you’re doing after 2 months in the lab. I mean, we did start working 30 minutes into our first day of lab, on our first day of school. Ask the stupid question, then ask why it matters. The sense of scientific curiosity is addicting.

III. Thou shalt not take the Bunsen burner in vain.

It’s nothing personal, just a friendly reminder to watch what you’re doing. It’s okay to mess up, but being meticulous is crucial to success in the lab. Every time I look at the fading scar on my wrist, I push myself to be a little more aware of my surroundings and the way I am handling the equipment. Maybe someday I’ll learn.

IV. Remember the time after plating and keep it holy… especially if they’re your lab partner’s plates.

Never grab an agar plate by the lid. There’s no worse feeling than seeing the pain on your lab partner’s face when their plate crashes to the ground. Working with a partner is a valuable experience. We share successes and failures, tears of joy and of pain, but never ever syringes or pipettes. No matter how disastrous your combination of skills may be, embrace them and you’ll be surprised at what they can teach you.

V. Honor thy professor and thy TAs.

Without a doubt a greater resource than access to an electron microscope or any infinite amount of 5 mL pipettes. Don’t be intimated by Dr. Schildbach’s wizard-like glasses of knowledge or Russ’s fearsome beard. Whether they’re giving you advice on how to fix your screw-up, or explaining the theory behind it all, absorbing this information is a privilege. Although it seems like Russ’s practical knowledge is infinite, Dr. Schildbach probably knows his facts a little better –take it in.

VI. Thou shalt not kill your old plates.

Stop hoarding plates and make it easy on the people who make it all possible. It physically hurts to see poor Laura carrying that giant box of old agar plates. Being a considerate human isn’t necessarily included in the course description, but it’s an important part of the experience.

VII. Love your phage.

 It’s a little weird but it works. There’s nothing wrong with being slightly attached to these tiny nonliving parasites. Treat your phage with care, and they’ll give you the results you want. Maybe it’s just about being careful, but when you put so much work into enriching, diluting, filtering, streaking these guys, it’s hard not to be proud.

VIII. Stay optimistic, no matter how many mistakes you possibly make.

Sometimes you just have to embrace the absurdity of the mistakes you make in lab. If I can walk into lab happy about only being 5 days behind, then anyone can be hopeful. It’s an inescapable part of the job. If you can’t deal with failure, you’re in the wrong place.

IX. Celebrate the small successes; be excited about what you’re doing.

Enjoy those moments where, as I like to say, “you feel like a scientist.” Words could never explain what I felt the moment I proved that my phage titer was no longer contaminated and merely behaved in a characteristic way at high concentrations. You might not care about this, but ask me about it and I’ll have a blast telling you the details.

X. No really, “It’s fiiiiiiine.”

This expression is a favorite among our hardworking and ever-helpful laboratory TAs. While the average person might find comfort in this vague verbal reassurance, I happen to find this state of limbo quite unsettling. By now I have finally come to realize that Russ is right. There’s almost always a solution. The important part is that we keep on learning, keep on trying, and keep on applying our knowledge to the next problem we encounter.

For a guy who’s made pretty much every mistake in the book, at least I’ve grown from it. As Dr. Schildbach once put it, I’ve had a very “enriching” experience throughout my time in phage lab. I couldn’t be more excited to come back second semester and “enrich” myself further.

Posted in From the Phage Hunters

DNA Prep and Electron Microsopy

By Jinny Huang

Phage hunting is by far the most interesting class I have this semester.  It requires hands-on work rather than sitting in a small classroom or huge lecture hall for an hour trying not to fall asleep.  It is also much different from high school labs that I took, where the directions are super vague and they make you try to puzzle out the lab as you do it.  Also, the lab notebook guidelines are much more relaxed, and I no longer have to write all my data in pencil and then retrace it in pen in order to not get points taken off for inaccurate data.  I can proudly say this is the only class I’ve never skipped once the whole semester.

I actually started one class period later than the rest of my peers, so for the first few classes I was behind.  I managed to catch up during the streaking portion, because my phage was quite easy to isolate so I didn’t have to streak as many times as other people.

So far, my phage isolation process has been going pretty well.  I managed to isolate one distinct looking phage, calculate my MTL and HTL, and start the DNA prep and EM.  I just analyzed my DNA prep today and it seems as though my DNA is pure and I have a decent concentration.  I was actually pleasantly surprised by my DNA analysis results because I accidentally messed up a few of the steps of the DNA prep.  I also started the electron microscopy today; however, the centrifuge was broken so I have to wait until next time to finish it.

 

 

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Raising the Minimum Phage

By Michael Shang

Ah, Phage Hunting. That two and a half hour class/lab, filled with CiDecon art, rushing to snag Top Agar before it runs out, flagging down Dr. Schildbach and the TAs before someone else gets to them, and gathering as many plates, pipette tips, and M. smegmatis tubes as possible before having to turn on that infernally hot Bunsen burner.

It’s been an interesting year so far, to say the least.

From the first day of class, frantically gathering soil from the ground in front of FFC, five minutes before class (and breaking my spoon in the process), to the fabulous 5-cent disposable glass pipette tips (I was equally astounded by their inexpensiveness…and felt way less guilty for going through so many of them), phage hunting has far exceeded my expectations.

“Oh, that looks cool!”

“THAT’S MY NEGATIVE CONTROL IT ISN’T SUPPOSED TO LOOK COOL!”

(A typical lab conversation)

We started with direct plating (lol, did anybody’s work?) and making our enrichment culture. After filtering and performing serial dilutions, the entire class listened as Dr. Schildbach told us what we should notice — obviously, the plaques should decrease in number as the dilution becomes more pronounced.

And obviously, I should totally panic when my plates showed the exact opposite trend.

Fortunately, cooler heads prevailed, and I found that the reason for the weirdness was just the sheer amount of phage in my sample-that same sample found in the hard, dry dirt behind concrete near FFC. Go figure. So anyway, I just diluted further and everything was right as rain.

Yet for all the merriment and all the amusement, tragedy has also struck my phage hunting. After six — SIX — streaks, I proceeded to attempt to make my MTL. But lo and behold, new morphologies cropped up! In addition to the standard inverted bullseye morphology, there were new dots, about ten time the size of the tiny bullseyes that cropped up. At first, I asked myself,

Then I realized that I had nothing to worry about- after all, the big dots were quite possibly just the appearance given by a bunch of phage concentrated in one location. To assuage my fears, I both continued in my MTL journey as if nothing was wrong and streaked the big dots, to see what would crop up.

And then, the next class, I saw that they were TOTALLY DIFFERENT.

That means that there are at least two different phages with which I’m working right now! So as of now, I’m still trying to isolate the phage with the inverted bullseye morphologies-unlike most things, having the smallest amount of phage (that is to say, just one morphology) is ideal. Once I do that,  I can finally make that MTL, make an HTL, and eventually even image it!

Well, that’s all I have for now. Stay tuned for updates on raising the minimum phage!

Posted in From the Phage Hunters

Cute…?

By Jane Miglo

Ever since I dug up my dirt sample with a plastic spoon from the FFC, I’ve been contemplating various names for my virus. Due to my obsession with House of Cards and the persistent image in my mind of phages as ruthless beings at the time, I seriously considered naming my phage Francis… maybe Francis Underwood. After all, viruses are supposedly coldblooded, feeding on their host, viciously ripping them to pieces as soon as replicas of hundreds of the same savage creatures are ripened and ready to attack.

But of course, during electron microscopy, it was determined that my expected malicious phage was . . . cute?

At about 100 nm in diameter with an 85 nm tail, my phage is minuscule — certainly not worthy of a domineering name. Not to mention that the tail is shorter than the body . . . how does the phage propel itself efficiently towards the bacteria to devour it in the first place?

Few things go according to plan in phage hunting — just as you think you’re getting good plaques, you realize it’s your negative control . . . Michael Shang.  Or as you enter the lab preparing for the final step in the purification process (FINALLY!), crisis ensues — contamination. Or, when given the opportunity to discover a new bacteriophage, it comes out “cute.”

But in those moments, the rare, rare moments when all goes well, all is well. For instance, when your 11th streak comes out successfully, or when Miguel’s plates do not contain contamination, or when you don’t have to turn on your fire to create a sterile environment for a good hour into class.

All in all, the phage hunting is my favorite class. Every time I step into the lab I am reminded of how much I love science. The mere act of pipetting brings me joy. And, alas, I have come to terms with my little phage.  Persistence and determination brought us together — the name, however, needs work.

 

 

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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