“Phalaphel” Emerges

By Suborna Panja

After 4 months of nonstop plating and re-plating, my phage “Phalaphel” has emerged! While the idea of discovering my own phage seems exciting, there has been an incredible amount of frustration. To all those future phage hunters out there:

1. Don’t be afraid of discouraging results. It happens to all of us.  For example after multiple rounds of streaking to isolate a pure phage, I ended up discovering I was unsuccessful when I plated my MTL. As a result, I had to do the entire streaking process over again.

2. You will become very familiar with contamination. Pretty much everything we work with has to be sterile. Even the smallest bit of exposure to non-sterile items can contaminate your plate.

Once I got past the endless amount of streaking and repetition, all the hard work finally paid off with a successful MTL and then later HTL. The HTL is the key to determining tons of characteristics about the phage. So once I got to this part, most of the repetition was over.

Nothing is more rewarding than using the HTL to see my DNA concentration and then finally my phage! I admit, because of the constant amount of errors I have run in to, I was partially convinced that I would have no phage. I thought that perhaps I screwed up at the last step and had nothing. But to my excitement, my phage was clearly there!
Phages look nothing like I thought they would look like. Perhaps I had this preconceived notion that bacteria looks like blobs of DNA enveloped in some sort of plasma membrane. Well, in fact, that’s not the case with phages. Phages have round heads with tails coming out of them. The size of the head and length of the tail varies per phage; my phage happens to have a 50 nm head, and 230 nm tail. The first time I saw my phage, its round head reminded me of a falafel, hence, I named my phage “Phalaphel.”

Well, the end is not near, it is here. It’s been a long and unexpected journey filled with many scientific obstacles, but persistence is the key to success. Phage hunting has definitely been one of the most challenging projects I’ve ever participated in. The idea of not only characterizing something that has never been discovered before, but also characterizing something that you can’t even see seems a bit impossible. But you’ll be surprised what you’ll find from a simple sample of dirt.

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Tears upon T–Just Kidding; There is no Crying in Phage Lab

By Michael Shang

It’s been an eventful month and a half. When we last left off, a new morphology had cropped up in my MTL dilutions (despite the previous step having a seemingly pure population and after it had already been subjected to around 8-ish streakings), and I was determined to find out what caused it. Weeks later, much information has been gleaned . . . but none has helped me move forward. Alas, after a semester of lab, and after coming in every day, Monday through Thursday, to squint at my plates and curse the Phage Gods once again, little progress has been made.

And yet, the information I’ve found is invaluable — the phages that make the new big plaques take two days to incubate, as I found out from coming in every day (e.g., I would plate the big ones on Monday, come in on Tuesday and notice that they made tiny pinpricks on the plate, and then come in on Wednesday and see their size explode to the usual “big” size). And, perhaps most important, I’ve found that yes, the two different morphologies are indicative of two different phages, rather than one phage behaving differently.

That information came, funnily enough, from EM–electron microscopy–which is supposed to be one of the last steps we take in the process of phage hunting. Instead of doing EM on an HTL, I ended up doing it on my MTL.  And the results are simultaneously encouraging and discouraging. Because the tails are 20 nm apart in length (190 nm vs 210 nm), the two plaques are definitely indicative of two different phages. The good news is that it confirms my suspicions; the bad news is that it means I have two phages. Another interesting thing: even though there seems to be so much more of the “small” phage, when I went to do EM, the big one was easily visible on the screen; finding “smalls” was actually quite hard to do (it didn’t seem like there were many of them).

Alas, after sticking with my small inverted-bullseye-morphology-phages for an entire semester, I’m forced to conclude that I must switch to the big ones. The decision was not an easy one, and I almost shed a tear — kidding, kidding (I exhausted my reserve of those a long time ago).

Even if I don’t get graded for it, though, I still want to figure out what’s going on. Even though I’ve had a myriad of woes, I think this is the coolest thing about science: when stuff doesn’t go your way and weird stuff pops up, you could be on the brink of discovering something. Anyway, let’s hope that, next semester, I’ll have more luck.

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The End.

By Meg Chen

“Please collect a few spoonfuls of dirt in a ziplock bag and bring the sample with you to the first day of class.

It’s been a long, yet quick journey.  The path from a random soil sample to a beautiful, unique phage feels so unreal.  But now that I come to think of it, it must have taken much longer than three months for the first researcher(s) to experiment with different procedures for isolating plaques and develop the most effective one.  For example, who came up with the idea of streaking a plaque?

One of the most important things I learned along the way was lab technique.  Early on, I would stay past 5 every day, checking my work at each step.  Yet I would still end up with contamination.  I figured that since there was no way to keep every single phage out of the microcentrifuge tubes, I resorted to methods to eliminate outside phages, such as cleaning my tools with CiDecon before putting my phage solution in and working closer to the flame.  One day, I actually worked dangerously close to the flame and burned a hole through my gloves (though the fire went out quickly and did not get to my skin).  Even though I successfully eliminated outside phages from my tubes and plates, I realized that I would have to find ways to minimize contamination instead.  For example, instead of reaching into the container to pick out microcentrifuge tubes, I learned from Dr. Fisher that I was supposed to dump them out and close them quickly.

I slowly grew accustomed to the method of sterile technique through trial and error, and by the third week, I was out of the lab by 4 most of the time.  I still remained skeptical that my plaques all came from phages in my enrichment plating and not from the surrounding air, and I saved 2 plates for each round of streaking and dilution series so that I could go back and re-streak if I run into contamination another time.  After putting top agar on my negative control plate, I would shut the lid quickly, sometimes shattering the outside of the petri dishes.  I always took one more tube to pick up the top agar with than I actually needed in case I accidentally touched the tip of one tube to a foreign surface.  When our whole class experienced contamination due to the old smeg, my plates with top agar and phage buffer alone did not have any contamination even though I had been slightly less careful.  This was when I realized that I had been too paranoid all along, and that the chance of my phage coming from the air is lower than what I originally expected.

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Webbed Plate of Lies

By Megha Telur

I can honestly say that Phage Hunting has been my most enjoyable class this semester. Everything about the class just clicked; research, lab work, and biology. I aspire to one day be able to do genetics research at Hopkins, so being in a UTL biology lab was an absolutely amazing feeling. It seems strange to many but I love being in the lab (I also love long airplane flights!) it’s where I feel comfortable and strangely enough, at peace. The wonderful huge open windows added to the charm of my second home, the UTL.

As much as being in a Johns Hopkins lab thrilled me the first few weeks of phage hunting were overwhelming. I found that I was extremely dependent on my lab manual and the TAs to get through basic procedures. With time the process became more and more intuitive and I was able to make judgement calls on my own. I knew when I had isolated my phage,  I picked plaques to streak, and I knew when additional plating or diluting needed to be done. Nonetheless, no matter how much progress I had made, nothing could prepare for the ultimate test of patience: the webbed plate of lies.

I calculated and diluted several times to no avail. My web plates never came out right!!! Many times there was hardly any lysis much less complete lysis of the bacterial lawn on the agar plate. After weeks of no success (I plated web plates at least 4 times) and discussions with Dr. Fisher and Dr. Schildbach , I finally flooded my partially webbed plates for an HTL. It was determined the initial concentration of my phage was so high (I couldn’t successfully calculate an initial titer) that the risk in flooding partially webbed plates was minimal.

Thankfully, the risk paid off in the end. Not only was I able to quantify enough DNA, but I was able to complete the procedures on time. This experience was a quick but effective lesson in independence and strategic decision making. I realized that the manual doesn’t have all the answers and deviating from the standard procedure is sometimes necessary. If there is substantial evidence and solid reasoning creating your own path may actually lead to the best results-especially if it means avoiding a dreadful webbed plate of lies.

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A Lesson in Patience

By Isabella Zellerbach

When I went to my first Phage Hunting class, I was over the moon. We were following a lab manual that made sense, doing science-y things, and I got to use all sorts of equipment that made me feel like a real live researcher. And I was good at it! I got my enrichment culture to work, had two distinct phages that were really different, and even managed to isolate my phages in just a few streaks. And then disaster struck.

A new phage decided to pop up on my plates and I had to re-streak. And re-streak. And re-streak. And I was streaking until the end of September. I finally got my phage isolated and moved on to my MTL and things seemed like they were taking a turn for the better. I calculated the titer, got all my ducks in a row, and did the calculations for the six web plates to create the HTL. Then that mysterious, unknown contamination hit the lab.

Even though I had six beautiful webbed plates underneath all that contamination, there was no way I was going to be able to flood the plates and create an HTL. So I re-did the six plates and waited to see if the contamination was gone. Luckily, it was! And so my HTL was born.

After that, I had a fairly smooth ride. My EM came out nicely, my DNA prep was a solid number, and my QC gel was bright and vibrant! It just took me until the very last day of class to get there, but I made it in the end. The entire process definitely made me re-evaluate my patience level and I’d like to think I came out of the experience way more patient than I went going in.

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To Phage or Not to Phage?

By Stephanie England

Summer before coming to Hopkins, so many things were on my mind. From trying to get to know my roommates via Facebook to making sure all my paperwork was in, the question of classes came to mind. Being one of the hundreds of pre-med kids, I wanted to try to step into the waters of research before everyone else rushed in. Phage Hunting came up on the list of recommended classes and it piqued my interest. However, so many other options were present and then I got to the freshman seminars and then timing conflicts. Overall, phage hunting was of least importance to me and it was going off the list if times conflicted or if the dreaded message came that I got wait-listed. Morning of registration came around, and luckily for me, my ideal schedule is what I got. Yet, I couldn’t help but wonder if taking phage hunting was the right decision or if I could have better used my two credits.

Now that the semester is coming to a close, I can definitely say that taking phage hunting was one of the best decisions I ever made. This lab is my favorite class and I’ve gotten to meet some of my closest friends here. The roller coaster of emotions that this class kept me on was something I never expected. Initially thinking that my enrichment failed, but then seeing plaques come from my one dot (that I had thought to be an air bubble) on my 100; this was a foreshadowing of the doubts and fears that would be dispelled from the reality of getting actual results and success.

It’s been a bumpy road, but the skills learned in this class are more than just commendable. Proper aseptic technique was definitely achieved, I mean, when there was class contamination from the old smeg and we all tested our PB and TA and not one of us contaminated our plates? Talk about impressive! Feeling the camaraderie after realizing that I wasn’t the only person who didn’t make it in time for the phage Olympics was just one of many moments that proved phage hunting a great decision. Every time I was able to streak without breaking the gel in the bottom, was such an accomplishment. I could be failing my math class in the morning, but then go to phage lab in the afternoon and just seeing plaques without contamination, would turn my day around for the better.

Later on, going in for electron microscopy was motivating, making me think that I was in the final stretch. Then a feeling of dread came when Patrick made a mistake with my sample and it had to be redone. Foreshadowing much? I hoped not. It was redone and a flood of relief came. Seeing my phage on the EM pictures, and knowing that all the streaking, all the serial dilutions, and all the hours spent were not in vain, was a moment where I could have shed a tear of happiness. My little buddy gave me such hard times in the past, but I’d do it all over again now that we’re at the end and see the results.

Now that it comes time to choose classes for next semester, phage hunting was the first class I signed up for and had to work other classes around this one. The thought of sequencing and working with DNA and genes is exciting to a nerd like me, and then being able to do our own experiments with the phages? It’s going to be great. My phage may not have made it to the phage Olympics, and it may be an equally (if not more) bumpy road next semester, but there is no doubt in my mind as to what I want to do. If this class is half as good as it was this semester, then I’d be satisfied. To Phage or Not to Phage? There is no question anymore.

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The Call of the Phage

By Gregory Konar

For those of you who picked up on the title playing on “The Call of the Wild” by Jack London, kudos to you, you must be either a writing sems major or a very well educated other person!

But I digress, already, this post is not about Jack London or literature at all, this is about the wonderful and shockingly populous phage!

This here is a picture of a very sinister looking phage, perhaps he is participating in No Shave November? There are many other phages just like him, thousands of millions of billions of phages exist on the earth at any given moment. In fact, as you read this blog post, you might have typed on a cluster of phages hidden in bacteria on your keyboard! Eww, go clean your keyboard please! All good now? Alrighty, let’s continue on this phage journey!

Phage Hunting is an absolutely amazing class that for me runs Mondays and Wednesdays from 2:30 to 5:00. Yeah, big time commitment for a 2 credit class, but trust me when I say that it is worth it. It is single handedly the greatest class that I have taken this semester! (and no, I am not saying this just to appease the almighty powers that I call Dr. Fisher and Dr. Schildbach)

So, how do we answer the call of the phage? Well first, we had to find a phage!!!

(wise words of advice!)

Soil was our medium of choice for finding the phage, so off I galumphed searching for the perfect patch of dirt, tedious tedious work! It took me a whopping 5 minutes to locate and dig up soil necessary for the lab, and it ended up being pretty darn nice soil!

I tried 2 different methods of coaxing phages from the soil sample: Direct Plating and Enrichment. Direct Plating was like making a pizza, the agar plate was the dough, and then I added the sauce in the form of Mycobacterium smegmatis (the friendly cousin to tuberculosis) and the cheese was the dirt. Enrichment was like steroids, we juiced up the concoction with lots of food and goodies and let it fly. Low and behold, only the enrichment worked, and the pizza had nothing to show for itself.

Once the enrichment provided some putative phage plaques (no, not the bad plaque that forms on your teeth), I went forward and plated and replated and replated the phage 4 times until the plaques were all nice and stable in morphology. Then the titering began…

And as chemistry cat so eloquently puts it, I needed the concentration of phages present on the plates. So the plates were flooded and liquid stocks of the phages were created. This was done once on a small scale to get the MTL and then again on a much larger scale to get the HTL. From the HTL the real fun began!

The HTL unlocked a whole new world of possibilities! From there I did a whole host of things ranging from DNA Preps to Restriction Enzyme digests to Electron Microscopy! The possibilities were endless, and everything was amazing and fun and eye opening! My phage baby  had a 48nm head and a 160nm tail, which I thought was pretty darn awesome!

Konar_EM

I haven’t ever used an electron microscope before, so this was a new experience for me!

As of the time in which I write this blog, I am busily preparing for the Phage Olympics on top of an RNA presentation, Chem Test, and panic over class registration. So, it will be a fun and great experience on Monday at the Olympics, hopefully my baby will be in tip top shape for that and will bring home the gold! Let’s see if it can answer the call of the phage!

Oh, did I mention that its name is Cell-Fie?

Until next time! Phage out!

Posted in From the Phage Hunters