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.

em_420-33-1

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

Posted in Uncategorized | Leave a comment

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.

Posted in Uncategorized | Leave a comment

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.

em-1-jpg

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.

Posted in Uncategorized | Leave a comment

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.

fullsizerender-8

Posted in Uncategorized | Leave a comment

The “Finding-The-Few-Spots-In-The World-Without-Phages-In-Them” Award Goes To…

Coming from a high school where getting A’s and quickly, efficiently working through labs was the norm for me, this class definitely was a little bit of a reality check. When assignments were dependent on intellect and consistency, hard work and studying was usually gifted with good grades. When labs required precise measurements, steady hands, and a calm demeanor, I was usually rewarded with accurate results. However, in great contrast, I’m infamous at my school for having the worst luck in everything else, what we call “RNG,” or random number generator. When I needed a 4, I would get a 67. I even recall my dad telling me why he’s not fond of investing in the stock market, because “his father’s genes don’t carry luck.”

This class requires some of that luck that I seem to lack, at least at the start. Come day one, I got my dirt sample, and was ready to do the direct isolation procedure, expecting to see plaques the next day in lab. When I did come in, I was already pretty sure that there was nothing there. However, I’m a very stubborn person, so I guess I called something that was not a plaque, a plaque, and tried to streak it. Not only did those plates get contaminated to add insult to injury, but I happened to basically incise the top agar when I was streaking. Like most, I also didn’t really believe that like a billion phages exist in each plaque, and that when you touched them with the streaking stick, half of them latch onto it, and that when you make a streak, you’re laying out almost all of those half billion phages onto the plate.

I happened to try four different dirt samples, looking for phages, before I finally adopted some of Sabrina’s plates. Some students had plaques on day one, and I felt so utterly left behind. However, that’s one of the strong points about the class. You get to work at your own pace, and don’t get graded down for things that aren’t really your fault. And technically, I wasn’t really behind at all. There are always going to be chunks of the class at the same place that you are, so you aren’t alone in that sense either. Phage hunting in my opinion, is one of the best classes that a freshman can take. It’s so welcoming in both procedure and people, and it’s a great way to learn about more advanced lab procedures that you haven’t learned in high school.
Now, that I have Sabrina’s adopted plates, I’ve been streaking for about four days now, and I’m just trying to perfect my streaking technique so that I can have three generations of a single type of phage in my plates. Coming in at the beginning of class and seeing that there are actually plaques on my plates never gets old. It’s like Christmas every Monday and Wednesday, and I think that by the end of the semester, I’ll be able to look back and say that I was really satisfied with what I was able to accomplish in this class.

 

Posted in Uncategorized | Leave a comment

If At First You Don’t Succeed…

Try, try, try again.

A polluted river. What a perfect place to get a virus living in the dirt, one might think. The person who thought that was me. Because I had gone to Hampden previously and had seen the sign near the river warning people to not go into the river because it contains harmful runoff, I thought that this area would be crawling with phages. I collected my dirt, making sure not to step into the river for fear of contaminating myself with whatever lied within. I went back to my dorm, confident that there were millions of little phages ready to lyse some M. smeg the following Monday.

I was wrong.

My first attempt at direct isolation ended up in plates that looked just like the control with no plaques on it and I sure was bummed. Maybe I just got unlucky? Maybe the phages were just tired and didn’t feel like lysing the bacteria? I knew the latter was clearly not true, but maybe I had just been unlucky. So then I tried direct isolation with the same filtrate, thinking that this filtrate that I had inside my microcentrifuge tube must be home to many phages.

I was wrong. Again.

I held back my anger as I looked at my second round of plates and saw exactly what I had seen last time; nothing. Just more plates that looked like the control. I felt like I had wasted so much time, plating and filtering and shaking these tubes, and for what? For nothing! It was with a heavy heart that I took another sterile tube and went back behind the UTL’s, begrudgingly scooping up dirt, knowing that I had to start all over again. All I needed was one plaque.

I returned to class with this new dirt sample and was told to do a different procedure; enriched isolation. Direct isolation, the procedure I had done the two times previous, had a smaller phage yield. Enriched isolation would increase my chances of getting a phage by a large margin. For the first time in a while, I had a real feeling of hope as I mixed some enrichment broth and 7H9 with my dirt. I extracted and filtered the supernatant and did serial dilutions up to 10^-5, plated them, and left class with a smile on my face. I knew that next class I would have plaques on my plate ripe for the picking.

I was wrong. Again. For the third time.

IMG_3749.jpg

This time, however, it wasn’t that I had no phages. The issue was the I had too many phages. All of my plates, even the 10^-5 plate, were basically all cleared, indicating a very high phage concentration. I scratched my head because I didn’t know how to feel. I guess this was a good thing? I mean, at least I had plaques. I decided that maybe streaking some of these plates would help dilute these phages. Maybe, next class, I would have some plaques!

I think you’re probably catching onto the theme here…

If this class gave credit for having plates that are as clear as they were before putting them into the incubator, I would have an A+. The streaked plates I had been so excited about were clear and one was contaminated. After yet another defeat, the amount I wanted to hang up my lab coat and leave forever was at an all time high, but not because I had not succeeded. It was because I had to do serial dilutions again. But this time, I had to do ten rounds. The thought of that made my stomach turn. There were so many ways I could mess up and contaminate my filtrate and have to restart, especially because I was still not very good at the procedure. Facing my fears and all of the odds favoring me messing up aseptic technique, I began the process of doing ten serial dilutions. After completing the serial dilutions, I plated the following plates: 10^0, 10^-1, and 10^-6 through 10^-10. As sweat dripped down my face from the stress and from the bunsen burner’s proximity, I placed my plates into the incubator.

Oh, what a feeling.

Plaques on 10^-6.jpg

Plaques. Plaques everywhere. On my 10^-6 plate, I had a beautiful array of clear dots, followed by fewer on my 10^-7 plate and only 2 on my 10^-8 plate. My dilutions had actually worked. All of my failure, all of my problems, were all worth it. These lessons that imposed themselves on me are definitely some that I can apply to my future in research, and, in life.

Sometimes, things don’t work. A hypothesis is refuted, a plaque doesn’t show up. The reason we experiment is to learn and to try new things and to fail. In my eyes, a failure in the scientific community may be just as valuable as success. Failure allows us to step back and view what we have done and see what could have gone wrong or what we can do to improve our procedures. Failure is a blessing in disguise, and it’s important to not fall victim of the nasty appearance that we perceive failure as having. Sometimes, following up on an unexpected finding or something that went wrong can be more beneficial than what one was looking for in the first place. I am happy to say that my failures have helped me learn more about laboratory procedures, and I have definitely learned that patience, in the scientific community, is definitely a virtue.

Posted in Uncategorized | Leave a comment

Phun Stuph Phursure.

Getting more Phun Part 2…

My enriched isolation i mentioned in “Not so Phun Part 1”, yeah. That didn’t work out. But, thankfully, I think I found what was going on: The procedure indicated that we had to open the enriched isolation tube every 24 hours for air to get inside. however, we did not, so anaerobic bacteria started to grow, and most likely denatured my phages. The group of people doing the enriched isolation with me also did not yield any phages. My lab partner, Jake, found phages, BUT it was because he had used a flask with an open top, so that air could get in. I did not use a flask at first, because there weren’t any left.

Proof of Adoption:

AdoptionJake'sPlate.jpeg

After determining what went wrong, and why I could not yield a phage, I had the choice of going through the enriched isolation process again. However, I instead chose to “adopt” a phage from Jake, in hopes of saving time. I labeled 4 plaques I identified from his old plate (he had not streaked these plaques yet), and streaked them on agar plates. Hopefully, these streakings will confirm that these plaques are indeed phages when I check back in two days.

Well, two days after, my plates indeed had plaques… but it was horribly infected. Luckily, there were some noticeable colonies that did not seem to have touched the contaminants, so i streaked from those, and hoped for the best. I also streaked from an extra not-so-contaminated plaque and filtered it with a 0.22 micrometer filter. After plating all those new streaks, I came back five days later to completely contamination-free plaques. The method worked, and I can finally go onto streaking for my generation 2 phages.

Contaminated example:

14699745_1244535262286967_2035421568_n

No more contamination!!:

Posted in Uncategorized | Leave a comment