The Hunt for the Blackrock Virus

Phages are viruses of bacteria, they are nature’s antibiotics, and students work to isolate phage each year on our campus in the AMSR program.  Elif K. (’13) wanted to focus her phage hunting on medically-relevant bacterium and, after a few conversations, she decided on Legionella, the causative agent of Legionnaire’s Disease.  We obtained an avirulent Legionella strain from Dr. Sunny Shin (University of Pennsylvania) and Elif went to work. Interestingly, this bacterial strain was genetically derived from the original Philadelphia organism that caused the historic major outbreak in 1976.

Elif BlueElif worked most of last year developing the recipes and protocols for Legionella cultivation and virus isolation.  After screening nearly 50 environmental samples and months of negative results, she triumphed.  Her patient tenacity paid off and she is now in the process of characterizing the Blackrock virus of Legionella.   The name Blackrock, she says, is a dual tribute to the exposed schist mountain ledge that is behind Berkshire School and the charcoal agar plates used for Legionella cultivation. The bacteria gives an off-white color to the black plates (see below) and, when infected, tiny holes in the off-white “lawns” are observed.  After submitting this work to INTEL this fall, she will prepare her results for publication in a peer-reviewed virology journal.  As this may be the first stable virus reported for Legionella, the phage and its use in medical applications or treatments that prevent Legionella growth may also be patentable. Congrats to Elif!First Plating May 222013

No one said it was easy…..

After a lengthy and relaxing summer, the members of the Phagehunting class began to get ready for an exciting year in AMSR. This year, we would be finding novel phages, identifying them,naming them, and hopefully having its DNA or RNA sequenced. As Berkshire School students,we are very fortunate to have an on-campus laboratory in which we can carry out this research.

Our mentors, Dr. Burch and Mr. O’Rourke began the year by giving us different packets ofprotocols, lessons, and laboratory technique that we all had to read in order to enter the research world. For the first two weeks of school, we reviewed this information carefully in order to make the solutions correctly, collect good soil samples, isolate phages, and eventually find a novel phage. It seemed like a lot, but with the help of our mentors, we were able to transition into the laboratory and begin our research.

The first step was to create all of the necessary buffers, solutions, and agar in order to grow Mycobacterum smegmatis, or “smeg.” Smeg would serve as the host for the novel phages that we would be finding. In other words, the viruses that we were hoping to find should be able to infect smeg.

Once we were ready to go, we collected our first soil samples. During the first couple of weeks,none of us had found anything. Then we identified the culprit that prevented us from findingnovel phages: our phage buffer’s pH was way off!

So far, we have not found plaques, but we have learned invaluable laboratory techniques and skills that will help guide us in the right direction. In order to find phages, we have began to enrich our samples with smeg from the very beginning of the soil collection phase. Hopefully, this encourages smeg to grow, thus increasing the possibility of viral infection. Virus Collection Tools

By: Jeffrey Erazo ‘15

Visualizing phage with negative stain EM

This slideshow requires JavaScript.

It’s true.  A picture is worth1000 words.  Just before Thanksgiving, the AMSR students traveled to the Wadsworth Center to tour the multimillion dollar facility that houses some of the most sophisticated electron microscopes on the planet.  The Wadsworth Center has a rich history of leading the field microscopy.  In fact, they were the first center to have a one million Volt Electron Microscope.  One student, Elsie G. (’13) got to sit at the helm of this two-story ancient but still operable microscope (shown below).

High-titered lysates of viruses prepared at Berkshire School were spotted on to tiny carbon grids and stained with heavy metal-containing buffers that reveal the structure of their novel phage.

 

It was a terrific day.  We have several nice structures to go along with our interesting plaque morphologies (large,  tiny,clear, and turbid).  In the next two weeks, AMSR phage hunters will isolate viral genomes and prepare for high-throughput sequencing.  Stay tuned….

Edeline L., Class of 2014, Phage Hunter

On Friday the Fifth of October, the new Math/Science building at Berkshire school was dedicated to two extraordinary former Berkshire faculty members, Mr. Bellas and Mr. Dixon. The keynote speaker for this event was Carl Zimmer, a lecturer from Yale University. Mr. Zimmer’s speech was very insightful and it compelled me to think about living things in a different way. To me, the most interesting part of his speech was when he talked about the patient that Dr. Alexander Khoruts treated a few years ago.

Dr Khoruts is a doctor from the University of Minnesota that specializes in intestinal medicine. A few years ago, one of Dr Khoruts patients was suffering from chronic diarrhea in which no medicine seemed to be able to treat. She had lost a significant amount of weight and was, basically, at death’s door. Dr Khoruts realized that something drastic had to be done if he was going to save her. He took her healthy husband’s stool, mixed it with saline and delivered it into her colon. Within two days, she was having healthy bowel movements and has never once relapsed. When Dr Khoruts took a sample of the bacteria in her intestines, he observed that not only had bad bacteria vanished from her gut, the types of bacteria in her gut were the same as that of her husband.

This example that Mr. Zimmer gave made me realize just how big a role bacteria play in keeping us healthy. Bacteria are the future of medicine, just like how antibiotics were 50 years ago. Maybe twenty years from now, when you are suffering from an upset stomach, popping a pill filled with bacteria will be a norm.

One reason why Dr Khoruts’s treatment was successful was due to the work of phages, viruses that infect bacteria. Phages located in the solution given to the patient were able to not only infect and kill bad bacteria but were also able to leave the good bacteria alone to give them the chance to colonize the women’s intestines. Thus, this is a fascinating subject to do research on; as this selection process by phages could help scientists develop specific treatments for illness. This is why I am excited to be studying phages, as it seems that this is the direction in which science is headed in.

~Edeline

Update: Edeline has two samples that contain phage.  Stay tuned for more information!

Elif K. Class of 2014, Phage Hunter

AMSR So Far 

Science is a game. Anyone who seeks answers to questions about life, earth, and universe is a player. The rule is simple: Think about a possible answer, test it, accept if you cannot disprove. Biology is a science that focuses on life. Anyone who is curious about life can play the game of Biology. I started playing this game when I asked my mom who the guests were at the wedding ceremony of Adam and Eve, at the age of 3. My curiosity toward life increased as I grew up. I wanted to play this game more professionally and I decided to become a part of the Advanced Math Science Research Program at Berkshire School as a phage hunter.

We started the year by getting to know our lab and basic lab skills and techniques. The first thing we learned was how to take off our used gloves and put them back on if needed. I was amazed to see how such a simple act could have a certain “technique”. We continued to discover the lab by learning about all the different equipment in the lab. It was so much fun to learn about the functions of all the high-tech equipment that real scientists used. It was even more interesting when we started using them. We actually tried gel electrophoresis just to familiarize with it. Moreover, we got to use other equipment when we were preparing reagents and buffers that we would use during our phage hunting experiment. I was happy to learn all the techniques and excited to ‘hunt some phages’.

We started off by collecting some dirt from around our campus. We made working phage buffer and added it to our dirt samples. A working solution is a diluted solution that is ready to use. After vortexing, incubating, and spinning the mixtures, we filter sterilized the liquid supernatants and transferred them into sterile tubes. We labeled each tube. Then, we plated the supernatants to previously inoculated Mycobacterium smegmatis petri dishes. “Smeg” serves as a host for some phages. We incubated the dishes and waited for three days. However, we didn’t find any phage clearings on our plates. Therefore, we repeated the same plating procedure. We made a spot test for the possible phage plaques that appeared in some petri dishes as slight clearings. And this is where we left off.

Even though we haven’t been able to isolate our phages yet, it’s been a good introduction to a professional level of science and I have enjoyed it a lot. I cannot wait to isolate and name my phage, and perform my own experiment with it. Our first trial of isolating phages wasn’t successful and we haven’t gotten the results for the second one yet, but it is okay. We can’t always succeed right away in science because as I said: science is a game.

~Elif

 

Update:  Elif has very interesting plaques to analyze now. Looks like she may have isolated a lysogenic virus. Cool!

Lars R., Class of 2013, Phage Hunter

The Berkshire phage hunters are off to a great start by any
standards.  We are well on our way to each isolating and studying our
own bacteria phage. Before we delve into any of the details or
specific procedures, there’s some useful background info.  Bacteria
phage (shortened to phage) are the most abundant life form on earth.
They are found everywhere, from inside the human body to the extremes
of the Antarctic, underneath a two mile thick ice sheet.  Phage are
viruses that attack and kill bacteria in order to replicate
themselves.  They are therefore easy and useful to grow and study
because of their huge abundance and strong resilience, not to mention
their potential for treating diseases like TB without antibiotics.

On to what we are doing:  for those of us who decided to hunt phage
for our AMSR project, we are currently trying to isolate our phage.
The process started on the first day of classes when all of us
re-upped our skills from basic chemistry (using precision scales,
mixing reagents, measuring at the bottom of the meniscus, etc.) by
creating the reagents and buffers that we will need all year.  After
we were sufficiently out of the summer mind-set, and into the learning
attitude, we finally began collecting samples.  Each of us chose sites
around campus and collected our samples of dirt.  After returning to
the lab, we re-suspended our samples and filter sterilized them in
order to get liquid containing massive amounts of many different
phage.  The idea then was to add bacteria (M. smegmatis) for the
phage to attack, then plate the mixture in a nutrient agar dish.
After incubating for a while, the plates should reveal clearings in
the bacterial lawn (called plaques) that indicate the presence of a
virus.  Later on, we will verify the presence of the virus, and begin
studying it. This is still a ways off though, as no one yet has had a
positive plaque on any of their dishes.    Due to the occasional
blunder and the unreasonably short class periods that we have to work
during, we are being delayed at the plating step.  This has led some
of us to enriching our samples, or finding entirely new samples to
start over with.  Even despite these minor setbacks, I am optimistic
about the year ahead

~Lars

Update: Today was a good Friday for Lars!  He got plaques on an enriched sample.  Stay tuned!

Elsie G. Class of 2013, Phage Hunter

The Advanced Math Science Research program is unlike any other course I’ve taken here at Berkshire School. It provides hands on learning experiences, as well as an outlook on a scientist’s working etiquette and atmosphere.

As a Phage Hunter, my mission is to isolate bacteriophages from dirt samples. Some of my samples were collected directly from Berkshire School, such as the mountain: a resource integrated into the daily lives of the Berkshire student body. Thus far, I have collected dirt samples from the Elbow trail, as well as the stream in front of the Maher’s House, and my back yard in New Jersey. I choose these specific places because of the dampness in the soil and the ability for viruses to thrive in these warm, moist places. Once I plated my samples and incubated them over night at 37⁰C, I was ready to check for clear plaques, which occur when virus kills a bacterial lawn. While scanning them, I came upon two plates that contained clear plaques. Excited to see that we might have a virus in my sample Dr. Burch and I decided to do the spot test. During this process, you must obtain a liquid sample of phage by picking a plaque from one of your plated samples. Once I had my liquid sample, I began to transfer my liquid phage sample to the corresponding labeled spot on my agar plate. This test is mainly to see if my top agar was infected and to see if the plaques reappeared, confirming the virus. Though my spot test was negative, it made me wonder what I would name my virus when the time came that my spot test would be positive. Being that a virus wasn’t present in any of my samples, we decided to enrich my backyard sample. This process enriches a sample with phages capable of infecting a desired host, M. smegmatis. If an existent phage in my sample is capable of infecting the bacteria, then this will be shown when plating the samples by creating plaques.

This program has allowed me to take information and apply it in a lab based environment, putting the knowledge I have learned in this program to work. I am not just a member of the Advanced Math Science program; I am a Phage Hunter. Plus, the embroidered lab coats are very cool.

~Elsie

Update:  Elsie found her phage after analyzing soil from an herb garden located on campus.  Her phage is named “Nilo”, her Father’s nickname, and has a really beautiful structure.  Stay tuned for more.

20121018-210552.jpg