Superlab

Hi everyone! I apologize for the delay in posting—I had an immunology exam due this afternoon and a physics exam yesterday, so I’ve been pretty busy studying the past few days, which means I have a lot to catch you up on. On Monday we had our third Monday meeting of the year, in which Professor Wilson talked to us about plant anatomy, and how to identify different kinds of plants and their physiology based on their physical characteristics. At the end of the lecture we were presented with seven different plants, and using the information we learned during the lecture, tried to identify what they were—which was actually really tricky! One of the plants, a Psilotum (the first image at the bottom is a picture of one) looked like a bunch of sticks covered in small knobs, but it was actually a kind of fern that diverged from the rest of ferns about 340mya and doesn’t have any leaves (I definitely wouldn’t have guessed that). Another plant, a Dioon (the second picture at the bottom) looked like a giant fern, but it was actually a type of gymnosperm. The leaves look soft but they’re actually really hard and sharp, and full of mucilage. Dioons have been around since the time of dinosaurs, and they’re the kind of plant that typically comes to mind when I think about movies like Jurassic Park. It was a really interesting lecture that made me rethink what I (little) I know about plants so far, and provided a good segue into Wednesday’s lab period.

In lab Wednesday, we had to work on a number of tasks. Unfortunately, the transformed bacteria we plated last Friday didn’t grow any colonies (which was really strange, and makes us think that maybe there was something wrong/we did something wrong with our competent cells), so task #1 on Wednesday was transforming new cells and re-plating them. I went in to look at them today, and we have colonies growing on the plates, but they’re all black… so we may have even more problems tomorrow. At the same time, we worked on task #2, which involved the culture-dependent portion of our experiment, taking bacterial colonies and performing PCR on them to amplify a specific sequence of DNA that will help us determine what kind of bacteria they are. Tomorrow in lab we’ll run a gel to see if the PCR went well, so I have my fingers crossed! Our third task, which relates to what we learned on Monday, was taking sections of our bamboo plant, making stained slides of them and then viewing them under the microscope to find structural features of the leaf that might make it habitable to various bacterial species. Something we learned about bamboo during this process, which I was not aware of, is that bamboo is made of 10% silica, making our sample both really difficult to cut through with a razor, and nearly impossible to stain (it made me realize just how powerful panda jaws must be!) We learned that bamboo stems have a leaf base, calling sheathing, wrapped around them, which also made cutting them difficult, because the two kept separating. Below are pictures of our bamboo stem and bamboo leaf section slides—note that the bamboo leaf looks like tiny bamboo stalks up close!

Hello again! As we approach the halfway point in the quarter, which I believe is officially Wednesday (but as of now we’re halfway to fall break!), we’re getting to the point where we’re beginning to identify the bacteria in our bacterial phyllospheres, which is really exciting—we can finally learn what lives on bamboo! Unfortunately, since we’re almost halfway through, if some part of our experiment isn’t working by the end of the week, we’ll have to abandon it and focus on other aspects of the project. Luckily for my partner and I, everything is looking great so far! On Friday, we focused on two separate tasks, simultaneously working on the culture-dependent and culture-independent components of the project. For the culture-dependent portion, we looked at the slides we made and stained on Wednesday with bacteria taken from the colonies we’ve been growing on agar the past few weeks. Under the microscope, we looked for various identifying characteristics of our bacteria: what shape it was, how individual bacteria grouped together, and what color it stained—which indicated whether it was gram positive or negative. Although we can’t identify the species of individual bacteria based on these stains (since there are probably thousands of species of bacteria that are gram-negative cocci), but it helps in preliminarily identifying what the bacteria isn’t. Later this week we’ll begin preparing the bacteria to be sequenced so we can get a better idea of their identities.

Along with looking at our stained slides, we also worked on the culture-independent portion of our experiment. On Wednesday, we performed ligation on our bamboo DNA samples, which involved inserting the various sequences that we cloned using PCR into plasmids, so each plasmid should have a different sequence, representing the entire phyllosphere of our leaf. On Friday we transformed these plasmids into competent bacteria and plated it on new agar plates, 18 hours later I went in and moved the plates from the incubator to the cold room, and didn’t notice any bacterial growth, but hopefully by class on Monday we’ll see something! For now, I’ll post pictures that my lab partner took of some of our bacteria viewed through the microscope. We have so many different kinds of bacteria that after class on Friday we hadn’t finished looking at all of them under the microscope, so we ended up coming back in until 11:30pm. Taking pictures was probably the hardest part, but at least we have something to show for our work!

I’m back, with results! Today we ran a second gel after re-doing PCR over the weekend. Our gel looked much better, but unfortunately there was still a faint band in the negative control lane, indicating that unfortunately we still had a little bit of bacterial contamination in our sample. We waited to see the results of the other pairs in our group, since each of us ran samples from the other partners, and luckily one group ran a gel with a clean negative control! That means that we were able to move on to the next step of our culture-independent procedure: ligation, which is a way of linking strands of DNA together. Since all of the bacterial DNA from our leaves was all mixed together, we need a way to separate strands from the different types of bacteria to sequence their DNA and determine their species. We’re going to be inserting our different bacterial sequences into plasmids, which are circular strands of DNA found in bacteria. We’ll then insert these plasmids into competent bacterial cells, and use these cells to sequence our original bacterial DNA. We started the ligation process today by preparing our bamboo sample, and will be working on it over the next couple of days.

Simultaneously, we’ve been working on the culture-dependent aspect of this project. Our plated bacteria are growing really well, so today we took colonies of bacteria from the agar plates and made slides out of them so that we can gather data about their shape, arrangement and gram (positive or negative). We did smears of bacteria onto slides, and then stained the slides using a variety of colors (purple, yellow and red—which got everywhere). We had a small panic attack when the decolorizer stain removed all of the sharpie labels from our slides, but luckily we fixed it before we lost three hours of work. These stains will tell us whether our cells are gram positive or gram negative—if it’s gram positive it should stain purple and if it’s gram negative it should stain pink or red. Gram positive or negative gives information about the plasma membrane of our bacteria, which is a first step in classifying their species. Below are some pictures of the slide staining process, enjoy! I’ll keep you updated with what happens when we actually look at the slides this Friday!

Hello again! Today we had our second Monday session, but instead of a lecture, we had our first journal club. One of the really great things about superlab is that it not only prepares us for the practical aspects of research—thinking and working independently, learning new techniques and perfecting old ones—but we also learn how to participate in lab group activities besides research, such as journal club. I’ve been working in one of the biology labs on campus since my freshman year, and every week we have a journal club where one member of the lab presents a paper that’s relevant to their research, usually talking about the broad concepts and main findings of the paper, and explaining the important figures and tables. As seniors, we’ll be participating in small journal clubs like that with the rest of our lab group, but at the moment the approach of one person presenting a single paper isn’t feasible for a class as large as superlab. Instead, we all read a paper, Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere (E. Yashiro, R.N. Spear and P.S. McManus. (2011) Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere. Journal of Applied Microbiology 110, 1284-1296), and answered a series of questions about it. In class today, we flipped a coin to see if the Tuesday/Thursday or Wednesday/Friday group would present the answers to their questions about the figures and tables in the paper. As it turned out, my group wasn’t chosen, which means I’ll be presenting figures from another paper in the second journal club in a few weeks. The article was really interesting—it explained why we’re using both culture-dependent and culture-independent techniques this semester (you get more bacteria, and a larger diversity of bacteria) and described the phyllosphere of apple leaves from different orchards in Wisconsin. I don’t have any data from our experiments to report on today, but I’ll keep you updated when I have lab on Wednesday!

Here’s a link to the paper in case you’re interested in reading it: Journal Club Article 1

Hello again! So yesterday we ran the bacterial DNA that had been amplified using PCR on a gel to see if 1) we actually did everything correctly and didn’t contaminate our samples, and 2) If our plant samples had bacterial DNA in them. The picture of our gel is below, it’s kind of hard to read, but we got DNA in our positive control lane (which is what we wanted to see), but we also saw a line in our negative control lane (far right), which indicates that there was some kind of bacterial DNA present in our negative control, indicating contamination, which is disappointing. However, all of our other samples look good, so our preliminary data indicates that both bamboo samples and the dicot sample were positive for bacterial DNA, but that conifer didn’t have any (apparently conifers tend to have less bacteria than other plant types), so that’s a good sign. Since our negative control was contaminated, we had to redo PCR over the weekend, and sometime in the next few days we’ll re-run our gel and hope that the negative control isn’t contaminated! On a positive note, our spread plates of bacteria seem to be growing really well, particularly a plate of neon pink colonies, which is exciting!

Fun bacterial fact of the day: based on our work with bamboo plants, Professor Okeke suggested that my partner and I read an article in PNAS, “Evidence of cellulose metabolism by the giant panda microbiome,” (L. Zhu, Q. Wu, J. Dai, S. Zhan and F. Wei. (2011) Evidence of cellulose metabolism by the giant panda microbiome. PNAS 108, 17714-17719), which discusses the bacteria present in the digestive system of the panda. Apparently, pandas’ preference for bamboo changes during different months of the year (it’s lowest during the summer), and during that time they eat different parts of the bamboo plant. This change in bamboo preference corresponds to a change in the microorganism community of the panda’s GI tract. Perhaps the phyllosphere of the bamboo leaf has something to do with this change in dietary preference?

Gel Data

Hi everyone! So today was our third official day actually in lab, and at this point our project is well underway. To try to identify the different kinds of bacteria present on our bamboo leaf sample, we’re using both culture-dependent and culture-independent techniques. Culture-dependent, as the name implies, means that we’re trying to culture our bacteria samples on agar plates—we’re trying to grow (hopefully pure!) colonies. We’ll then use various techniques, such as staining, to try to identify our bacteria. At the same time, we’ll be performing culture-independent techniques to try to identify microorganisms on a genetic level. By extracting and amplifying a specific sequence of DNA from bacteria, we can compare their different genetic markers to determine the phyllosphere of our leaves. For our first journal club (next Monday!) we were assigned to read an article from the Journal of Applied Microbiology, Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere (E. Yashiro, R.N. Spear and P.S. McManus. (2011) Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere. Journal of Applied Microbiology 110, 1284-1296). The article discusses both culture-dependent and independent methods of examining the bacterial community of apple leaves, using techniques similar to what we’re doing in lab, and mentions the fact that they were one of the first groups to directly compare the effectiveness of the two techniques (culture-independent produced more data on a wider range of previously unknown bacteria). Since this article was only published a year ago, it’s exciting that we get to follow in their footsteps and be one of the first groups to compare both techniques on a previously undocumented community of plants—who knows, maybe we’ll discover a new species of bacteria!

Today in lab we worked on both culture-dependent and independent techniques. The bacteria we plated last week grew into hundreds of gorgeous colonies (see pictures) of all different colors! We had bright yellow, dark orange and neon pink colonies! Our task for the day was to spread individual colonies onto new agar plates so that we can grow pure colonies of distinct bacterial strains. Before spreading, we began working on the culture-independent portion of our project, which involved extracting DNA from the bacteria on plant leaves using an intricate protocol of vortexing and centrifuging. Overnight, specific sequences will be amplified using PCR, and on Friday we’ll get to run the samples on a gel and find out if we’ve done everything right so far, so I’ll keep you updated on how that goes! For now, enjoy pictures of our rainbow bacteria!

Bacterial Colonies from Bamboo Sample 1 (1:10 dilution)

Hi everyone! My name is Carson and I’m a junior biology major here at Haverford. I’m going to be blogging about my experience in superlab, an upper-level lab course, this semester. If you’ve been following the superlab blog over the past year you may already know a little bit about the class, but if not, I’ll give you a basic introduction to what superlab is all about. Superlab is a yearlong, 7.5 hour/week lab course designed for biology majors to prepare us for our senior thesis research, and, if we so desire, eventual research careers. Every Monday we meet to discuss the theory behind what we’re doing in lab, go over lab techniques or go over papers in journal clubs. During the rest of the week we have six hours of lab time, where we work on various projects. Unlike traditional labs, which introduce procedures and are designed with specific results in mind, superlab is more of an independent learning experience. The goal is to get us to frame a hypothesis and to design experiments, based on procedures we learned in previous years, to test that hypothesis. It’s designed to help us engage with the project and really get us to think about it as if we were working in a research lab, especially since there’s not one specific result we’re trying to achieve. Throughout the year we learn how to present our research in various formats, including writing lab reports and making posters.

The year is divided into four quarters, and each quarter we work on a different project with a different pair of professors. This quarter we’re working with Professors Iruka Okeke and Jon Wilson, examining the phyllosphere (the microbial community) that lives on plant leaves around campus. Everyone in my section of the class has a partner, and then pairs of students were divided into three different groups of approximately six students. Within our groups, on the first day of class, we had to come up with a hypothesis that we’ll spend the rest of the quarter trying to answer. My group asked the question: is microorganism diversity and population dependent upon plant species or local environment? We predict that location, rather than plant species, will determine the phyllosphere, but we’ll have to wait another six weeks to find out! We started out by choosing a location to gather plant leaves. There are seven of us in our group (one group of three) so we decided that we could collect four different plant samples. We needed to gather plants from the same area to assess the effects of local environment, so we looked for an area with a lot of plant diversity. After a good half hour of searching, we found an amazing spot between Comfort and the duck pond. In a five square foot area we found bamboo (a monocot) next to a pine tree (a conifer) next to some kind of small bush (a dicot). Unfortunately, there were no ferns or ginkgos in the area, which would have represented all five major groups of vascular plants, but we figured 3/5 was still pretty good. Using sterile aseptic technique, we collected two samples of bamboo, a sample of the conifer and a sample of the dicot. My partner and I will analyze the bacteria on the first sample of bamboo, while the other groups will look at the other plants, and at the end of the quarter we’ll compare results. I realize this post is getting long, but before I stop for the day I want to give you a basic idea of where we’re going this quarter: last week we extracted bacteria from our leaves and set up 7-day cultures of various dilutions to grow our samples. Once we successfully grow our bacteria, we want to isolate and grow individual strains, so that we can eventually identify the species. Tomorrow we get to see how our bacteria are growing, so I’ll let you know how that goes!