Well, this past week Greg, Malisa, and myself have spent more time in lab outside of our designated TThu 1 – 4 slot than during that time slot! We have been busy doing more PCR’s – we have gotten even MORE successful results using just inner primers for our subculture DNA extracts! If there is ANYTHING I learn from superlab this semester, it is preparing PCR reactions and sterilizing loops for restreaking and plating. We have been cooped in lab, late at night, ligating and transforming our successful PCR products. Almost all of our ligated products were successfully transformed (only 3 were unsuccessful…tomorrow we’ll see whether Iruka wants us to try retransforming them again) and we have just finished setting up liquid cultures to let our wee little white colonies grow! Furthermore, we have been playing plant anatomist, dissecting and staining samples of our species of interest, the Norway Spruce. Greg, Malisa, and I have become pros at carefully slicing pine needles and stems in all directions – I think it’s safe to say we could all become surgeons with our stead hands (all thanks to Jon Wilson’s tips. of course). After slicing, we stained each sample in a variety of stains in order to identify different tissues such as xylem, phloem, cuticle, etc. It took us a few tries to get the staining right (we either didn’t keep the sample in the stain for long enough or overstained it by leaving it in too long…we had a few samples overstained with safranin that looked like the devil’s eye…) but alas, got some decently stained samples and imaged them for our records. Until next time!
Archive for the ‘Uncategorized’ Category
Hey everyone! So I realize I haven’t blogged in quite some time, though in my defense, we had to do some EPIC planning for our April Fool’s day joke (que in LOTR music). In lab, we’ve reached the point where virtually everyone is at a different point in lab and no two groups are doing the same thing. We (Greg, Malisa, and myself) are slowly but surely catching up to speed – we had some contamination issues with our original DNA extract from the norway spruce pine needles. Our isolated cultures looked GREAT – we got a variety of colonies including orange, white, reddish/pink, and cream color! Sadly, our slimy lawn of clear liquid didn’t yield any culture, but I heard that another group did actually obtain clear colonies, which is pretty cool. We did a ‘dirty’ DNA prep on these isolates, essentially boiling and lysing the cells and extracting DNA in only a few steps (no Quiagen kit required!). We prepared a PCR for all of our isolates, and when we checked via gel electrophoresis, low and behold we got 1 band! Considering our bad luck with original DNA extract PCR, we were THRILLED to see atleast 1 culture’s 16s sequence get amplified! Oh! I almost forgot to say – we did our very first Gram staining! Gram staining was a nice change from all of the PCR and culturing techniques we have been focusing on. It was fun to prepare our slides using the different dyes and then actually being able to look at our individual bacteria. We got a nice diverse group of bacteria ranging from gram positive spherical bacteria to gram negative rod shaped bacteria – no spiral shaped bacteria though, which was upsetting. I believe our plans in lab for this week are to continue working with our PCR samples (Malisa was awesome enough to come in over the weekend and do a ligation of our successful PCR), though I must say I am pretty excited to disect plant anatomy! I personally never learned plant anatomy (not even in AP bio!), so everything I learned today in lecture was completely new. Not so surprisingly, I thought the meristems and plant vascularization were pretty interesting. I was so taken back by thinking about a plant having stem cells – I never think of plants in a scientific way in so far as they too must grow and develop from one cell. Alas, I shall never think of plants in such a naive way again…thank you, Jon Wilson! Well, that is all I have for now…I will let you all know how plant dissection goes tomorrow!
After giving our cultures long incubation times, we finally were able to see colonies growing! It is amazing how diverse and abundant bacteria thrive on these small pieces of leaves. We re-streaked all of the visibly distinct and individual colonies onto agar plates of their own and grew those up. Two weeks later, Harry and I are proud to announce that we have 23 beautiful and different organisms. We almost ran out of letters labeling them from A-Z! Having this many colonies also means that we will be doing many of the protocols from now on x23!
We have now come to the point in lab where every group starts to work at their own pace. The biggest deciding factor of what step a group is on is the result of the PCR (polymerase chain reaction). The purpose of this step is to amplify pieces of pure DNA so they can be cloned and analyzed in the future. So far, we have run two PCRs and have come up with negative results. When we ran our reactions on electrophoresis gels to visualize the DNA, we ended up getting bands (DNA indicating bands) in our negative controls both times. Troubleshooting is now our plan of action! We are currently in the process of running our third PCR reaction with plans to use nested primers if the need arises.
Last night, Harry and I spent time in lab working on a different protocol, Gram staining. We stain hand-made microscope slides to pigment the bacteria so we can visualize the cells under the microscope. We use a variety of dyes to account for both gram-positive and gram-negative bacteria. Gram-positive bacteria are bacteria cells that have one membrane and are stained with crystal violet. Gram-negative bacteria have two membranes and are stained with safranin counterstain. In order to account for both bacteria types, the protocol had us using first the crystal violent on all slides, then washing it off with water and staining with safranin. After quiet some time of tedious but deliberate work mixed with a good playlist and some visitors, we ended up with 26 beautiful slides (3 extra slides were made from the controls).
Our next step is to run our PCR on a gel and hope to see bands so we can continue on with the cloning process.
Hey! Well, sadly, I don’t have much exciting news to report about Thursday’s lab. We ran a gel to see if we successfully amplified our PCR product, but unfortunately, there seems to be contamination – either from the milliQ water in our negative control sample or from the PCR primer mix we used. Sadly, we have to run PCR again and hope for contamination free product! We are running both the old PCR primers and the inner nested ones in order to discern where the source of contamination came from. It seemed to be a general trend in the Tuesday/Thursday lab group that their sample was contaminated, so it could feasibly be either option. Looking at the colonies from our cultured plates was quite fun! We got to see some beautiful mold spores (not what we want, but still cool to look at), some interesting thick, slimy clear liquid on the surface of one plate, some orange colonies, some white colonies, and some yellow colonies – all with different morphology. We were really curious about this one colony, that was perfectly dome shaped and clear – however, we learned when we went to actually restreak the colonies…that those colonies were actually bubbles in the agar. Slightly embarassing, but also rather funny – we were really excited about those colonies too! We also had a few white colonies which were actually embedded in the agar, which meant they were not from our sample, but from bacteria that were present when the agar was poured. Luckily though, we did restreak atleast three different types of colonies and now we are just crossing our fingers and hoping both the restreakings are successful and the PCR!!
Keep you updated,
Greg and Ashley here! Actually, Greg just really creepily showed up behind me as I was about to write this…but I”ll include him anyway. I think Malisa is upstairs in the SciLi doing physics =(. This monday, we learned a bit about microbiology techniques, particularly how to isolate a specific specimen using different environmental and media conditions. Tuesday was DNA isolation day! As fun as this sounds, all we basically did was a series of vortexing, centrifuging, and mixing different solutions in order to get rid of plant DNA, lyse bacterial cells, and isolate only bacterial DNA – Greg and I appreciated that the lab manual this quarter actually told us what was going on in each step, so each step made sense and wasn’t mindless mixing. After isolating the DNA, we prepared our PCR reactions to amplify the DNA – Greg and Malisa were probably annoyed with how obsessive I am about sterility and keeping all PCR equipment and such contamination free…and yes, this includes wiping down all automatic pipetters with EtOH. We also got to check our cultured plates on Tuesday and we were excited to see some growth! We saw some irregularly shaped yellow colonies, white colonies, orange colonies, and strangely enough, clear colonies! SO MUCH EXCITEMENT!!! Keep you posted!
Greg and Ashley (and Malisa)
Working in the field is very dependent on the weather, and the upcoming forecast was rain! We needed to collect our samples before the rain hit and washed all of our bacteria away. So, with our samples tucked neatly away in sterile baggies, we were ready to begin taking our cultures. The most important aspect of this protocol was to keep everything sterile! Our goal of the day was to separate all living bacteria from the plants and grow them up in our own cultures. It was very important to avoid contamination in this first step so that we don’t end up mixing our plant bacteria with bacteria from our hands, or even from the air in lab. The procedures used were ones that we are familiar with, so the protocol did not take very long. The biggest and most important step of the day was to shake then sonicate our leaf samples to separate the bacterial cells and suspend them in water with Tween (detergent).
Before we plated our samples, we diluted them to 1 in 10, 1 in 100, and 1 in 1000 dilution samples. The purpose of the dilutions was to ensure that there will be an appropriate amount of bacteria on each plate. We need to be able to see small individual colonies of bacteria, so we would need about 30-300 cells initially. If we had too little amount, the cells of bacteria would be too small to see and culture, and it we had too many cells, then the cells would grow in a lawn across the plates and the individual cells would again be impossible to see. After dilutions, we spread our samples aseptically onto agar plates to induce bacterial growth. These plates contain cyclohexamine to kill any fungal cells so we had to wear gloves and a lab coat at all times since this drug is also toxic to humans.
Each different strain of microbe will grow differently in different environments. Since these cultures were taken from plants that live outside, we are incubating them at room temperature, underneath a window for about a week. We do not expect to see colonies until maybe Tuesday, and it will be exciting to see what ends up growing!
Hi, Katie and Harry here reporting in from the semester 2, quarter 2 junior Superlab! We are embarking upon this crusade of our last superlab quarter with a mixture of excitement and nostalgia. It’s hard to believe that we are going to be starting our theses next semester. On the other hand, our proficiency in laboratory techniques and lab notebook upkeep since 200 lab has been truly staggering. This quarter, we are going to be documenting our progress through this blog. We are fortunate enough to be mentored by Jon Wilson and Iruka Okeke while we study the types of microbial organisms that can be found on the surfaces of leaves throughout our campus. The project will conclude with a collaboration of all the lab groups resulting in a semi-comprehensive record of the microbes that live on campus and where we can find them.
Unlike superlab projects in the past, this one began outside the laboratory on Campus Tree Tour with Jon. We spent time walking around the arboretum observing the different and unexpected species that co-exist with us here on campus. In accordance with Jon’s teachings, we kept a record of the WHO, WHAT, WHERE, WHEN, WHY AND HOW of our observations during our small field trip. These notes were of course accompanied by small artistic (in some cases) sketches and pictures taken meticulously with our iPhones. Along with learning about the arboretum that we live in, we also got to get a little exercise in during lab!
It was soon our turn to explore the world of botany on our own! Back in the lab, we faced the task of developing a hypothesis relating to variations in the density and diversity of the bacteria on the surfaces of leaves. Our lab section broke up into two super groups, one focusing on comparing plants that vary spatially while the other group focused on plants that vary phenotypically. After some deliberation, we found a way to integrate the fascinating concept of plant immunity into our project; we decided to determine the relation between the density and diversity of bacteria on leaves and the degree of venation of the leaves, placing us in the latter of the two super groups. This led us to our next objective: choosing among the endless variety of flora throughout the campus merely two specimens. We knew right away that these two specimen should differ as drastically as possible in terms of their vein density.
With Jon’s help, we decided to sample a conifer (pine) with low vein density along with a dicot (flowering plant) with high vein density. We then ventured off into the pinetum in search for our samples. This gave us the opportunity to apply our field training independently. The two lucky plants that we chose were an Ivy plant (yet to be specified) and a Japanese Arborivitae (cypress family). We are looking forward to cultivating the bacteria that reside on these plants and continuing our experiments!
That’s all for now! Over and Out!
Hey everyone! While today certainly was no where near as beautiful weather as Tuesday when we surveyed the land, we were all lucky to beat the rain and collect our samples before the rain hit! (I don’t even know if it’s still yet to hit or has hit already and I haven’t noticed). After collecting samples, we all cultured our specimens (that we collected aseptically to prevent any outside contamination) and plated them – now we have to wait a week to see what kinds of bacterial growths we will see on our plates! I’m hoping we get an array of interesting bacteria that are all from our leaves and not from our hands! Malisa, Greg, and I tried to be as careful as possible outside, but it is so much more difficult to be ‘sterile’ and not contaminate anything when you are outside and there are microbes everywhere…all of the time (well, at least much more so than you would hope would be in a lab)! Collecting was quite fun, although a little stressful and tedious when trying to not contaminate our samples and take good, detailed field notes in our notebooks. The best part of lab, for a pyromaniac like myself, was flaming our spreader and the caps of some of our sample tubes. If all I did today was flame spreaders and plate bacteria, I think I’d be happy…it’s one of those techniques that I weirdly love doing. It’s almost as fun as using the frogger plater that we used last semester with Alya and Karl – it involves plating many samples at once…and sterilizing it feels like holding a torch because so much EtOH gets caught on fire (the frogger is quite large).
Ok, well that essentially sums up our lab work for today….I’m off to do more work!
Hey everyone! My name is Ashley and I am a junior biology and philosophy double major here at the lovely Haverford College. I think that is all you really need to know about me because I’m just here to tell you about the important stuff – Biology superlab! I’ve been asked to blog about my experience this quarter in superlab, which is a lab course taken by upperclassmen biology majors (and chemistry majors planning to fulfill a biochemistry concentration). Biology superlab is designed so that students get first hand experience using techniques and equipment commonly used in graduate labs and research facilities; however, what really makes superlab special is the independence the professors give us. This independence, in my eyes, truly allows us to think about what we are doing, why we are doing it and how we would go about solving future problems/questions. What makes superlab special is that we aren’t just blindly following protocols, but actually learning how scientists think, how questions are formulated and answered thoroughly (that includes how to present our data, and most importantly, how to record our data =) ), and how fields of research progress step by step. In short, superlab is preparing us not only for senior research, but also for possible careers in research.
This particular quarter of superlab we are investigating the wild and wonderful local phyllosphere of our beautiful campus. As Jon has informed us, “microbes are everywhere, all of the time!” – and that includes on the plethora of leaves, buds, and flower pedals that decorate our campus. Honestly, I’m actually stunned by the amount of microbes that live on plant leaves – my mind literally has difficulty visualizing over 10 million bacteria inhabiting a square centimeter of leaf area (and we think NYC is crowded…). Naturally, in order to even begin our studies on the bacteria that live on plant leaves , we must venture out of the lab and into nature (no matter how humid and buggy) to survey the Haverford Campus. While each group is doing their own specific experiment, we are all essentially looking at the types of bacteria on different plant leaves; today, as a class we walked around campus and put the field methods to use (sort of); we surveyed everything from the magnolias, the weeping willows by the duckpond, those MASSIVE giant seqouias and dense walls of bamboo that line the nature trail (fun fact – despite many runs along the nature trail, I never noticed either the bamboo or giant sequoias…which is slightly embarassing) Sadly, I think my sad attempts to annotate everything I saw, smelt, (heard?), and touched turned to silly scribbles in my notebook that I really hope Jon and Iruka can read. I felt more like an artist examining something I wanted to draw and paint later than a scientist! (Monet must have had to made specific field maps for his series of paintings of lakes and ponds at different times of day). Actually, I take that back – I felt like Aristotle. Marking the specific location, examining the plant and determining whether it was a monocot, dicot, conifer, etc., and really making an effort to ‘get to know the plant’ in order to understand ‘its nature’ was pretty fun. Of course, we are taking Aristotle into the 21st century and doing even cooler stuff: examining an environment that are imperceptible to our senses. Ok, well, this blog post is really way too long for one blog post..so I’ll stop babbling on. I’ll let you all know how Thursday goes, when we actually collect our specimen!
Just wanted to let you know that the results are in!
We have observed many genes that play a role in heat response.
The most interesting genes are the ones that also play a role in photosynthesis. This infers that the response to light stimuli and the response to heat shock follow a rather similar pathway.
This light response/heat shock response pathway was inferred by several articles in the scientific literature. The fact that we were able to repeat previous findings gives validation to both our experimental results and to the implication that the two stimuli are connected.
Pretty exciting, eh??