Monday, July 28, 2014

Katherine Vaccaro's Internship, Summer 2014 - Part 5

Hello everyone!

Last week was a busy one. After lots of troubleshooting, we finally got results for our single cell ciliate isolates! 
TA-DA! Ciliate DNA bands in gel 
6 out of 8 cells worked (not including our positive sample) which is fantastic news! I cut the bands out of the gel and saved them in individual tubes. Later, when we have a few more samples, we will be purifying the samples to extract the DNA out of the gel for cloning and transforming. And if all goes according to plan, we will be sending out our samples for sequencing and I will have concrete results telling me the species of each ciliate I picked out!

Identifying our ciliates is especially exciting, since Marie Head and I came across an interesting organism while searching our samples for ciliates:
Mr. Mohawk, Unidentified 
Meet our strange little unknown friend! I’ve been calling him “Mr. Mohawk”. Very scientific, I know. After staring at him through the microscope for several minutes, Marie and I decided he had some tintinnid-like qualities and we grabbed him for DNA amplification. Worth a shot, right? When all was said and done, we had 6 out of 8 ciliate samples work, like I said before. The cool thing is, this guy was one of them. The band he produced was bright and clear (Band on the top right of the image of our successful gel)!

When talking to Dr. Snyder about this little guy, he suggested that perhaps he is an invertebrate pluteus (larvae) that had ingested a ciliate, resulting in ciliate DNA being amplified during PCR, but he agreed that it is entirely possible to have found a previously undiscovered ciliate! Marie and I are in the process of asking around our department to see if anyone has any insight into what this little organism might be. We sure are hoping that he is a ciliate, and we are very curious to see what sequence comes back.

So have I assisted in discovering a new species of tintinnid ciliates, or have I just found a (super cute) invertebrate pluteus who had a ciliate snack before we grabbed him from our water samples? Either way, I was part of a fun little mystery, and I got to play detective while trying to determine our little mohawk friend’s identity! Fingers crossed for a new species :)

Unfortunately, my internship ends in two weeks, so I won’t be able to report back to Deep-C on the results of “The Mystery of Mr. Mohawk”, but I still attend school here at UWF so I can continue to assist Marie and Dr. Snyder even after my time with Deep-C ends.

My time here in the lab has flown by! It seems like only a few weeks ago I was getting ready for our research cruise. Now I am getting ready to say goodbye! I still have 1 more blog post to write before my time is up, so stay tuned for my last glimpse into the world of an intern in Dr. Snyder’s lab at UWF!

Posted By:
Katie Vaccaro, University of West Florida

Herbert Johnson's Internship, Summer 2014 - Part 4

Hello everyone!

I have been conducting a primary literature search gathering data on the magnitude of nitrogen fluxes to and from the Gulf of Mexico to characterize the nitrogen budget in the Gulf of Mexico. I will also use the data visualization program Ocean Data View (ODV) to plot water column profiles of nutrient distributions to gain insight into the location of the sources and sinks of nitrogen to and from the Gulf.

I hope that you all are learning a lot and having a great experience with your internships!!!!

Posted By:
Herbert Johnson, 
Florida State University

Thursday, July 24, 2014

Cynthia Kane's Internship, Summer 2014 - Part 2

I can’t believe the ten weeks are coming to an end! It seems like just yesterday I was on the boat collecting samples! The last blog post ended with PCR and gel electrophoresis, so I’ll just pick up from there. After the gel was run successfully, the bands of DNA were cut out of the gel using a scalpel and stored in the freezer. When they were ready to be used, the samples were cleaned with a DNA cleaning kit called Qiagen. Basically, the kit was used to dissolve the gel, so that only the amplified DNA was left.
DNA Cleaning Kit
After the DNA had been cleaned, it was cloned and transformed, in preparation for making clone libraries. The DNA was first mixed with a vector and a salt solution (to maintain the pH) and allowed to incubate at room temperature to allow the cells to replicate. The solution was then added to electrically competent E. coli cells and then shocked to induce the uptake of the vector by the cells. Afterwards, the solution was incubated at 37 degrees Celsius (human body temperature) for an hour, and then the solution was plated on agar plates for the bacterial colonies to grow overnight. In order to select for only the transformed colonies, ampicillin, kanamycin and x-gal were added to the gel plates. Only cells that had taken up the vector would be able to grow on the plates, as the vector contained ampicillin and kanamycin resistance, while normal E. coli cannot grow in ampicillin or kanamycin. The x-gal allowed further selection of colonies by causing colonies that did not transform properly to appear blue/purple, rather than white.

The cloning and transforming part of this process gave me a lot of trouble, as many of the samples that I plated did not form colonies during the overnight incubation. In fact, only samples 16 and 36 developed enough colonies to be transferred to well plates. The cause of this difficulty remained a mystery for several weeks. We thought originally that it was the transformation kit that I was using, so I switched to a new kit. We tried using different agar plates, in case something had gone wrong in the preparation of the plates. When this still did not solve the problem, I used chemically competent E. coli cells instead of electrically competent ones, to see if there was a problem with the machine used to shock the cells. I reused two samples that had not previously worked, samples 28 and 34. After the 18-hour incubation, it was found that all six plates (three for each sample) had grown colonies!

Top left: me plating the transformed cells
Top right: a successful cloning reaction (sample 16); the white dots indicate successfully cloned and transformed cells, the blue dots indicate cells that did not take up the vector. 
Only white colonies were used.

The final step in the DNA analysis was to prepare the DNA for sequencing. To do this, a well plate of 96 wells was used, and one colony was carefully removed from the plate with a toothpick and placed into each of the wells. LB media was added to the wells (a solution to encourage the colonies to grow more overnight). The well plate was incubated at 37 degrees Celsius for 12 hours and then divided into three shipping well trays. A 50% glycerol solution was added to these trays to act as a buffer for the DNA during transport, and they were then sealed, labeled, and sent away for sequencing.

The empty well plate, along with the agar plates containing the transformed colonies
Me pipetting the LB media into the well plates
 The well plate in the incubator. The toothpicks were removed after one hour and the well was sealed with a tape cover for overnight incubation.
Unfortunately, due to the problems we had with creating the clone libraries, the sequencing for the samples will not get to the lab until after I leave. While this is disappointing, as it would have been nice to see the sequences firsthand, I understand that this is also an important part of research; learning how to deal with challenges such as this, which are just another part of research. Also, Joe Moss, my lab instructor, said he would send me the sequences when they are finished, so that I can see the fruit of my labors!

Posted By:
Cynthia Kane, University of West Florida 

Wednesday, July 23, 2014

Erica Levine's Internship, Summer 2014 - Part 3

As this internship draws closer to its end, it’s interesting to look back at where we started and where we've come. We were able to start with a relatively unorganized collection with no form of digital record and make it into the phylogenetically organized and cataloged collection it now is. We have almost completed entering all of the jars into the digital database, and should have all 500+ previously identified items entered by the end of the week. With our remaining time, we are using genus and species keys to identify and label new specimens as they come into the collection. We will also start cataloging the specimens that are stored in larger containers and tanks too big for the shelves.

While there is still some work to be done to catch up with the identification and cataloging of all of the collected specimens, the work we have accomplished over the past month will make it much easier for others to continue expanding the collection and find items already stored at the FSU Coastal and Marine Lab. While there were a few snags along the way, working to overcome the problems and get as far along as we have has allowed me to learn new things about working in a marine collection as well as about general problem solving skills. I have enjoyed my time in this collection and look forward to any future opportunities to continue used the skills I developed while working on this internship.

Posted By:
Erica Levine, Florida State University









Monday, July 21, 2014

Christopher Horruitiner’s Internship, Summer 2014 – Part 2

From June 19th to the 28th, I was on a research vessel called the R/V Weatherbird II. The objective of our cruise was focused on identifying hardbottom habitat along the outer shelf of the De Soto Canyon’s eastern margin (Figure 1). The Chief Scientist on board was Dr. Stan Locker, and his team was primarily interested in using a Teledyne-Benthos C3D interferometric sidescan sonar device to map the sea floor and give us a better understanding of benthic habitats and paleoshorelines. My team from Valdosta State University and I were interested in capturing whole water samples across the water column using a CTD carousel, which also measured the Deep Chlorophyll Max, sound velocity, temperature, etc.
Survey Areas - DeSoto Canyon's Eastern Margin

Anastasia Nienow and I loading samples from the CTD.
On this cruise, we collected pigment samples to later analyze with High Performance Liquid Chromatography (HPLC) and discreet Niskin bottle samples from several depths across the water column to later be analyzed by FlowCAM imaging cytometry, and Scanning Electron Microscopy (SEM) to see how populations (primarily diatoms) change with depth and with season and hopefully we can compile that with a data set going back to January 2011 to identify/understand the temporal-spatial patterns of phytoplankton populations in the DeSoto Canyon region of the northern Gulf of Mexico.

While onboard, the science crew and I had to work two four-hour watches per day, where we helped monitor data acquisition, keep track of navigation, and took a log of activities. We also enjoyed three amazing and nutritious meals a day made by the onboard chef Thomas Lee, who took the care to make me separate vegetarian meals.

With only 8 or so hours of scientific obligations per day on the cruise, there was more than enough time to enjoy living out at sea in the Gulf of Mexico.

The whole crew came onto the deck to take a look at the formation of a water spout.  You can see the tendril of another to the left.
Dolphins were a regular phenomenon whilst on the R/V Weatherbird II.
And I also enjoyed my first real sunset (as well as my first real night sky and my first shooting star(s), which were impossible to photograph at night).
A pod of dolphins!  There was even a calf as well.  We thought they were made curious by the sonar device, but even after it was turned off they continued to show up.  
I am looking forward to analyzing our samples using the new lab equipment!

Posted By:
Christopher Horruitiner, Valdosta State University

Rachel Holladay's Internship, Summer 2014 - Part 4

Hello! Phases II and III of the ROV construction have been delayed while we are waiting for the rest of the parts to arrive. We have, however, tentatively scheduled some field testing in Bay St. Louis once more progress on the prototype can be made. This testing will also allow us to try out the manual that I am currently developing that will assist the intended audience, high school students, to easily collect data.

As a computer scientist, I have little to no experience with using CAD (computer aided design) programs. However, wanting to model the ROV as well as provide some detailed drawings and schematics, I decided to download SketchUp Make and fiddle around. While it isn’t as powerful as many, more professional CAD services, its relatively small learning curve made it incredibly appealing. Below are some of the sketches I’ve made, including a full mock­up of the intended end­result and a heavily stylized idea of the ROV swimming along in the ocean.

 

Posted By:
Rachel Holladay, Naval Research Laboratory

Friday, July 18, 2014

Sam Holladay's Internship, Summer 2014 - Part 3

Over the past few weeks I have continued work on the adaptive climatology iPhone app. I have continued to conduct a great deal of research into the app, and began work on the user interface. However, I have encountered some problems with designing the app totally from scratch: learning all the iPhone development on my own is difficult, and unfortunately Web resources aren’t the best. After some consulting with my advisor, I have decided to try a new tactic and focus instead on the most important part of the app: establishing communication between the user and our server, so the user can send in information for us to use in our model.

When facing adversity, I have found that determination, repeatedly attacking a problem, usually works after a while. If repeated attempts do not work, then trying the problem from a different angle helps. I hope that working on this different facet of the app will yield better results. Even though I have been greatly frustrated by setbacks, I have still learned a lot about Objective-C and iPhone development, and hope to continue with progress.

Posted By:
Sam Holladay, Naval Research Laboratory

Tuesday, July 15, 2014

Cynthia Kane's Internship, Summer 2014 - Part 1

Hello everyone! Things here at the University of West Florida have been pretty busy - but super fun as well! About a week after I arrived, I went on a research cruise to collect water and sediment samples for analysis. The trip was very education and also very wavy; it took some time to get used to the swaying of the boat. Here are a few pictures of the cruise (also see Katherine Vaccaro’s first post - she has some really good pictures!)

Left: my wonderful partner, Katherine Vaccaro setting up our lab station before we left  
Right: The cruise was great for collecting water samples - and fish! Here is Dr. Jeffrey holding a wahoo we caught while out at the one of the deeper stations

 Us taking water samples from the SeaBird CTD machine

After the cruise, things started picking up; we began doing lab analysis work, including PCR, gel electrophoresis, DNA purification, and clonal library creation. It took some time to get the hang of all the different tests and tools, but it was also really cool to be able to gain first hand lab experience. I’ve included some photos below, mostly of my PCR work. PCR stands for Polymerase Chain Reaction, and is used to amplify very small amounts of DNA so that it can be analyzed.

Me making the PCR samples. 
The “hood” I’m working in is UV sterilized between each sample to prevent contamination.

Left: The gel visualisation machine (on the left) shines a UV light through the gel, causing the DNA to glow so we can see whether the PCR worked or not. 
Right: A successful PCR! The bright bands indicate that the correct fragment of DNA was amplified.

Posted By:
Cynthia Kane, University of West Florida 



Herbert Johnson's Internship, Summer 2014 - Part 3

Hello Deep-C comrades!!!

Since my last blog entry, we have moved forward with running water column samples for nitrate concentration as well as running persulfate oxidation of total dissolved nitrogen (TDN) to nitrate. We oxidize TDN to nitrate by pipeting a sample into a glass test tube and adding a persulfate oxidizing reagent (made up of sodium hydroxide and potassium persulfate). Next we autoclave the prepared samples with reagent for 55 minutes. After the samples have cooled, the nitrogen concentration of the samples are then measured using a chemiluminescent analyzer.

Additionally, I have been spending many hours learning ocean data view (ODV), which is a free data visualization program that you can use to plot oceanographic data sets, including publicly available data sets such as World Ocean Atlas. The software allows users to create maps, cross-sections and nutrient profiles. ODV even allows the users to create their own variables relevant to their specific research. It is an invaluable resource for chemical oceanographers or anyone studying ocean chemistry.


I hope that everyone is having a great time with their internship!

Posted By:
Herbert Johnson, 
Florida State University

Monday, July 14, 2014

Katherine Vaccaro's Internship, Summer 2014 - Part 4

Hello again! We have had some exciting things happen in the lab! We recently got our lovely FlowCam back after being upgraded! It now has a nifty autofocus feature and produces beautiful pictures of every single microscopic organism that flows through the camera (Hence the name FlowCam!) One of the graduate students, Marie Head, has been showing me how to run water samples through the machine and it is absolutely amazing! 
The mighty FlowCam and all of its beautiful pictures!

The goal of using the FlowCam is to get an idea of 1) what is in our water samples and 2) what kind of ciliates we can find. The pictures can be categorized into libraries based on previously identified species, and we can see what kind of ciliates we should be finding when we try to isolate them! Unfortunately, somewhere between the FlowCam and the collection bottle, our organisms didn’t quite make it. We suspect they may have gotten stuck in the syringe or perhaps stuck to the plastic tubing. Luckily, the FlowCam only takes 5 mL samples so we had plenty more water to look through. While we aren’t able to use the FlowCam-checked water samples to isolate, the machine still provides us with vital information about the communities we can find in the Gulf of Mexico!

Posted By:
Katie Vaccaro, University of West Florida

Katherine Vaccaro's Internship, Summer 2014 - Part 3

Hello everyone! I have been busy at work with the samples we collected from our cruise a few weeks ago! I am in charge of looking at the samples under a microscope and isolating whatever ciliates I can find, especially tintinnids. Once I am able to isolate individual ciliates, I place them in microfuge tubes and run a nested PCR to get DNA. Because I am working with only 1 cell, a single round of PCR isn’t enough to produce the amount of DNA needed! So I run a second round of PCR to further amplify the DNA. This was a new procedure for me, and I found it really fascinating to learn! It sure does take a while though :) Usually the two PCR cycles take up to 5 hours!

After I have enough DNA, I have been running the DNA through a 1% agarose gel to separate the bands and hopefully get good results! But, like I said, working with only 1 cell is more difficult and often I don’t get great results. But when I do, it is so, so rewarding!

So here I am, chugging along through samples from the Gulf of Mexico, finding individual cells and taking their DNA. It’s demanding, but I am having so much fun! Hopefully for my next blog post, I’ll have exciting results to share!

Until next time!

Posted By:
Katie Vaccaro, University of West Florida

Emily Goetz's Internship, Summer 2014 - Part 1

Hi! My name is Emily Goetz, and I am an undergraduate student interning at the FSU Coastal and Marine Laboratory this summer. Through the Deep-C internship, I will be contributing to the development and improvement of the Atlantis ecosystem model for the Northern Gulf of Mexico. Atlantis serves as a tool to model interactions between organisms and the physical environment and geography, while simultaneously factoring in anthropogenic effects and potential conservation efforts on ecosystems. While a preliminary model is already in use in the Gulf of Mexico, it is lacking in some basic information and has glitches that make it less user-friendly. This summer, I will be helping Dr. Stephen Gosnell of FSUCML to fill in the gaps of species-species interactions, to develop further the Atlantis model specific to the northern Gulf of Mexico, and to create a more user-friendly instruction manual, which will allow outside parties to understand and use the model. I really look forward to using my background in biology, geology, and GIS to work on this project. Additionally, simply being at the marine lab fosters relationships with scientists in related fields and provides opportunities to assist with additional field and lab work. This summer, I look forward to both working on the Atlantis project as well as being exposed to additional facets of marine biology and conservation.

Posted By:
Emily Goetz, Florida State University 

Friday, July 11, 2014

Erica Levine's Internship, Summer 2014 - Part 2

As working in the collection progress, I am picking up a lot of useful information about how to deal with problems that arise. First, we ran into problems with setting up the database in a way that would make it easy for anyone to enter new collection objects or search for existing specimens. Next, we discovered that some of the labels we currently have are not suitable for long-term storage in ethanol with the specimens. Since the labels we had would not work, we had to design new labels, figure out what ink and paper would be best to use, and how or where to print the labels. So far, Ryan and I have explored Specify and how to get it to do what we want for the database, and we've investigated how to design labels for all the jars in the collection. With a little help from the program’s technical support, we are a few steps closer to being able to catalog and label all the specimens. By the end of the week we hope to have the database entry forms ready to go so we can start entering all of the jars into the database. Once we can order the correct paper and best printer for making ethanol resistant labels, we’ll be able to start the process of relabeling all existing jars in the collection. While the going has been slow, we've finally made some good headway and will be able to jump into organizing the collection even more soon.

Posted By:
Erica Levine, Florida State University










Wednesday, July 2, 2014

Rachel Holladay's Internship, Summer 2014 - Part 3

Construction has begun! With the arrival of the standard SeaPerch kit, I was able to assemble the electronics, thrusters and modified PVC frame. I’m waiting to mount the thrusters until I have the other components that will sit alongside them. Thanks to our local SeaPerch guru, David Young, we have been able to play with an already assembled SeaPerch, which has been extremely useful. After using the NRL pool for some base testing, we realized some important buoyancy issues which lead to some minor redesigning in the PVC base.

The process of the rest of the parts being ordered turned out to be a more lengthy process than I originally expected, but they have been ordered and are on their way! A critical piece of the Sensor Suite, the custom made PCBs, has already been happily delivered. While waiting for everything else to arrive, I’ve been exploring all about Arduino, so I am very excited to get my hands on that! In addition, fellow intern Sam and I have been creating a first draft of the graphics that will be used on the OS Kit App.

Posted By:
Rachel Holladay, Naval Research Laboratory