First Blog: From the Bottom Up

The Earth Sciences internship is under way! Two weeks in, and we've dived straight into things. Fernanda and I are working with Dr. Shellie Bench, a post-doc who studies phytoplankton on the Western Antarctic Peninsula (WAP). This summer, we will be studying the composition, size, and productivity of the phytoplankton community through microscopy and DNA analysis of the samples. Shellie had collected numerous samples off the WAP during her trips to Palmer Station, a residential research facility on Antarctica.

http://www.tampabay.com/specials/2010/reports/antarctic/maps/index.shtml

The first few days were a lot of catching-up. Shellie gave us articles and textbook readings so we could understand why studying phytoplankton in Antarctica was important, as well as the scientific methods we will use to draw conclusions. As it turns out, phytoplankton affect just about everything else on the food chain. These microscopic, free-floating autotrophs feed zooplankton, which feed just about everything else. Differing preferences for phytoplankton and zooplankton can cause populations of large animals, such as elephant seals and Adelie penguins, to change behavior or even decrease in population. The WAP is experiencing the most warming of any region in the world, and this scary fact raises many questions that we hope to answer: What does receding ice mean for the phytoplankton population? How will organisms of higher trophic levels deal will these changes?

During the second week, we began to analyze samples under the microscope. Because phytoplankton contain pigments such as chlorophyll-a and phycoerythrin that fluoresce under varying wavelengths of light, we were able to identify and assess the abundance of different species of phytoplankton. Common species include diatoms, phytoplankton with a hard outer structure, dinoflagellates, which have flagella, and cryptophytes, which were identified with their yellow-orange fluorescent signature. Learning how to use the microscope properly was more difficult than anticipated. Looking at samples through the 1000x magnification required meticulous adjustment of the microscope focus. However, after a few days of practice, we began to get the hang of it. The images under the microscope were no less than beautiful - from perfectly round centric diatoms to bright-red clusters of chloroplasts, the wonder of these small creatures were captured by the camera, which was connected to the microscope. Later on, we will relook at these images and record species and count data. Recently, we also prepared concentrate samples and looked at them under the microscope. Phytoplankton under white light was just as stunning, and we were able to see more of the structure (such as the hard, silica-based frustules of the diatoms).

http://www.daviddarling.info/encyclopedia/D/diatom.html

These two weeks have been filled with new knowledge, hands-on experiences, and science. However, there's even more excitement coming. Next, we will be analyzing DNA and RNA sequences of the samples taken throughout the bloom period. By using powerful DNA analysis software such as MG-RAST, we will be able to confirm our microscope findings with sequence-derived taxonomic data. Last but not least - a huge thank you to Shellie who has really done a outstanding job giving us a crash course on phytoplankton, the WAP, and methods! We will keep you posted with new findings.

Second Blog: Out of the Fields and into the Lab

In the past few weeks, we wasted no time by jumping right into the heart of research - field and lab work. Before performing analysis on actual samples from Antarctica, Shellie familiarized us with DNA extraction and quantification procedures. But there was one small problem - where could we find phytoplankton samples to "practice" on? None other than water samples from the San Francisco Bay!

The Palo Alto Baylands are the perfect place to collect water. It's only about 15 minutes from Stanford, and contains water directly from the San Francisco Bay. Equipped with a cooler, several 2-liter bottles, and a 5-gallon container, we scooped as much water as we could carry. The water itself was very murky from suspended soil, but also teemed with phytoplankton and bacterial life. Upon arriving back at the Francis lab, we took the temperature and salinity of the different samples.

The next step was to filter the samples to remove the dirt, but keep the organisms. Our first approach was to let the water settle for a day, which allowed most of the dirt to collect at the bottom. Then, we filtered the water through a mesh filter that proved to be too large for the extremely fine soil particles. The 10 micrometer and 0.2 micrometer filters were the sizes we ended up using - the 10 μm filter removed the dirt, and the 0.2 μm filter captured the biomass. Water was moved through the apparatus by a vacuum hand-pump, and often took hours to be filtered completely. The filters were sliced in half and kept for DNA analysis; the now-filtered water was discarded.

Before last week, I had never done a PCR reaction before. Shellie explained the basic gist: we had to break apart the cells and isolate the DNA. Thus began a long process of freezing/thawing, beating with micro-beads, incubating, and dousing the samples in different buffers. I loved getting to work with liquid nitrogen and micropipettes - it was definitely reminiscent of AP Biology but took lab work to a whole new level!

This week, we followed another protocol to quantify the DNA before sending it off to be sequenced. This was important because sequencing can't happen with too much or too little DNA. Shellie introduced us to two methods: Pico-green and Nanodrop. Nanodrop was by far the easier protocol - a machine analyzed a 2 μL drop of sample and determined the DNA content through spectrophotometry. Pico-green was similar, except "standards" of DNA had to be prepared in addition to the samples. A μg DNA vs. Fluorescence line of regression was computed using the standards, which had known amounts of DNA. Therefore, the amount of DNA in each of the samples could be calculated, using the line of regression and the measured fluorescence.

Being in the lab was exciting but also somewhat nerve-wracking - when working on such a small scale, being precise and eliminating sources of contamination are key. These are some of the skills I hope to hone over the summer, until I'm ready to analyze the real samples!

Up next: identifying/counting Antarctic phytoplankton species from microscope images, PCR reactions/quantification on Palmer Station samples.

Third Blog: The Final Countdown

The last segment of the internship was indisputably the most exciting yet!

In the lab, we dove straight into PCR reactions and gel electrophoresis. Previously, we had extracted and quantified DNA. The final step before sending the DNA off to be sequenced involves checking for quality and contamination. The PCR and gel reactions allow us to conduct a visual quality check. PCR involves binding primers to the segment of DNA we want to replicate and using DNA polymerase to actually do the replications. Thus, we end up with high-density DNA that can be run through a gel. When working on gels, we would often go through several pairs of gloves in one day – ethidium bromide, the substance used to make the DNA show up under ultraviolet light, is a carcinogen and requires special care. Making the gel and loading the wells was also part of the procedure. Because the agarose gel contains tiny pores, the DNA separates by size and we were able to see bands of DNA. The bands tell us whether the DNA is the right size and whether it has been contaminated. Unfortunately, bands showed up in our negative control (in which there is no DNA), so there will need to be troubleshooting to find out where the DNA contamination is coming from. Otherwise, the bands we were getting on the gel are relatively strong.

For the past few days, Fernanda and I worked on translating our experiences and results into a poster for AGU and a PowerPoint presentation for the rest of the interns. Drafting and revising the poster and presentation introduced us to the scientific process – apparently, real scientific papers undergo similar processes before being published! I had a great time reflecting on my summer and discovering the other exciting fields of science that other interns explored. The final presentations were supplemented by the lab and farm tours, where we got a first-hand look at different work environments and scientific endeavors. I particularly enjoyed the pizza we made at the farm! Overall, it was a week full of bonding and discovery.

Next steps for my project involve more DNA sequencing and analysis of the sequences, especially for the 2014 season. We have already started to analyze the sequence data MG-RAST has given us. Eventually, we will be able to compare the results of this data with the results of counting/sorting of specimens on microscope images. Combining these methods allow us to obtain a more accurate picture of what is going on in the Antarctic.

Lastly, I would like to thank Dr. Jenny Saltzman, Dr. Shellie Bench, and Elizabeth Morin for making this internship the best it can be. Cheers to a summer well spent!

To read about Catherina's experiences in 2013 - https://sites.google.com/site/earthscienceshighschool/history-of-life-blogs/catherina-s-blog