Teenage Mutant Phytoplankton

Alright, alright well this week was certainly interesting. As of last Friday, our last diatom cultures, the Thalassosira, reached maturity after being exposed to the oil. (I don't think I mentioned it in my last post, but we decided to use our batch of Thalassiosira to compare to the Chaetoceros since they're both diatoms and we had the culture ready to be used.) Looking at these bottles, I can tell the difference between each of the cultures is going to be much more minute.

After measuring and calculating out the cell density, I found the average density of the standard (aka, the control) cultures was 1.73x10^6 cells/mL.  The culture that had been exposed to one drop (or 166 ppm of oil) increased in cell density to 4.11x10^6 cells/mL. After what we saw with the Chaetoceros cultures, this increase not alarming in the slightest. What was surprising though was that our culture containing three drops (about 500 ppm) decreased significantly in cell density to 9.77x10^5 cells/mL. This trend wasn't consistent, as the ten drop (1660 ppm) culture showed another increase from the control at 2.44x10^6 cells/mL. Likely, the inconsistency can be contributed to either a foreign contaminate or a lack of proper aeration.

But this off-trend pattern of cell density wasn't the only alarming trait of these cultures. At 1660 ppm, we noticed some of the cells had oblonged themselves and attached to other cells. We also noticed strange "ghost" cells forming in the oil drops. Below is first a picture of a normal cell group within the 1660 ppm culture, and then an example of the stretched cells and the translucent cells at the 40X objective.

A researcher at the Dauphin Lab had some interesting theories about why the cells did this. The translucent cells could be formed from dissolved organic carbon (DOC) produced by the Thalassiosira. "In a culture, DOC congeals through random interactions such that the "sticky" DOC particles aggregate into larger particles collectively called "Transparent Exopolymeric Substances" (aka TEP).  TEP is reactive and will cause aggregations, especially with crude oil droplets," according to Mr. Jeffery Krause. He also said that depending on how we aerated the culture, the phytoplankton could have reacted with the oil. He didn't specify how, as he's only been at the lab since 2012 and didn't study the Deepwater spill personally, but he did refer me to a "Gulf of Mexico Initiative (funding agency) research consortium" that solely researches with those types of interactions.

Overall, I'd say this part of the experiment did not reveal anything about my hypothesis, but it did help to deepen my understanding of phytoplankton on a cellular level. 

Today, I'll be going back to Mr. Soderblom's to record data on the green phytoplankton we began experimenting with last Friday and have now probably reached maturity.

Signing off till next time, this is Erin Butcher.

They Kept on Growing

Our Chaetoceros cultures have reached maturity in one week. Just looking at the bottles, I can tell right away that the culture with the most contaminate, 2500 ppm or 10 drops of oil to be exact, did anything but die. The color is slightly darker than the 250 and 750 ppm cultures, indicting that instead of complete annihilation, the oil has caused an abundance of growth. 

Very carefully, we took samples of each of the contaminated bottles and placed one drop on individual slides. We captured pictures of the tiny cells, now darker in appearance because of their oil-heavy meals, and I used these pictures to calculate cell density. (For this, we calculated the area in a single field of view or FOV on a microscope at 10X, and then found out how many FOVs would be in one slide cover and multiplied the amount of cells in the FOV we captured in our picture by the ratio of a FOV to the slide cover.)

Below are pictures of the 2500 ppm culture at 10X and 40X respectively.

From the data, I was able to compute that the cell density of the 2500 ppm culture was on average 2,510,000 cells/mL. Compare this to the control cultures, which averaged a cell density of 860,000 cells/mL. That's nearly a 1:3 ratio (actually 1:2.97, but close enough).

The 250 and 750 ppm cultures came out to 1.24x10^6 and 1.31x10^6 cells/mL.

This data disproves my hypothesis of the 2500 ppm of oil killing all the plankton, but it does illustrate something amazing. A concentration of 2500 ppm of crude oil is an impossibility in a real spill. With the constant current of the ocean dispersing the oil over its surface and the lack of any boundaries (besides the shore) containing the oil to one section of the sea, the oil would never be able to remain all in one area long enough to reach a concentration that high. So the fact that the diatoms were able to not only survive but thrive under these unrealistically strenuous conditions shows that these organisms are perfectly capable of coping with spill conditions without the need of any clean up whatsoever. (Whoa.) 

Moving on, after we recorded all the necessary data, we had to discontinue our experiment cultures. The reasoning behind this is that with every day these cultures are allowed to grow, the chance of an uncontrollable contaminate such as a bacteria or virus polluting and overtaking the cultures becomes more probable. This would lead to confounding evidence as we wouldn't be able to determine whether cell death would be due to the oil or another contaminate.

For the next round of experiments, Mr. Soderblom and I decided to move away from diatoms to a green or brown phytoplankton. This is because, while diatoms are the most populous group of plankton in the Louisiana Bay area, they are not the select food source for zooplankton. Diatoms, unlike other species, have a sodium metasilicate shell covering their cell walls. This makes them harder to digest and somewhat  toxic to zooplankton when consumed in large quantities. Green and brown phytoplankton, however, are eaten by almost every species of zooplankton and even constitute the diet of other creatures of the ocean, such as fish, sponges, sea stars, and even whales.

So the two species we have decided to culture for the next round of experimentation are Chlorella vulgaris and Tetraselmis suecica (I'm not exactly sure on the Tetraselmis species, but I'm pretty sure it's that one). Both are between 10 and 12 micrometers in width, making them easy to observe, and the Tetraselmis is motile, making it fun to watch. Below are pictures of the Chlorella and Tetraselmis respectively at 10X and 40X.

We plan on exposing these buggers to oil starting this Friday.

Signing off till next time, this is Erin Butcher.

An Unexpected Turn and Experimentation

Well, as stated in my last post, Mr. Soderblom and I had hoped that the Cyclotella species would be the diatom to survive the best, as it is the easiest to observe, but alas, not everything goes as planned. Unfortunately, all of our Cyclotella cultures turned clear after one week of allowed growth, aerated or not, meaning a majority of the cells died due to unknown causes.

Our Chaetoceros cultures, however, have taken really well. Their bottles are a darkish yellow color, even the cultures that ended up taking 5 days to ship, meaning they are thriving quite well. The aeration seems to have aided in growth rate, so we will be using this method in our experiment.

We officially began experimentation today!! To start, we expanded our best culture (the one that took 5 days to ship, surprisingly) by diluting it with more medium (in a 1:1 ratio) to allow it to grow. In the end, we had 1 liter of the expanded culture. Then, we separated the culture into 5 bottles, each getting around 200mL. 

Two of the bottles we designated as controls. The other three were contaminated with the oil. Now after having sifted through some research, I found that oil concentrations in the ocean water of the Louisiana Bay were about 160-260 ppm near the time the drill was capped off. We decided that in order to get noticeably different results for each bottle, we would have one bottle be within the range of the actual spill, and two with more exaggerated levels. In the end, the bottles held 250, 750, and 2500 ppm of crude oil (or 1, 3, and 10 drops from a pipette). I hypothesize that the 250 ppm won't cause much difference, the 750 ppm will have accelerated the growth rate, and that the 2500 ppm will precipitate, turn clear, and kill all of the plankton. The oil sat on top and looks really sludgy. (I thought it was funny the researchers who gave us the sample labeled it "sweet crude").

We took pictures of Chaetoceros yesterday. They're notably smaller than the Cyclotella at about 8 micrometers by 4 micrometers. They also aren't very neatly-shaped. (The third is from Google and apparently, they enjoy sticking together but it's hard for us to tell if ours are exhibiting that behavior). (At 4X and 40X objective respectively).

We also took pictures of the Thalassiosira and plan on using this species if we have the proper amount of time. (at 4X and 40X)

As you can see, they're small and hard to count, but we'll just have to do our best.

Signing off till next time, this is Erin Butcher.