Thursday, November 19, 2009

Experimentation Complete

Filtering has finally been completed. In total we filtered ~91 liters of sample! Our next primary objective was to extract RNA from one of the filters and quickly convert it to cDNA to prevent degradation. We can then ship the cDNA back to LSU to sequence.

While challenging, the information we will learn from this type of analysis will be very valuable. Sequencing the 16s rRNA fragment directly allows us to determine which species of microbes have remained viable when trapped inside Taylor Glacier. The normal method of just sequencing the 16s rRNA gene (which is located on DNA, not RNA) only tells us which microbes were present in the glacier at one point in time. Any microbes which died in the glacier thousands of years ago would no longer be viable, but may have have left there DNA behind. In other words, with DNA alone, it is difficult to tell which sequences came from viable cells as opposed to those sequences which came from dead cells.

Our extraction turned out to be a success as we managed to extract ~25ng of RNA from one filter!!! From this RNA we reverse-transcribed the 16s rRNA molecules to cDNA which is now on it's way to LSU to be sequenced!

Additionally, we measured the amount of ATP in our samples with the luminometer (as described last month on the blog). This experiment was successful as well, indicating about 10,000 cells/mL. This reinforces some direct microscopic cell counts we did earlier which yield roughly the same amount.

Sunday, November 8, 2009

Filter, filter, filter...

After some initial difficulties with filters clogging Amanda and I have finally gotten into a good rhythm of cleaning ice, melting ice, pre-filtering melt water, centrifuging sediment, and filtering. We are managing to put about 10 liters of melt through each filter!

Wednesday, November 4, 2009

Analysis Begins...

Hey Guys! I'm back in McMurdo and back in the lab ready to go. Its nice to be back and enjoy the comforts of being inside, out of the wind. Here's an overview of what I am doing in the lab:

I want to concentrate about 150kg of our sample ice down onto filters for further experimentation. Several tests such as DNA or RNA analysis rely on relatively large amounts of cells to work efficiently. Our ice samples only have a few thousand microbial cells per mililiter. This may sound like a lot, but it's actually quite low. For example, one gram of soil commonly contains several million cells per mililiter. By filtering several gallons of melted sample onto a filter, I can thus concentrate the microbes to levels which will result in a reliable signal.

First, I will need to decontaminate the outer surfaces of our samples. The ice has been handled extensively during collection and it's imperitive we remove any foreign chemicals or microorganisms we inadvertantly added.

Here is what happens when you don't decontaminate samples. These are microscope images of filtered ice. The samples have been stained with a dye which glows bright green when it binds to DNA. As you can see on the left, the sample that was not decontaminated has lots of foreign material. The sample on the right was cleaned prior to filtering.

To decontaimnant the ice, we melt the outer surfaces away from the ice using sterilized water. This is the apparatus I setup to sterilize the water. Normally, we would just autoclave large carboys of water but unfortunately, McMurdo does not have any autoclaves large enough to do so on station. As a result, I must sterilize the water by filtration as shown above. On the far left is the carboy of water to be sterilized, followed to the right by a large parastolic pump. The wooden box in the middle is a portable laminar flow hood which is simply a fan which pushes air through a HEPA filter. This is to help prevent contamination from any microbes normally floating around in the lab. Finally, on the far right, is my collection bottle in which the filtered water is collected.

A variety of glassware which has been washed and autoclaved. As you can see, one cannot take contaimination lightly in a microbiology lab.

This is the ice cleaning station we created. We set samples on the grill right next to the laminar flow hood and pour the clean water all over the sample to melt away the outer contaimination.

Amanda uses forceps to manipulate and position the ice sample on the cleaning station.

A close-up of one of our samples. It looks pretty awesome once you clean them off.

This is the leftover sediment which was washed off the outside of our samples.A dear, dear, friend of mine which I haven't seen since I left Louisiana. I was very happy to find a six-pack in the station store.

Monday, November 2, 2009

A brief update..

We have completed sampling and I will be heading back to McMurdo tomorrow to begin analysis.
My main goals will be to concentrate a large volume of sample onto filters. More details and pictures to come once I return to McMurdo.

Sunday, November 1, 2009

Geaux Tigers