Before you even have a chance to really get to know your classmates, you are led on a dark walk to a stinky Cedar Pond, where the brave venture chest-deep into the cold, sulfuric muck. They stomp around in the soft, slimy, decomposing leaf litter and sediment, releasing bubbles of methane gas. The methane is trapped by one person (Dan Buckley, in this video) in a stopped funnel. On the count of three, Dan removes the stopper and Steve Zinder holds a flame as the methane shoots out of the funnel. Voila! Volta!

I mentioned in my last post that one of the many wonderful things about the Microbial Diversity Course at the MBL is the quality of the invited speakers. Another favorite of mine was Jorg Overmann, who wrote the book on Molecular Symbioses and is the director of the DSMZ (I did not know that!) He introduced us to his favorite bugs, the consortium known as Chlorochromatium aggregatum.

A really nice summary of what we know about them is here on the Small Things Considered blog. Basically, there is a flagellated Beta-Proteobacterial rod that is encrusted with non-motile green sulfur photosynthetic symbionts (epibionts, to be precise, because they are attached to the surface.) Their relationship is complex, but what’s overtly cool about it is that the rod can chauffeur the epibionts around in search of the right conditions for photosynthesis. One can (and we did) see their cool scotophobic behavior in a light microscope. It looks like this (run towards the light!):

Source

Jorg said that I should be able to find these consortia in the nearby Trunk River, so hello, of course I will try to enrich for them! However, he strongly discouraged my reliance on their enrichment for my independent research project in the last half of the course, because they are quite elusive and grow very slowly.

So, let me backup a little here. The first three weeks of the course you attend lectures in the morning and then you are in the lab until late at night. Those weeks are spent attempting to culture a diversity of isolates from environmental samples that you collected in the first few days. You are also introduced to all of the super-cool equipment housed at the lab, to which you will have full access. For the last three weeks of the course, you draw upon your new-found knowledge, the support of those around you (students, instructors, TAs), and the abundant resources at the MBL to work on an independent research project. Most people work on AN independent research project, but I apparently am not most people. I worked on FOUR. This is the first (and favorite) that I will describe in a series of blog posts.

Setting up the enrichment is easy. You just take some sediment from the river, put it in a bottle with some spring water supplemented with sodium sulfide, making sure that there is no head space and no way for oxygen to get in. Then, incubate it at room temperature with a light-dark cycle and wait.

Then you wait. I waited for 23 days. Some of them had purplish top layers of sediment and some had greenish top layers. Jorg said that Chlorochromatium aggregatum will form a biofilm on the surface of the glass. A biofilm formed! On the surface of the glass! It was purple.

Then you play with your biofilm. First of all, before I proceed, I did NOT find any consortia, but I hope you will not be too disappointed, because what the hell was I going to do with them in the week that I had left anyway? My biofilm consisted primarily of two cell types (see below.) You can see big, oval cells that contain highly refractive sulfur granules, and you can see some smaller, dark rod-shaped things.

I decided to use one of our cool toys to see if I could physically isolate the presumed purple sulfur bacteria (encircled in yellow) from those other guys. I wanted to isolate them 1) to get a 16S PCR product to identify them and 2) to see if I could transfer them from the biofilm into new culture medium and grow them.

I used the PALM CombiSystem which is a laser dissection/optical tweezer type microscope. Basically, I could spread out my biofilm on a slide covered by a very thin membrane, then use a laser to cut out a circle around one or more cells on the membrane, and then use some mysterious quantum force to catapult the membrane+cell(s) directly into the cap of an Eppendorf tube (filled with culture medium or whatever.) What the… what!?!? That was supercrazycool! Somewhere I have a movie of it, but you can check this out to get an idea:

So, I was able to isolate these guys (they were Chromatiaceae, per RDP) for 16S PCR and I was able to get them to survive the transit into the Eppendorf tube. After two days, they were still metabolically active!

These figures are from my final report. I must admit that I wrote the bulk of it, while drinking beer, and during an all-nighter. So, I wont vouch for it’s quality and can’t recommend that you read it. These reports, regardless of the quality, are good to have around because current students pore over old reports for inspiration. Reading it myself after 3 years, I felt a strange combination of “Wow, I did that?” and “Eww, I wrote that?” But, my goal here is just to share a little bit of my MBL experience with the world, and I hope you enjoy it.