Siliceous and Delicious

Tried to quickly make a CLSM slide, but screwed up—tried to heat the slide up more to make it dry quicker, and ended up with bubbles in the epoxy. A nuisance, since I thought I’d finally mastered the technique.

I also noticed that the fluorescein in my vials wasn’t looking quite as neon-y as I had remembered it, so I thought I might go and get some more from Jacques’ lab. I ran into Jacques and Sébastien there—fortuitously or otherwise—and got some more fluorescein, in addition to some fluorescein sodium salt (which Jacques suggested I try, given that it will dissolve in water rather than having to use methanol), calcofluor (which is meant for staining cellulose), and some ruthenium red (which Jacques said was expensive, and according to wikipedia binds to tubulin). We chatted a bit about how my projects are going. He suggested that, if there was still organic matter in the fossil frustules, I might be better off not using methanol, as fluorescein might preferentially bind to the organic matter and the methanol might dissolve and remove that organic matter. He also made me realize, very painfully, that I’m still essentially where I was three years ago—solidly stuck in methods development and nowhere near collecting data. I was left with much the same feeling after an eerily similar conversation with Sébastien.

Fled Harvard for an hour to sit and have breakfast with Beau at Bloc 11. As always, Beau was tremendously supportive and did his best to try and get me to remember that I am making progress, and that I do have the skills it takes to finish this PhD. He’s right, of course, and he’s right that there’s no ‘magic recipe’—every graduate student has to fight their own battle and find their own path through the spiny thicket of graduate school.

I returned and rallied the motivation needed to face the afternoon’s CLSM session in the face of the morning’s frustration. When I showed up at the CLSM, it wasn’t there. Gone. No microscope, no computer. Someone using another instrument in the room nodded in the direction of the adjoining room, muttering something about the confocal having been moved. I found the microscope there, with nothing switched on, the computer disconnected, and parts strewn around in various states of disassembly. Evangelos was nowhere to be found in the building. And there had been no notice emailed out regarding the facility being moved, no indication on the web schedule that the tool was offline. Nothing.

The frustration over tools I have faced over the course of this PhD is phenomenal, this is just another example of why it’s taken me so damn long to get anything going. I am frustrated and disappointed. And any motivation I may had left in me to work today is gone.

Trawled the internet for help on using the other fluorescent dyes I took from Jacques’ lab this morning. Found the following passage describing a protocol for preparing a calcofluor solution from this paper:

Here, we have adapted the technique to environmental aquatic samples. Before staining, a stock solution of CFW was prepared as modified from an original protocol [http://www.mycology.adelaide.edu.au/], by adding 35 mg of CFW to 7 ml of sterile distilled water and a few drops of 10N NaOH to increase the pH to 10-11, because CFW does not dissolve well in neutral solutions. The final volume of stock solution was then adjusted to 10 ml with sterile distilled water, aliquoted in small quantities.