Ritualistic Procrastination - Bleb Monsters - Vindication At Last - Incipient Fatal Fungal Infection - A Call To Microbiologists!

In order to effectively write my committee meeting report, I am watching every episode of Mystery Science Theatre 3000 featuring 50s educational films ("A Date With Your Family", "Good Grooming" and "The Home Economics Story").

This is an example of ritualistic procrastination. I will continue to watch MST3K videos on YouTube until I begin to get bored, at which point the sweating panic of not having written my report will kick in. Then I will be spurred on to write, which will take all afternoon and evening. I am not in the lab this morning because the urge to set up just one more experiment would be even more impossible to resist than YouTube.

I think there is a certain amount of ritual procrastination that is an inescapable part of the writing process. Don't you?

Actually, there's a sidebar to the reason I'm not in the lab and it involves filamentous fungi. I would not really be doing experiments because I would be devising strategies to identify and destroy the fucking fungus that is growing in the incubator. I don't even know if I can talk about this yet ...

I did a great experiment the other day but when I went to do the analysis (single cell morphology) the controls were all funkster. Wha??? Shit. Now I don't believe anything!

I set it up again with freshly-thawn cells, this time with even more experimental goodness, extra coverslips just in case. In parallel I set up a big-ass RNAi experiment with brand new siRNAs and cells that I could tell were knocked down even before blotting, that's how much of a phenotype they have. Aw yeah. Two big money experiments right before my committee meeting. I am a rock star.

But oh no. The FSM does not want me to be data gathering when I should be writing this buggering progress report. Yesterday I was looking at my cells under the 20x in the tissue culture room. As one does. Although I am fairly sure I look at my cells about seventy-nine times more often than any of my lab mates. I have probably logged ten thousand hours of looking at cells under 20x. I am like a cell anthropologist. I look at them. I learn their ways. I know what is a happy, healthy cell and what is an unhappy, unhealthy cell. Seriously.

So there I am, looking at my cells prior to doing the experiments; this is several days after the weird controls problem. And then ... uh-oh. Cells are doing things that strike the chord of fear in my soul. They're not popping off the plate or even dying in the normal way. I know what a cell looks like when it's just undergoing random apoptosis. It happens, especially when you start messing about with serum-free medium and different matrix proteins. This is not that. I have seen this before.

Flashback, about two years ago. My cells are looking ... just not quite right. I thaw them out, they do okay for a couple passages, and then I see the Bleb Monsters. The Bleb Monsters are cells who look all spindly, with long retraction fibers that are dotted with phase bright blebs. These are presaged by cells who leave extremely long, thin tails behind them as they migrate around the plate. Eventually, these cells round up and come off the plate, often remaining attached to the substrate by one or more of the thin, blebby fibers for a while before completely detaching .The debris accumulates in the media, which becomes full of phase dark blobs.

A hefty double whammy of fungicide and antibiotics slows the process but doesn't stop it completely. I chuck all my media and make new. Still happening. Can I trust my cell morphology experiments when there are Bleb Monsters in the culture?

I show my lab mates. "What's wrong?" they ask. "Cells look okay to me." My brow furrows - I am seeing a problem no one else seems to see. Hm, I think. Maybe it's nothing. I do my experiments, with a growing sense of unease. How can I confidently study migration and membrane extension if my cells are leaving huge chunks of themselves behind when they migrate and their membranes are weirdly blebby? I start surreptitiously looking at other people's cell cultures. Now I am seeing Bleb Monsters every where I look. Still, no one else seems concerned about this.

Holy shit, I think. I have a tendency toward hypochondria - am I displacing my stress-induced hypochondria onto my cell cultures now??? That takes the psychosomatic cake.

Six months of this. By now I have cleaned and autoclaved the incubators, which really ought to be done every few months but which in my lab only gets done when I insist on it. The problem is less severe but still after a few passages, I see Bleb Monsters accumulate. (In retrospect, I froze down many vials of cells before I identified the problem, which may account for the slow contamination.) No one notices anything funny in their cultures. Is it that they don't look carefully enough at their cells, or am I looking too closely at mine?

Finally, I show my Bleb Monsters to Wacky Microscope guy, who says, "Looks like endotoxin. Might be that the media bottles aren't thoroughly sterilized." Endotoxin! At this point I'm just glad someone else sees that my cells aren't normal! Again, I throw everything away and start fresh, with the oldest cells in the liquid nitrogen and all new media. Summer turns into winter. Finally, the Bleb Monsters disappear. (In retrospect, this also coincides with the time that I stopped working with a certain cell line I got from a collaborator - not coincidentally, in retrospect, this was the first culture in which I saw Bleb Monsters.) But, I have spent six months feeling like I am taking crazy pills, possibly winding myself up over an imaginary problem, and never really identifying the problem, which has now, as mysteriously as it arrived, gone away. And no one but me and the Wacky Microscope Guy has even noticed anything amiss, which makes me think I was just seeing Bleb Monsters everywhere I looked because I was so obsessed with them.

Fast forward again to this past week. Weird controls in morphology experiment. Fuck. Then, the Return of the Bleb Monsters. Double triple fuck with whipped cream and a cherry on top. But now I am not an insecure, mentally distressed G3 (third year PhD student) anymore. I am a scientist, dammit! I am, furthermore, the person in the lab who most often takes long, hard looks at her cell cultures - both as part of my research project and during routine cell culture maintenance. (The reason for this is largely that I enjoy looking at my cells, but the focus of my project is identifying subtle differences in cell morphology and behavior so it is actually very important that my cells are happy and healthy looking, or else my experiments will be inconclusive.) If there's anything amiss in cell culture, I am going to be the person who spots it because I am the one who is looking. I didn't trust my own instincts back then. Now I do. If I say a cell is fucked up, it's fucked up. Period.

"Lab mate," I say. "I think there's a problem in this incubator. My cells are looking fucked up." We investigate the incubator.

"Lab mate," I say. "What are these whitish crystaline entities on the ceiling and back wall of the incubator?"

"Oh, we think it's just salts," says my lab mate, who is in most ways one of my favorite people but who right now I want to bludgeon.

"Salt crystals?" I query. "Right. Why would there be salt crystals forming in the incubator? That shit is alive."

To test my hypothesis, I scrape some of the mystery "crystals" into a petri dish with some water and head to the microscope.

"You probably shouldn't scrape them like that," says my otherwise cool lab mate.

"Why?" I ask. "If it's salt, it's harmless, and if it's alive then we have to throw all these plates away anyway."

I image the scrapings at 10x, 20x, 60x, 100x, 150x. Long branched filaments, some with visible internal partitions, surrounded by grape-like clusters of small ovoids, some with visible nuclei. I don't have to be a fucking mycologist to know a filamentous fungus when I see it. Closer inspection of the incubator reveals not just a few but hundreds of enormous white colonies of the stuff all over the ceiling, on the undersides of the shelves, in the corners, on the walls - it's polluted. If this incubator was the Jersey shore there would be big Mister Yuck warning signs all over the beach telling you not to go near the water, and dead three-eyed fish littering the sand.

Jesus H. Christ. This is why I don't have to wait a few passages to see Bleb Monsters. The whole incubator is one big fucking spore spewing mysterious mycotoxins all over the place. I stick my head in and take a sniff. Smells like apple cider vinegar. Later, I regret doing that as the hypochondria asserts itself and I imagine filamentous fungi filling my nasal passages and lungs ...

Then I put the pieces together. About a week ago I plated some of the Cell Line of Death, but then the cells were ugly and I threw them away. Shit shit shit shit shit! Okay, so at least I know what it is, and I am finally vindicated re the six months of being Cassandra at the Incubator Gates two years ago.

Here, too, is where the story gets interesting. The control cells that have been exposed to our mystery mycotoxin are behaving in my morphology assay in a manner similar to the genetic mutant cells I have been studying (which have various cytoskeletal and migration defects).

Whoah, man. Whoah.

So I put it to you, loyal readers. Help a girl out if you can 1) identify fungi based on some photos and written descriptions or 2) advise someone on how to extract potential mycotoxins from media. If you help me, I'll put your name on the paper. No joke.

Haven't told any of this to my advisor, but I will - after my committee meeting. Which reminds me, there's something I really ought to be doing just now ...