Effing up

I knew that in the course of attempting to do some actual science I would fuck something up. Once again, ladies and gentlemen, Kristie does not disappoint. First mistake I made was to forget just how weird cholera bacteria are. They live in the environment, which is normally quite a bit cooler than body temperature (except for the last week or two), so you'd think that you could toss a Vibrio culture in the fridge and everything should be just fine. Not so. Vibrio cholerae is not only a genomic hoarder, with two chromosomes, but it's more thermopersnickety than a woman with a malfunctioning thyroid gland- it really doesn't like to be cold. So, I grew overnight Vibrio cultures for my fish infections last week and, not thinking, took them out of the incubator and popped them in the fridge while I finished up some other stuff and went to lunch. They were in the fridge for two hours at the most. When I got back, I took them out of the fridge and was spotted doing so by two of my fellow cholera cult (lab) mates:

"That wasn't Vibrio you just pulled out of the fridge, was it?"

"Uh...yeah." Uh oh...

"You know you might have just killed your cultures, right? Vibrio dies if you put it in the fridge."

Shit! "Oh, right. I totally forgot. Well, I guess we'll see if two hours is enough to kill them!"

Even though I  felt like a complete moron, I did still have the presence of mind to plate a couple of dilutions of each of my cultures, just to make sure I really didn't kill them. When I infected my fish later that day, I had no idea if I was adding live cholera or just polluting their water with dead useless cells. Dumb, dumb, dumb. At the very least, I figured out how to set up the fish infections on my own - which tank to take fish from, to make sure that they hadn't been fed first, which water/beakers to put them in, how to mix up the tricaine solution to euthanize the fish, etc. Plus all the other stupid little details that get forgotten by people who've been doing this stuff for a while - like where they keep the ethanol, how do I clean up the fish shit when I'm done, where the dissecting kits are...it's getting a little bit easier every day. Oh, and it turns out that I hadn't killed my cholera after all! Sometimes I think a lot of research is figuring out just how much you can fuck things up without ruining the entire experiment. That sounds smart-ass-ish, but I think it's absolutely true. You need to know how much wiggle room you have in your protocols - then you try to figure out why, which sometimes leads you to new side projects. It has been strongly and repeatedly suggested to me (by a favorite cantankerous former professor of mine) that a good student should be working on at least three projects simultaneously (at the time of my scolding, I think I was only juggling two things. And rather lamely, at that).

Tomorrow, I'm going to attempt my first ELISA assay (ever!), looking for cholera toxin in last week's fish water. If it doesn't work, then there's a good possibility I fucked up precipitating protein from the fish water. I really have no idea what the hell I'm doing! I'm also still stuck in cloning hell, but so is everyone else in the lab. We think that the UV transilluminator is frying all of the DNA we try to gel purify - even though I limit the gel exposure to 15 seconds or less, plus we add guanosine to the gel to act as "sunscreen". We're supposed to be getting a blue LED transilluminator soon, I think. Maybe that will help.

I still need to get some work done tonight, but to hold you over until my next post, here are some crappy iPad pictures of the lab.

The fabulous view from my desk.

My bench, replete with impressive-looking bottles of stuff I made, new pipettors, a brand new ear-piercingly loud timer, and ice! Woot!

How many new posts constitutes a habit?

I'm trying to get back in the blogging saddle and write more frequently, since my hellish first year is all but over. I won't promise to write every single day - a minimum of twice a week sounds more reasonable. Writing is still cathartic for me, even though I am finally getting a healthy daily dose of social interaction with the other grad students. I'm starting to feel less awkward and clueless all the time, but I still think that I cling to my seat a little too much because I'm afraid of my lab mates being aware of my presence. That thought/sentence hardly makes any sense, but I can't really clarify it. I guess the social phobia is still hanging on for dear life, despite my shrink throwing some Lexapro at me two months before turning me over to yet another new resident. That reminds me - I need to set up a fucking appointment with the newbie shrink before my Adderall runs out. I don't like having to be seen at all, but I have to play by health insurance rules if I want to remain somewhat functional. The initial appointment is the worst. All I want to have to say is, "I'm depressed, afraid of people, and I can't fucking concentrate. Give me some good drugs and I'll get out of your hair". Instead, they ask me to rehash my entire psychiatric history, even though I can see that they've already printed out the relevant information from my online chart. Blah.

I was gonna write about what I did in lab today, but now I'm too tired and cranky. I guess I lied when I said this was still cathartic. Better luck tomorrow, maybe. On Friday the 13th, no less.

And we're off to the races

I'm firmly establishing my own well-worn ass-groove in the leather chair that goes with my desk in the cholera lab. One thing that annoys me about my desk set-up is that the chair is fairly wide and it has armrests. The armrests are so wide that every time I scoot in toward my desk, one armrest or the other hits the side of the desk. Every. Single. Time. I've got half a mind to take a damn screwdriver to school and remove the fucking armrests. If it's annoying me now, imagine how I'll feel after five years of it. Maybe I'll wait until the PI has agreed to accept me as a full-time lab member and gives me a key to the lab before I start disassembling his furniture. Since I didn't put in as many hours during my rotation as I should have (to avoid failing my classes, not making it to prelims, and subsequently being kicked out of the program), the PI is letting me work in the lab over the summer on a "trial basis" to make sure that I will actually show up to work and generate some decent data. So I'm technically not a full member of the lab. However, I have no intention of being kicked out, asked to leave, or whatever. In my mind, I'm a permanent lab member - it's just not official yet. Unfortunately, I'm reminded of my provisional status whenever the PI sends an email to the lab, but only carbon copies me on it. I don't really understand why that arbitrary distinction has to be made in stupid two-sentence emails stating that we're having lab meeting this week at 2:00 pm - like we do just about every week. That bothers me. Alright, that's enough of the complaint department - I'm starting to feel really pissed off about my stupid provisional status, which will get me nowhere fast.

I had a good productive day in the lab today. It almost felt like it did when I worked in Jim's lab at Eastern, when the science determined what time I went home for the day- not sheer boredom. My PI is off this week, so I don't have to worry about him keeping a surreptitious eye on me. Yay! I feel like I made a butt load of media, but I don't think I actually did. Let's see, I made a liter of 5X M9 salts, 100 ml of 10X PBS, a couple of 90 ml dilution blanks, 100 ml of 20% (w/v) glucose, 100 ml of 1M MgSO4, and finally a liter of complete M9 minimal medium. The shelves atop my bench are becoming beautifully lined with bottles of stuff. Most of the bottles are identical, each containing exactly 100 ml of whatever potion I whipped up - just like everyone else's benchtop shelves. I'm telling you - I really feel like I've joined a cult. Thankfully, no one is sporting any forehead swastika tattoos and wild eyes or wearing all black outfits with gleaming new white Nike shoes.  All the perfectly filled and arranged bottles make me feel like I'm doing real science. M9 is a royal pain in the ass. I think that alone took up half my day - just autoclaving and cooling to room temperature. Fucking ridiculous. But, I had to make it because that's what I'm growing my WT cholera and ToxT deletion mutant cholera in overnight for tomorrow's zebrafish infections. I'm just trying out a very basic experiment to see if it works and to see how long the whole ordeal takes. I'll have two groups of four fish each in separate beakers of 200 ml sterile saltwater. I'm going to add my overnight cultures: WT (cholera toxin-producing) to group 1 and ToxT mutant (no cholera toxin production) to group 2 and incubate the fish at 28 degrees for 24 hours. Then, I'll euthanize the fish and collect the beaker water. The plan is to concentrate whatever protein is in the 200 ml of fish water by trichloroacetic acid precipitation - which I've never done before, so will probably screw it up. The multitude of proteins precipitated from the fish water will be used in a cholera toxin ELISA, which I will also likely screw up in some way. The idea is to see if cholera toxin is present in the fish water. In previous experiments, when naive fish were placed into water conditioned by other cholera-infected fish, the naive fish died. It was assumed that the naive fish were killed by cholera toxin that had been secreted into the water, but no assays were done to confirm this.

Oh, shit. It's almost 12:30 and I haven't had my nightly Ben and Jerry's Coffee Toffee Heath Bar Crunch ice cream gorge session yet. And I haven't finished the paper I'm supposed to read for my virology discussion tomorrow during lunch. And I'm still Jonesing for more of my second read of "Fifty Shades Darker" before I fall asleep. I just love trashy sex and romance novels (with a smattering of BDSM thrown in, as the case may be) - I am certainly NOT a fine literature critic, but I like what I like and, embarrassing as it may be, that's good enough for me. And Chris :)

I'm supposed to do what now?

I've been working in the cholera lab since the end of June now. When the PI said that he expects me to be independent, he wasn't fucking around! My research project is centered around developing a zebrafish model of cholera colonization and infection, so I'm continuing the failed cloning that I feebly worked on during my rotation. Also, now that I've passed prelims and thus should know what I'm doing (ha!), I've been charged with developing a protocol to test for the presence of cholera toxin in water that held cholera-infected zebrafish. The PI didn't tell me what strains to use (and what medium to grow them in) to infect the fish, if I should use log phase or overnight cultures, if I should do a time course, or anything. It's been surprisingly stressful trying to come up with a reasonable experimental plan. I'm sure the point of the "sink or swim" approach is that you learn better what not to do by falling on your face a few times, but this is pretty brutal for me at the moment. However, I don't have to run any ideas by him before I try something new, unless I need really expensive equipment or reagents (I don't even know what dollar amount he considers "really expensive"), so at least that's kinda nice.

 I have to rely pretty heavily on the other grad students in my lab - as well as the grad student, PI, and tech in the fish lab upstairs - for help. I hate being dependent on other people for anything. I'd rather kill myself trying to do something independently than to make my life easier by asking for help. I'm working on getting over this. Slowly. It's really strange working in a lab with other people. It almost feels like I've joined a cult. Seriously. Everyone shares all the reagents (down to the same tubes of Taq and homemade PCR buffer), and whoever uses the last of something has to make more. Sharing tubes of reagents is a little weird (why not just give everyone their own aliquots?), but replacing what you use up is not weird - I don't want to make it seem like I think it is. That is a very good thing! Everyone is assigned a certain chore like making bottles of autoclaved water, LB, autoclaving tubes, etc. The one group activity is stuffing tip boxes - which really sounded cult-like to me. I just picture us all standing at a bench in a row, stuffing tip boxes like synchronized automatons. Makes me shudder a little. As strange as all of this is to me, everyone has made it clear that I can do things the way I want to - if I want my own aliquots of PCR buffer, I can do that. But, I don't want to break away from the way the rest of the lab does things - at least not this early on. Everyone has been very accommodating and helpful though.

I apologize for such a boring blog post - I'm even boring myself right now. Hopefully I'll be back to my normal smart-mouthed self before long and I promise that I will write accordingly.