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Trash

From the LabLit short story series

João Ramalho-Santos 23 August 2010

www.lablit.com/article/617

Growing up I wanted to be an environmentalist, join Greenpeace, something like that. Now I study how cells work, creating a minor ecological disaster every single day

The cell culture room is empty. No wonder. I always avoid the middle of the day rush, everyone knocking everyone else to squeeze into one of the flow hoods, get to the centrifuge, microscope, incubators, water baths, supply cabinets. I treasure early mornings, lunch breaks, late nights and weekends, moments of eerie calm unless someone is finishing a project or trying to convince the PI she is working really hard. The PI who has the interesting habit of calling the lab at odd hours, just to check on who picks up the phone. Slim chance of that happening today, some holiday or other. Not that I’d know, being both a post-doc and a legal alien, two degrees of foreigner.

The UV light flickers off, the flow hood hums to life. Stuff warms up or thaws in the water bath while supplies are gathered, tasks prioritized. There has to be a method, a system, lest I be here forever. Or everything contaminates and the experiment starts again from scratch, ensuring even more endless odd hours.

Sit down, exhale. Go.

The filter tower slips out of its plastic condom. Sterility at all costs, even that of redundancy, a lesson learned the hard way. I need 370 ml of basal media. One 50 ml disposable sterile pipette coming up, plastic sleeve removed, pipette fitted into the automatic pipettor, ready to use. It takes a repetitive while, luckily the filter tower has a measuring scale or I’d mess up. Next comes serum, 100 ml of thick brownish goo. I fish for a 25 ml pipette, three 50 ml Falcon tubes of serum. Yes, 50 ml tubes, but I still need three, each has about 45 ml worth, never fill them all the way up if you’re going to freeze; that’s science at work right there. Add 5 ml of glutamine solution. Another 5 of pyruvate solution, 10 of non-essential amino acids, 10 of antibiotics to top it off. The filter tower connects to the vacuum pump, forcing the mix through the filter, into my new complete media bottle. Remove tower, cap bottle, put in water bath to warm up. In the refrigerator goes what’s left of the ingredients, into the trash the filter tower and its wrapper, two Falcon tubes, six pipettes plus their sleeves.

Step one.

Next I collect the five 10 cm plastic Petri dishes left in the incubator three days ago. When last seen the culture media covered a pristine lawn of lozenge-shaped cobblestones; a tight monolayer of cells, specifically fibroblasts. Today the pattern should be ruptured, dead cells floating about, the few live ones looking ill and unhappy. This is good. I added plasmids to the plates, and for three days the cells have been making five different viruses, killing themselves in the process. A small sacrifice for science. I need all five viruses for the experiment, all five plates have to look the same. Sometimes I secretly hope one would look normal, freeing me from this bond. A fleeting moment of cowardice.

The virus particles should now be floating in the culture medium – I just have to remove the debris, the gutted remains of my virus-making helper cells. Five sterile filters fitted to five disposable syringes forcing liquid into five 15 ml Falcon tubes. Five, hopefully functional, virus preps I leave on the tube rack. Syringes and plates and filters and all the wrappers into the trash.

Step two.

Now for the real experiment. There are twenty 12-well plates patiently waiting in the incubator. I prepared them yesterday, just before leaving. The cells in them are to be transformed into something they should never be, a cellular metamorphosis, if you will. How is this miracle to be achieved? Using different combinations of the five viruses, each forcing the cells to express one particular protein. What I want to know is if all five are needed, which combinations work best, if there are certain drugs that will help the process along. I make sure I have the drugs in my handy icebox. Can’t tell you want they are though, top secret. Actually even my PI doesn’t know about some of them; there is no reason for me to look stupid in meetings unless they actually work. Twenty times twelve. Two hundred and forty experiments. Well, not really, just a small exaggeration. I’m running duplicates, so it’s more accurate to say one hundred and twenty times two. Big difference, I still need to add oodles of stuff to a bunch of wells.

The first rule of adding is subtracting. I take the plates out of the incubator and remove the old media using a suction pump. The fluid is sucked up the tube and hisses and moans through the twisted plastic until it collects into a big liquid waste flask on the floor. Collecting the remains of many an experiment, nasty looking stuff. The flask isn’t even half full; I won’t have to empty it, wash it out, add some bleach. Lucky me, ruined many a clothing item doing this over the years. Next is adding a little bit of fresh media to each well – don’t want the cells to dry out. I want to manage it all with just one 10 ml pipette, but end up using three, as the first one clogs and the second touches the hood wall, ever so fleetingly. No matter: redundancy in sterility is the ultimate rule, no exceptions, into the trash it dives.

Now for the hard part. Excuse me, I really need to look up my lab book notes. One hundred and twenty times two 1.5 ml Eppendorf tubes, each pair containing different amounts of different viruses and drugs. I made a handy color-coded table yesterday. It’s a big table. I tack it to the flow hood to check out items as I add them. Yesterday I was a thinking scientist, planning all the experiments that will grant some modicum of immortality. Today I aim to be a mindless technician. Just add the right thing to the right place and move on. No way I can do all two hundred and forty at once though. I’ll run out of both racks and logical ways of numbering tubes. Scientist-me settled for sixty tubes at once, and this is what I do. I need sterile Eppendorf tubes and boxes of adjustable pipette tips, lots of them. The 2µl, 20µl and 200µl Gilson pipettors become my best friends for the next hour or so, their big 1000 µl sister coming in when it’s time to scoop up the content of each Eppendorf and add it to the appropriate well, appropriately labeled (both on the lid and the bottom of the plate itself – it’s surprising how often lids can be exchanged by sleep-deprived individuals). I hope I haven’t screwed any of this up as all the tips and all the tubes are swept into the trash. They don’t take up that much space though, cascading like confetti over the filter tower and Petri dishes and pipettes, already nesting in the trash can.

Step three.

Of course science is a continuum. In the incubator there are five more 10 cm Petri dishes with cells, in the icebox plasmids and transfection reagents, all that is needed to make another batch of virus. A few days from now I’ll be repeating this all over again. It would be called reproducibility if there were guarantees the experiment started today would work, which I’ll only know in a couple of weeks. Let’s just call it wishful thinking. A few dozen pipette tips and more Eppendorf tubes to prepare the appropriate mixtures: incubate. Remove old media from the five new plates. Add plasmid and reagent mix, incubate. Top off with new media, put back in incubator.

Step four.

The easiest I leave for last. Nothing exciting, I just need to passage and plate cells. Two types actually, the ones to make virus and the ones used for the transformation experiment. I could do this in my sleep. There are four T-75 flasks of the virus-making cells. The media comes off, and a 5ml pipette adds trypsin to two of them. The other two go directly into the trash. I know how this sounds, but it’s the logical thing to do, care only for the cells you need. Trypsin detaches the cells from the surface, two other 10 ml pipettes add some media to counteract the trypsin and transfer the cells into two 15 ml tubes, flasks joining their brothers in the bin. Spin down the tubes, ressuspend. The cells from one tube are divided into four new T-75 flasks. Two pipettes are needed, a 25 ml for new media, a 2 ml for the cells. The cells from the other tube are counted, the appropriate amount goes into five new 10 cm Petri dishes with media. A few more pipettes. For the other cells the procedure is the same, except they go into 12 well plates, instead of Petri dishes. The trash is getting crowded with more T-75 flasks, Falcon tubes pipettes, pipette casings. I dutifully press down so it won’t overflow.

Step five.

All that’s left now is to pray, even for us atheists. I’ll be checking out this experiment regularly. Maybe I’ll start two others, with some variations, but not more, I’m not that masochistic. If I get good results, confirmation will be on the way, saving time. If nothing works, oh well. Hopefully all two hundred and forty wells will turn into numbers, useful numbers. That is all I, and they, can hope for. At any rate, everything will end up in the trash, eventually. Trash is the only guaranteed result of any experiment.

**********

Growing up I wanted to be an environmentalist, join Greenpeace, something like that. Now I study how cells work, creating a minor ecological disaster every single day.

Wang will come in soon to clear out the trash can here in San Francisco, replace the plastic bag, haul away my discarded shells of science. As Clarissa used to do in Pittsburgh, and Elói in Portland. And as somebody else will do in Boston, starting next year. Asian, Black, Mexican, in Massachusetts who knows. Does this mean anything? The world as represented by those who remove my trash. And all I care about is that the bin is empty the next time I come in.

There are three groups in my wing, maybe seven people apiece. There are six wings on my floor. There are sixteen floors in my building. My building is one of four on the main campus. There are other campuses, blooming in cheap reclaimed areas around the city, once it became obvious that no more construction could be done on the main site atop the hill, that being one practical problem with the old ivory tower concept. I don’t even know everyone on my floor, I’m pretty sure my PI can’t name all the groups, all the research, all the science. All the trash.

I wipe the workspace clean. Diluted bleach, ethanol. Erasing any evidence that I have been here. The goggles go back on the shelf, the lab coat on its hanger. Shoe covers, mask, latex gloves and my crumpled cheat-sheet table the icing on the trash-can cake. If anyone else drops by today they’ll be unpleasantly surprised.

Wang better hurry.

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© 2010 João Ramalho-Santos

With a respectful nod to Alexandre Gaspar-Maia, Ph.D.

Other articles by João Ramalho-Santos