Thursday, July 12, 2007

The problem of biology: You have to keep your cells alive

When I was in the eighth grade, ambitious little budding scientist that I was, I decided that I wanted to show that cells evolved in response to environmental stimuli (ala natural selection) for my science fair project. That's right, no baking-soda and vinegar volcano for me, I was going to single-handedly prove Darwin's theory of Evolution, and set aside all that pesky debate.

Of course, I didn't know that researchers had been growing e. coli in a variety of different conditions and doing the same exact thing since the time of my birth -- those are some damned cool strains of e. coli now. BUT that's beside the point.

I ordered algae (a strain of cyanobacteria called anabaena that is primarily single-cellular in unit) from Carolina Biological, along with the media to grow it in, and I set up cultures in my bathroom (after scrubbing it down with isopropyl rubbing alcohol). My mother taught me sterile technique and I carefully tended to the cultures. I grew them under green light, red light, white light, and no light, and at the end of about two months of growth I used flow cytometry to measure their color -- and get an approximate measure of the chlorophyll/carotene ratio. Idea being that cells growing in green light would have fewer green-reflecting chlorophylls and more green-absorbing carotenes as compared to cells growing in red light or white light.

I got my data and was happy, in that naive eighth-grade sort of way, and the mentor I had goen to for advice on methods and background and so forth suggested a stunning alternative hypothesis that explained my data as well: I had, simply put, killed the cells through overcrowding.

I was shocked, and decided to leave that part out of my science fair backboard. It was understandable; I was, after all, in the eighth grade.

I bring this up because today, in my PCBio meeting, one of the presenters showed a fairly interesting piece of data. She was using a rather strange system to try and determine melanin's photoprotective effects. She created and measured the presence of harmful oxygen radicals the following way: Compound S (rose bengal, I believe) was photoexcited (had light shone on it) in the presence of bubbled oxygen. Since rose bengal is a dye, it does a lot of funny chemistry that I don't really understand, but the end result is the creation of oxygen radicals. These oxygen radicals were then detected in a two step process: first they bind to histidine with some other funny chemistry that I only sort of understand, and then that complex binds to a reporter complex, changing its emission spectrum from 440 nm to 360 nm. Without any melanin in the system, she saw exactly what she expected: Over time, the peak at 440 nm disappeared and was replaced by a peak at 360 nm. Experiments with synthetic melanin showed only a "filtering effect" namely, the melanin was absorbing the light before it could hit the rose bengal, so before the chain reaction could take place. Hence, no oxygen radicals and no shift.

Next she used eumelanin (real melanin) in the form of cells from the Retinal Pigmented Epithelium (a layer of skin just behind your retina, which... er... has melanin). What she saw happening was that the cells would clump up together and fall out of solution, along with much of the rose bengal. Since RPE cells phagocytose dead or dying cones and rods in your eye, she (and her faculty mentor) guessed that the cells were phagocytosing rose bengal, and that for whatever reason this was causing them to clump together and fall out of solution.

What struck me most of all was that she was letting cells sit at room temperature in PBS for 20 minutes. To me, that seems like a very foolish thing to do if you don't want to kill your cells. Mammalian cells like physiological tempetatures, about 12 degrees celsius higher than room temperature, and PBS contains no nutrients or proteins to buffer them and feed them. However, I guessed that no faculty mentor would overlook something as obvious as "maybe your cells are dying."

I walked up to her after her talk and suggested it. She was just as shocked as my eighth-grade self had been when my mentor suggested it. One of the program leaders, Michelle, backed me up and said that it made sense. She said she would look into it. If experiments on cell lysates and purified components don't resolve that issue, well, then I'll be damned.

Which all goes to show that a little bit of common sense goes a long way. And that chemists (apparently) don't really think about things like cells dying. Or, perhaps more accurately, some chemists don't really think about things like cells dying.


Abby said...

i was sort of hoping the cells would be zombified. sigh.

Duff said...

At least some biologists are studying living things instead of dead things!

You should have exposed some of the E.Coli to country music versus ROCK MUSIC to test the neo-Darwinian theory of SURVIVAL OF THE AWESOMEST.

Elizabeth said...

Hehe. If you exposed e. coli to emo and goth music, would they turn black?

Abby said...

yes; black like the endless darkness of their souls. also the medium they were in would turn blackish from the running mascara.