Part 2. An Update to "Biological Oxygen" and Shake Flasks January 2006

I've written way too much already about shake flasks; and I've put some of that on the web, like this essay you're reading right now, http://www.frogojt.com/biologicaloxygen.html .*

http://www.frogojt.com/FallingOffALog+NADH2.html is good; so is

http://www.frogojt.com/Richard1969.html

But just how convoluted the situation is with these shake flasks is still unearthing itself to me, even now in 2006.

"Convoluted", by the way, is not a good thing.

I resubmitted my play Modern Biology www.frogojt.com/mdrnbiol.html to the Magic Theater in San Francisco, in December of 2005, for the Magic's Understanding of Science Program, funded by the Sloan Foundation. Prof Clair Brown, my wife, has been a Principal Investigator for the Sloan Foundation for a long time [in her field, Economics.] Clair heard about the Understanding Science theater program that Sloan has with the Ensemble Studio Theater in NYC. She mentioned my biology play to a higher up she works with at Sloan, who told her about the homologous Sloan program at the Magic Theater in SF, to whom I submitted the play. I involuntarily commenced to rewriting the manuscript here and there, there and then.

One thing that got sprinkled into the rewrite were a few comments about shake flasks, like calling a guy "Ö another moron with a shake flask".

It occurred to me that I might go out and actually get a few shake flasks; maybe they'll use them as props in the play, or maybe I'd send one over to the Magic's Public Understanding of Science Program Director, thus reverse-engineering Konstantin Stanislavski's physical theater theory to drum up interest in my manuscript**. I'd get a case of twelve, say, couldn't set me back more'n a few bucks. So I called up the local scientific supply house here in Richmond, American Laboratory Supply Company, on Ohio near Harbour Way, and guess what? Never heard of shake flasks. And the boss, he's been there twenty years, he's never shipped one nor sold one, nor seen one. We're not talking about just some Erlenmayer flask, even if it's put on a shaker and shaken, as gets done on a regular basis in bio labs all over the world. We're talking here about the genuine item, a "Culture Flask", in fact a "Culture Flask with Sidearm", aka shake flask. Here's a picture of a genuine shake flask, the real McCoy. This one is from Wheaton Scientific, and it's properly called a Nephelo Culture Flask with Sidearm.

(The three outfits that make scientific glass like these shake flasks with sidearms are all located within a few miles of each other, in the neck of the woods in South Jersey where I grew up! No doubt they're there because of Port Norris sand, not far from Millville.)

To use one of these culture flasks, you tip the culture medium, including the microorganisms growing in the culture medium, into the sidearm; then you insert the sidearm into a turbidity measurement machine (a "colorimeter", or a spectrophotometer), and you measure the turbidity, every half hour or hour or so, and chart the growth curve.

Real pain in the ass. You know it takes hours, sometimes days, to grow a culture of cells.

Turns out that NOBODY carries these flasks in stock.

Must be that NOBODY uses these flasks in the lab.

EVERYBODY uses ordinary Erlenmayer flasks.

No sidearm.

Hence no measurement of turbidity; hence, no measurement of growth.

That's like cooking meat without sticking a thermometer in it and reading it now and then, to see if the meat's done or how it's coming along.

But the interesting part is that nobody even knows the phrase "culture flask".

"What's a culture flask?" they ask, when you call up a scientific supply house.

They have heard of a sidearm, because the sidearm feature is used in ordinary chemistry. A filtration flask, for example, is a heavy-walled Erlenmayer flask with an inch and a half or so sidearm that has a hole in the end, so you can pull a vacuum in there. There's even a flask called a trypsinizing flask with a sidearm that's almost a dead ringer for a culture flask with a sidearm, but that's not what we're looking for here either.

This lack of measurement: Did it transpire in an Orwellian fashion after the words disappeared? Did "culture flask" perish from the culture of the lab?

Nowadays, people use flasks minus the sidearms. They couldn't measure the turbidity even if they suspect they should be doing so. The flasks without a sidearm -- those ordinary Erlenmayer flasks, shaking on a shaker, the common ordinary shake flask being shaken in the lab -- is a dickless culture flask, having suffered, apparently, a peiotomy

They kept the concept --- they lost the FUNCTION. Maybe they kept the concept after they lost the function precisely because the concept incorporated the measuring rod, and the measuring rod was dicklike, and the scientists are all guys, so they felt protective. (This isn't a dumb sexist joke; this is a dead serious inquiry into the culture of -- gendered science -- which is briefly touched on in Modern Biology. One of the characters spits out the phrase "Feminist biology!", as an epithet.) Maybe they called it a sidearm because sidearms are sooooooooo masculine. Think about it; when was the last time you ever saw a woman with a sidearm, except for a policewoman?

It's an eight hundred pound gorilla in the room, this lack of measurement, this failure to be scientific, with all those hundreds and thousands of dickless shake flasks in all the labs all around the world, and all those thousand and thousands of journal articles about experiments performed on the cells grown in those dickless flasks. All of those trillions and trillions of cells are in a state of oxygenation, and hence in a state of oxidation-reduction -- the Redox State -- , that can only be described as unknown and unmeasured, and perhaps unhouseled.***

Now here's the science part, the real no-bullshit understanding of science part: I can't tell you -- it's not my job -- how important it is, in general, not to use shake flasks; or how many papers have been published that are going to have to be shitcanned or redone because they used shake flasks. All I can tell you is that in MY system, it mattered a LOT; it was the sine qua non. Listen up: Everybody else's system ain't that different from my system.

Those shake flasks are the quintessential stage prop; and they are the crux of the understanding of science. Modern Theater is physical; Modern Biology is, or should be, skeptical and quantitative.

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*Upon rereading the Biological Oxygen essay, Part 1, it turns out I never actually mentioned shake flasks at all. Like I'm about to say, infra, "the situation is still unearthing itself." The nonmention is notable.

**Stanislavski wrote that at the second rehearsal, the Property Master shall have, on the stage, the actual physical props that will actually be used on opening night; and that the Actors shall never say their speeches without handling these very physical props; and thus will one get a truthful performance.

***I even called the California State Public Health Laboratory, the new one in Richmond, CA, and talked to the Microbial Diseases branch. (I'm still waiting to hear back from the Enteric Diseases sub-branch.) GET THIS: To monitor the growth of a culture of microorganisms, they put in some fluorescent organisms, shine a light on them, and monitor the growth of the culture by the DECLINE in fluorescence as the culture grows (the State employee patiently explained to me that the fluorescence reaction requires -- duh -- oxygen.) See what I mean? They're monitoring the growth of the culture by measuring its asphyxiation. Huh? This is getting to remind me more and more of the Thai Scrabble players at the Scrabble tournaments, the ones who can't speak English. (The World Champ and the US Champ have at times been such men.)

Biological Oxygen (c) 2005 Richard Katz

Introduction

Not too long after I started working in biology laboratories, I got a job growing yeast. It turned out, a year or so later, that the answer to the main research question depended on how you grew the yeast, in particular, the air supply. In looking back on the yeast studies, it would appear that the air supply -- and the yeast oxygen demand -- was the most interesting part of the whole thing.

That was thirty-five years ago. In looking ahead, it appears to me that oxygen demand is the most interesting part of brain function.*

I. The Yeast

The lady scientist growing those yeast, back in 1969 or so, was as much of a scientist as anybody else. She had her degrees, her publications, her assistant professorship at Penn, her career path; and I never would have hooked up with a job as one of her technicians, not in a million years, if God hadn't invented nepotism. My girlfriend's mother was a full Professor who ran a big lab at the same biophysics Institute as the yeast lady. I had had a menial technician's job at Cornell, the summer before I met my girlfriend, Laura, the daughter of the Professor back at Penn. Not long after I had been to Laura's house to Meet the Parents (both of whom were full Professors, at Penn) her mom asked me if I wanted another job, like I had at Cornell.

The job turned out to be the worst job I ever had. The lab she fixed me up with worked with beef heart mitochondria; the biggest part of the job was butchering up beef hearts. Disgusting. I had had lots of experience with disgusting jobs, because I had grown up on a farm. Mud, pesticides, machinery -- didn't matter, you worked sunup to sundown and they paid you in the dark, on the farm, and you didn't complain because it was your own family farm.

After a few months, the beef heart mitochondria lady let it be known that I would learn more if I switched jobs and worked for her friend the yeast lady down the hall. I think the idea here is that technicians at an Institute are chattels, and you deploy them where they'll do the most good, but with a definite eye toward the technician's development. Not a bad system; a bit feudal and patriarchal, perhaps, but not a bad system at all.

I started work in the yeast lady's laboratory thinking everything was just fine, totally under control, nothing to worry about, certainly not the bubbles in the carboy growing the yeast. Everything was according to the way it's supposed to be in The Literature. But imagine that you're staring right at the problem, and you (and your superior) have absolutely no idea that there's a problem. The yeast lady was doing it all just right, but her yeast (and everybody else's yeast) have a much higher demand for oxygen than her equipment could supply.

Equipment? You mean that one cheesey little sparger? Hah!! You could have all the spargers in the world bubbling away, it turned out, and the oxygen needle wouldn't budge off zero. A culture of fast growing yeast demands oxygen at such a clip that you have to dose them with oxygen from a tank of compressed gas. Gases don't diffuse into or dissolve in liquids worth a damn, kinetically speaking, including yeast culture medium. Ask the fish in Lake Erie.

It had never occurred to the lady scientist growing her yeast that the yeast needed -- were demanding -- more oxygen. In fact, when you stop to think about it, it's a problem that compounds itself: The more the yeast demand and get, the more yeast you have, and the more oxygen they'll demand! They don't get better at it, there just get to be more of them. But you'd never know that, until you supply more oxygen; and all of that probably only occurred to me because I grew up trying to get a good yield growing foodstuffs on a farm. To all the city slickers, it was just a matter of if it ain't broke, don't fix it.

Now what's that got to do with thinking, or thinking hard? With the brain, you'll never know you could consume more oxygen, until you find someone somewhere who demands more oxygen. And we did.

II. The Brain

I always maintained a connection to that lab, right up to the present. Not to the particular laboratory run by the yeast lady; after she fired me, for being uppity and wanting to do my own experiments, her boss -- the Boss -- the Director of the Institute -- picked up my meager salary, and gave me a desk out in the hall. I improvised a lab bench, on a table I moved into the hallway. I measured the oxygen in a yeast culture, the same kind of yeast culture that the yeast lady and microbiologists all over the world use for growing experimental batches of yeast and bacteria, and got a reading of a solid zero. NO oxygen in the culture medium. That's like measuring the hemoglobin in your blood and finding that it's zero, that there's zero oxygen being carried from your lungs to the rest of your body. You're dead. The yeast weren't dead, though; as it was said on Monty Python's Flying Circus, they're only resting. **

One night I got the idea to construct an iron lung for a yeast culture. A friend of mine, Jim Geogalas, and I went into the lab thinking we would customize a pHStat so it would regulate oxygen instead of pH. The Boss came by on his way back to his lab down the hall. My buddy and I probably looked like we were two guys destroying an expensive pHStat, when in fact we were only, well, cannibalizing it. The Boss grimaced, the sort of expression that shows slight annoyance, mostly pity, and not a trace of what I would call criticism or reproof. ***

The Boss really swung into action. He is one of the pioneers in biophysics, so equipment modification was his long suit. Modification, hell, the Boss designed and built equipment just to be able to do this or that experiment, over the years. He'd been doing that since before I was born; he'd been one of the scientists who helped win the War. So he didn't bat an eyelash at what we were doing. He just told us to stop destroying the perfectly good pHStat, and come see him in the morning.

Next day he told me what it was I had been trying to do, and how one would do that electronically (they had just invented integrated circuits a few years before.) He sketched a schematic of how to do it. He told me to take that rough sketch over to the electronics shop, down the hall, and the boys in the shop would know what to do with it. A week or so later I got back a shiny little aluminum box. Armed with that box, I could dip an oxygen electrode in my yeast culture and the dissolved oxygen reading would be somewhere safely between 5% and 20%, because the shiny little aluminum box would give it a shot of oxygen now and then to keep it that way. If you kept a wide range like that, you could even keep track of the rate the yeast dragged the oxygen concentration back down -- the rate of respiration. Measuring that rate of respiration -- the rate of oxygen consumption -- led to the realization that, "My God, these poor things were gasping for breath, all these years!"

Now skip ahead thirty years.

The Boss is measuring oxygen consumption in brain, in situ. Here's a picture of Britton Chance and his Cogniscope. Notice the headset in Figure 0.

Now look at Figure 1, which shows the oxygen consumption at the prefrontal cortex of an ordinary brain (mine, in fact) answering ordinary social and moral questions. Note the excursions of the pen in the 0.5 to 1.0 micromolar range.

Now look at Figure 2, which shows the oxygen consumption at the prefrontal cortex of a trained brain -- a Division One Scrabble Player solving eight letter anagrams. Note the excursions of the pen into the five micromolar range.

What we want to do now, in 2005, is CREATE oxygen demand in the brain! If yeast can suck oxygen out of the water like a freight train climbing a hill -- power under the hood -- why not go looking for a way to get trainees' and students' brains to get up to that level of oxygen demand? Power under the hat. Brainpower!****

Richard Katz October 2005

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*Perhaps my published scientific findings from thirty-five years ago, about acquisition and loss of rotenone sensitivity in Candida yeast mitochondria (the main research question) will help cure Parkinson's disease in humans; perhaps not. "The Literature" doesn't really reach back that far (1971), and so it is very unlikely that any current day researcher's literature search is going to unearth the fact that complex I of the mitochondrial electron transport chain is poisoned by rotenone when the yeast cells are growing, and is insensitive to rotenone if they aren't. Rotenone can cause Parkinson's.

**To this day I don't know of a word to use to describe their state of oxygenation. The Literature usually just says the culture was "sparged" or "aerated" and gives the rate of airflow in liters of air per minute. Any auto mechanic would immediately recognize this setup as being totally halfassed. Modern fuel systems are sophisticated, and the modern mechanic measure oxygen with a sensor that's built in to the car. The Boss would probably use the word "hypoxic"; never asked him.

***I got the idea from then on that, at least with guys like him, really good leaders, it's okay to fuck things up as long as you're experimenting, or as he always put it, you're working. He'd ask you, "What are you working on?" and it meant what experiments are you doing.

****We can approach these higher levels of cognition during the process of imparting expertise via short unadorned digital video sequences accompanied by Quicktime text tracks. This is spelled out at http://www.frogojt.com/frogojt2004.html .