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GROWTH PHASE AND ROTENONE SENSITIVITY IN CANDIDA (formerly TORULOPSIS) UTILIS:
DIFFERENCE BETWEEN EXPONENTIAL AND STATIONARY PHASE by Richard KATZ
C. utilis is insensitive to rotenone during exponential growth in a batch culture. When growth stops and the cells enter the stationary phase (in this case due to carbon limitation), rotenone sensitivity appears after a short lag. [note: Torulopisis utilis was renamed Candida utilis, about the same time this research was published.]
This is a picture of the rig we used to do the experiments on yeast and rotenone. I'm looking into the refrigerator where a fraction collector is patiently collecting samples of the culture. The graphics guy at the Johnson Foundation took the picture and gave it to me, gratis. The materials and methods section of the research paper (FEBS Letters, Vol 12, No. 3, Page 153 January 1971) says, in relevant part,
The culture vessel used was a 1.0 liter cylinder, approximately 5 X 50 cm, equipped with a sparger, stirrer, and Teflon-covered oxygen electrode (Yellow Springs lnstr. Co.) immersed in the culture medium. The electrode output activated a solenoid vaIve when the culture oxygen concentration fell to approximately 15% saturation, giving a pulse of oxygen sufficient to raise the 02 concentration to approximately 25% saturation. With this oxystat (designed by Dr. Dieter Mayer) the rate of oxygen consumption by the culture was monitored, by recording the rate at which the oxygen concentration fell from 25 to I5%. Cells were siphoned continuously from the culture throughout the experiment and collected in a fraction collector, maintained at 3.0 deg C, at a rate of 5 ml of cells in a 12 min interval.
Dieter's oxystat is the custom little aluminum box pretty high up and on the left side of the "bench". That wasn't really a lab bench; it was a table parked at the end of the hallway, just before the elevators and opposite the stairs. It's surprising that you would walk into a world famous biophysics research institute and see a scientist [an undergraduate, in fact] working in the hallway. But this was the Johnson Foundation. And this was Lou Kahn's Richards Building at Penn. Everybody worked in the hallways. The hallways were made to work in; you got to know everybody that way, by design; you got exchange of information and ideas that way. Even the library was a hallway. You can't see the glass culture cylinder, because in this particular experiment (which was never actually published), in addition to measuring the culture's respiration, rotenone sensitivity, pH, and turbidity, I was going to measure the entire culture's pyridine nucleotide fluorescence (in response to excitation by UV light of 240 or so nanometers. In the foreground are two gas cylinders, one of oxygen and one of nitrogen. I would gas the culture with nitrogen and measure the fluorescence, and then with oxygen, to calibrate the fluorimeter. Then I would measure and record continuously the fluorescence of the entire cylinderful of growing yeast cells, at (as I recall) a wavelength of 340 nanometers.
Now don't get the impression that I knew what I was doing. In a sense, I knew what I was doing better than anybody else on earth; it was my series of experiments. I was the world expert on what I had discovered, about the relationship of rotenone sensitivity and the actively-dividing-ness of yeast cells. But when I said to Dr Chance (Britton Chance, the Director of the Johnson Foundation for Medical Physics and Chairman of the Dept of Biophysics and Physical Biochemistry and Benjamin Franklin Professor of the University of Pennsylvania, now Emeritus) that I wanted to measure total pyridine nucleotide fluorescence, in vivo, in a soupy yeast culture, Chance didn't bat an eyelash. He told me what pieces of equipment I would need (all of them sophisticated pieces of electronics and optics, most of it custom stuff that that had made there at the JF shops). I scrounged all the stuff from different labs, and from his lab, and hauled it down to my table at the end of the hallway. I made a stab at setting it up to measure what I thought I wanted to measure. Dr Chance came down the hall about 11 PM, going home after a hard day, and in about ten minutes he had all that stuff hooked up.
Now I'm going to get to the point here: Nobody had ever done this before. But I Didn't Know That. I had no idea. I said I wanted to make that physical measurement; Dr Chance said, Go get one of these, two of those, and a brace of those, and then one of this kind of cable and two of some other kind of cable (I don't remember the details now); and that's the kind of place the Johnson Foundation was. You made measurements. And they were sophisticated measurements, where you were measuring just a few photons, really, and getting the difference between two tiny measurements and getting a result. And I knew so little about it, to this day, really, that I have no idea if there were other places that operated like that. Naaaah, couldn't be. That would mean there would be a bunch of guys like Chance. And I've never (in the intervening 27 years) met anybody like him. Quick as a flash, he hooked all that stuff up. He said, "So here's how you calibrate it, you just ..." and he did this and he did that, and then he left. On his bicycle (he lived just a few blocks from the lab, in West Philly, near 41st and Pine. He also had a place out in the country, and probably a few other places too; he had some old money, and an Olympic Gold Medal in yachting, and at the time I thought he was a Nobel prize winner who just hadn't been awarded his prize yet. I hope somebody writes Chance's biography some day, like a series of long New Yorker magazine articles, based on interviews with him and on his notebooks. He remembers all the details, and what he doesn't remember is in those notebooks [which he always had Bound, in leather. Class.] And then get the back story from wherever a good writer would wring it out of: You'll know the writer got it right, if it's got a deep and penetrating tragic essay on John C. Lilly. The Dolphin Dude who believed in [of all things] ketamine. )
So the total pyridine nucleotide fluorescence increased more or less exponentially with the increase in the number of cells. Frankly, I can't remember what relationship there was between the onset of rotenone sensitivity and pyridine nucleotide fluorescence. You see, it's not like the yeast run out of NADH, or run out of fluorescence. What I think we were measuring was How reduced are the Cells? When they would run out of ethanol (their only carbon and energy source) they (all of them, en masse) would go from being pretty much totally reduced to pretty much totally oxidized. In fact, they would become pretty much totally oxidized when they ran out of oxygen!! That just blew me away. That was like finding out what Monsieur Jourdain found out from the Philosophy Master in Moliere's le Bourgeois Gentilhomme: That he'd been speaking in Prose, all his life! Electrons are flowing ; we are all of us, mostly, reduced.
I though I had found out something pretty basic about Life doing these yeast experiments, like that it mattered whether the organisms were growing or not. Most of the labs I went to and checked out didn't actually know if the bugs they were working with were growing or not. They certainly didn't know if they were "aerobic"; most people just shook them in an Erlenmayer flask, or sparged them with air. If these scientists had ever measured an exponentially growing "log" phase culture with an oxygen electrode, they would find that the oxygen concentration was zero. The bugs were asphyxiating, the microbiological equivalent of ischemia. That's why I sparged with pure oxygen; that's why Dieter designed the oxystat, and that's why Dr Chance molded the Johnson Foundation to make that sort of thing happen. If you measure stuff precisely, and you pick the right horse, you might find something out.
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July 1999 - Update - Dr Chance is now assaying cognition in humans, using a phased array detector for NIRS (= near infrared spectroscopy) for non-invasive detection of hemoglobin concentration/volume in the brain cortex. This technique could be used to determine whether the brain is adequately mobilizing its resources for cognition to be taking place during FrogOJT Rapid Skill Acquisition employing digitized video.
back to FrogOJT Systems page
March 2007 Update
It occurred to me that I never actually checked an actual shakeflask on an actual shaker to see if a moderately dense culture of microorganisms, Candida yeast for example, exhausts the available dissolved oxygen and keeps it exhausted. To make a long story short, I checked it out, here in The Garage; and indeed, the oxygen is exhausted at a Klett "OD" of about 275 with Candida guilliermondii. I fully intend to publish that result in Nature, with Jasper Rine and Betty Ishida.
Doing these "aeration", or shaker, experiments made me think of something else:
My brother, Gary Katz, invented a 3D version of the Periodic Table of the Elements, called the Periodic Roundtable. You can read all about it at www.periodicroundtable.com . This was back in the 1970's, like the Candida yeast experiments supra. I never grokked what was so special about Gary's version of the Periodic Chart, except that it wasn't a chart, it was a round wooden wedding cake, and it was symmetrical as hell. A while back, I even made a Quicktime movie for it of the GetSkilz/FrogOJT genre www.frogojt.com/PDRM0072disassbackwards.mov (requires Quicktime).
Not that long ago, maybe five or six years ago, Gary wrote an article for The Chemical Educator (Chem. Educator 2001, 6, pages 324 to 332) about his 3D Periodic Roundtable, and about other versions of the Periodic Table of the Elements that have appeared over the years. By the Doctrine of Fair Use, let me quote just a snippet of my brother's scholarly work concerning the Hydrogen of the Periodic Table:
We now know that period one elements,
hydrogen and helium, are fundamental to the chemical
evolution of the universe. The present day (detectable) mass of
the universe consists of about 98% period-one elements, but
only 2% heavier elements .
The big-bang cosmology implies that all higher matter
descends from period-one elements. The observed cosmic
abundances of hydrogen and helium are real facts, regardless
of the validity of the big-bang theory. In the last thirty years,
the case for this theory has matured and is now included in the
curriculum of first-year courses. We therefore cite the cosmic-
abundance results as evidence that the first two elements
constitute a uniquely autonomous period, disjointed from the
grouped elements below them. This might alter the conception
chemists have of the first two elements, focusing attention not
just on terrestrial chemistry but also on the roles helium and
hydrogen play as the progenitors of all the higher elements.
Thus, the first two elements in the table are above all the first
elements in the cosmic synthesis of elements. It would be
proper to display this information as part of the periodic table,
and it would also resolve the placement of the first-period
elements at the head of groups one and two.
The first time I read this Hydrogen part, I missed it. It went over my head, or it went in one ear and out the other. The whole Hydrogen bit, I think my brother has been preaching it for a while now, that hydrogen deserves a very special place on The Chart (perhaps at the top right corner), and atop The Periodic Roundtable wedding cake. It's easy to miss, because it's mixed in with a lot of physical chemistry.
Hydrogen is ... get this ... the majority of the atoms in the Universe, by a lot. That's pretty special. Largely unappreciated. Not taught in school.
Hydrogen is also what makes a molecule reduced, as in "we are all of us, mostly, reduced."
The vast amount of hydrogen on the sun throws off a lot of energy; a small part of that energy becomes the energy in the hydrogen of the reduced molecules on earth. That's how we get by.
The fact that this is going to sound like incomprehensible gobbledegook to most people with a high school diploma and higher, testifies to the need for a Periodic Roundtable in every science classroom.
NB My brother's website is, as of this writing (March 2007) fossilized; frozen in time; one of those bits of cyberspace that was created by someone other than the owner, usually for hire, sometimes gratis, back in the day. It's going to be updated soon; who knows, it might even have my movie put on a page. That would be cool, because the movie makes the point about hydrogen real well (you'll have to download and watch it to see what I mean; the actor hesitates a bit before placing the Hydrogen/Helium layer on the wedding cake. It doesn't belong, does it? Yes and no.) But that's not the point. The point is that when it was created, my brother didn't himself think the cosmic significance of hydrogen was worth mentioning. Check it out. It's cosmic.
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