The problem of the meniscus

Where firefly flashes and the growth of stems are introduced and problems with apparatus.

The main event this week was getting to work on building a system to measure stem growth. Yes, stems. I know, I wrote about roots before, but I have two projects, one below, the other above. Get over it!

For months, I had been aware of, and thinking hard about, some of the problems involved with the stem growth project. For one thing, stems grow best in the dark. When a seed germinates deep in the soil, there is no light for photosynthesis, and the seedling stem’s job is to elongate without delay or demur to bring those incipient leaves above the surface where they can start capturing light and fixing carbon (i.e., feeding the plant). There is only so much carbon food stored in the seed and the seedling will die if this growth trajectory falls short, which is, as they say, “strong selection” to make this growth process effective.

What happens next is different – with need-to-find-light panic over, the stem slows down and starts doing things that also involve strength. All those leaves need to be held up against wind and gravity. And while the end of the growing season will arrive too soon, there is still a lot more time than that held in the reserves of one seed. How much light is enough to turn the do-or-die growth of the dark seedling into something more sedate? It turns out to be a graded response, with a little light causing a small change and a little more causing a bit stronger change, and so on. And what is “a little”? When I was doing my Ph.D. some of the folks in a neighboring lab were doing experiments to answer this question and they would joke that the sensitivity went as low as a tenth of a firefly flash. But that was actually about right in terms of the light quantities involved.

Now, for my experiments here, I want the best growth I can get, not even one firefly flash. In other words, I need total darkness. I don’t mean the odd dim bulb in the corner, but total darkness. Not only will this give me the fastest growth, it is the most reproducible. If I allowed a little bit of dim light here and there, how many firefly flashes is it? To make my experiments reproducible, that is to report them to a would-be reproducer, I would have to measure how much light got in when I opened and shut a door, or whatever. This is even more of a pain than keeping things strictly and totally dark.

But working in the dark is a pain. Fortunately, the labs here have a small dark room, one with two doors so that the inner, experimental room stays dark while the outer door is opened. The first thing that I ordered, I think even during my first week, was an IR night vision monocular. What this does is to convert infrared light into visible light. This lets you see in the dark and it was only a little embarrassing ordering a product that is clearly designed for war games and other nocturnal nefarious purposes. Although the people in that neighboring lab I mentioned would work in the total darkness unaided, I am unhappy about the idea of fumbling about in the dark, with solutions in small dishes and razor blades. Even better, studies (yay studies!) have shown that pretty much like the rods and cones of human vision, plant photo-receptors also pay no attention to infrared. Safelight I can rely on.

The crux of the project is to measure the growth of small pieces of stem, say 5 mm long. The stems need to be cut into pieces and floated in a solution because the experiments will involve investigating what happens to their growth when this or that compound is added into the mix. Difficult to do that to intact stems. Also the sides and cut edges of the stem make ideal landmarks for measuring its growth. The measuring will be done by taking pictures. For simple experiments, I can take one picture when the experiment starts and a second one when it ends, say 2 h later, and find out how much growth happened in between; for more complex experiments, I can take pictures every 10 min, or every 10 sec, whatever. The pictures can be taken under infrared light, by an appropriately sensitive camera. Happily, the native state of nearly all digital cameras includes sensitivity to infrared, only this is customarily blocked by manufacturers to remove interference with the visible signal. Along with a suitable room, CPIB also have a suitable camera, at least in principle.

These were the kinds of things that I had been thinking about and talking over with Darren to plot out a work space and key gear for this project. And this week, we decided to have a play. Talking can get you only so far. Certain things needed to be tried. Could the camera give the right magnification range? Could we get a good image through the dish? And so on. Because we can easily mask the visible light reaching the camera, we could work in regular room light but the images recorded were as if we were in infrared only.

The stems we were going to play with had failed to grow, I hope because they were too young. But the roots had grown so we used those, since we just needed thin, cylindrical planty bits. I like the idea of using a six-well multi well plate (Figure) because they are a convenient unit and would let me do six treatments at once, probably enough. Darren and I experimented with attaching the camera to a macro lens, to a compound microscope, or to a dissecting scope. The latter two gave far too much magnification, giant megaliths! But the macro lens seemed about right. No problem about light sensitivity, there was a huge signal, even from the rather small group of light emitting diodes we were using, and even when we put some foam over them to diffuse the hotspots.

Common or garden six-well multiwell plate

Common or garden six-well multiwell plate

But. Ahhh there is always a but. One thing emerged that I had utterly failed to anticipate. The meniscus. Unless you took Greek and learned that ‘meniscus’ comes from the word for crescent moon, the first ‘meniscus’ you probably encountered was in chemistry lab, when you were told to measure the volume of liquid in the graduated cylinder from its bottom (figure). The bottom is the well behaved flat bit, hence useful for measuring. When I have water in the six-well plate, or in any small dish, there is a meniscus, and the problem is that the stem pieces are, as romance novelist might put it, inexorably drawn. When stem pieces go there, as go there they will, they are no longer flat but at some odd angle and the water curving into the meniscus forms its own lens, adding further distortion. Even this simple playing around was clear—for accurate measuring of stems—the meniscus must go.

meniscus-2

Stay tuned for next week’s installment: To kill a meniscus. And in the meantime, you are all welcome to send me suggestions.

2 thoughts on “The problem of the meniscus”

  1. Homo Neanderthal says:

    To remove the meniscus, you can either use a surfactant (often a detergent but YMMV) – presumably not wanted here; or you can use the standard microscopists technique – a cover slip. While that might sound silly for a plant and a coverslip sounds too small, this is the solution to the meniscus problem for all sorts of live samples from tissue culture upwards.

    If you are worried about gas availability/exchange then there are other solutions. For example, the hanging drop (for floating things) or hanging column (for anything) – where you put your sample in a small (or big) drop of liquid and put a coverslip (or slide, or dissecting dish or bucket depending on the size required) on top. If the sample floats or sinks, you can see it without distortion (and can fill up the cover slip with liquid to your desire).

    “Ahhh..” you say, “but the sample will be crushed – mais non?” – here we can use the leetle grey cells and (if necessary) put a pair of columns (glass from broken slides works well – Plain, Doric or Ionic – your choice) on either side of the slip. If you need to anchor these in place, or sterilise the culture, I recommend a drop of molten vaseline (nicely hydrophobic).

    The meniscus is not evil, it is just drawn that way..

  2. Tobias says:

    Thanks Homo! Those are all reasonable suggestions. But added to the fun is that I am working in total darkness, with a night vision goggle hanging on my head, offering me sight through one eye, at one plane of focus. This makes me need to avoid tricky manipulations. I found that by using a teflon washer as my dish, because the water does not wet it so much, the problem was solved. Smile.

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