Teaching a sprinter to run marathons (Anatomy of lab work)

I've always been an endurance athlete.  Probably because I'm just plain slow, and you can hide that a lot better in a 5K than in a 100-meter dash.

This Douglas-fir seedling has lost its chance to take over the world.
Or at least, the surrounding native forests. 

For the most part, after more than a decade of semi-competitive running (though over the last few years it's been more about the pleasure of stretching my legs over a few miles than about lowering personal records), my brain has been molded into that of a distance runner.  I like to stick things out, I like a certain amount of pain (I can picture Mom and Dad making their 'self-flagellation' gestures... thanks for the emotional scarring, guys! :), and -- according to Dad, anyway -- I have a reasonable degree of mental toughness.

But in some things, I will always be a sprinter.

To some extent, I espouse Hemingway's "Live hard, play hard" dogma: I have a tendency to throw myself into science and work feverishly for a few weeks or months, then wear myself out and collapse in a fit of TV-watching, elaborate-meal-cooking relaxation.  (Err, I guess that's not really relaxation... more like throwing myself into something else, equally intensely.)

In addition to seedlings, we also collect any sporocarps (mushrooms,
the fruiting bodies -- a.k.a. the physical structures that release spores,

the reproductive part of a fungus) that we find in the field.  These are

brought back to the lab for DNA sampling, photographed, and dried.

I've never been very good at pacing myself when it comes to work.  So it's no surprise that, where we said we'd collect 15 seedlings per canopy type, we came home with closer to 30.  And now, with bags and bags of samples piled up in the freezer, I'm feeling some perturbation, which is currently manifesting itself in nightmares in which I open the door to the cold room and am instantly toppled by a tidal wave of Douglas-fir seedlings that come pouring out.  (Silver lining: This is a much better dream than last week's, which featured an unfortunate encounter with a band saw while cutting sample tubes for the second half of my field work.)

As I know from experience, there's only one cure for this dream-inducing stress: Getting to work! With one sprint into the field over, it's time for the sprint to finish up lab work before the next phase of my project.  Here's how it's done.

Basically, we excavate all our seedlings in the field, digging deep around them to try to scoop out their entire root system.  Depending on the type of soil they're growing in, we're often able to shake off a lot of the dirt in the field.  (K. is much better at this than me!)  We're conservative, though, because fungi are found colonizing the very tips of roots, and we don't want to break any of these off by accident.

We bag up the seedlings, haul them back to the lab, and then soak the root systems in water to loosen any remaining dirt.

Step 1: Soaking the seedling in some tap water for about an hour.

Step 2: Gently washing the roots with more water.
Note: The seedling changes with every picture. :)

A prepared seedling (this one about one year old) waits in a
water dish for further examination.  You can see some light-
colored knobby looking things on its roots, right? 

After the seedlings have been cleaned, they're ready for some close examination.  First, I record the coloration of the seedling -- that is, how dark green it is -- which indicates its health.  As any gardener knows, a yellowing plant isn't doing so well.  Here, a yellowing Douglas-fir seedling is probably lacking some fungal partner that helps it gather a critical nutrient.  I also estimate the seedling's age by carefully counting scars on its stem -- an imperfect method, but speedier than trying to slice and stain sections of the tree's tiny trunk.

Since you can see the roots with the naked eye, it doesn't take too much power to get a close look at any fungi that might be colonizing the roots.  So I use a low-powered microscope called a dissecting microscope to examine the root system.

Examining a seedling under a dissecting microscope.

I scan through the root system tip by tip, using a counter (the thing with the colorful tabs on the right-hand side of the scope picture) to record the number of tips of each morphotype.  A "morphotype" is a category of appearance, an approximate way of grouping tips by species.  See below for a couple of examples.

A "clumper".  You can see the branching structure of the root
system here in brown.  The white is a coating of fungal hyphae --
hyphae are the fine hair-like structures that fungi produce.  The
yellow bits are pieces of still-attached dirt.  Some fungi are sticky!

Another "clumper".  Some species of fungus induce lots of
branch-formation when they infect tree root tips, producing
clusters like this one.  You can see a lot more attached dirt
here.  Also note the air bubble: This particular fungus is hydro-
phobic: That is, its surface repels water.  So although the roots
are submerged in water here, you can see clinging air bubbles.

One of the prettiest morphotypes.  This fungus produces a
really smooth coat, giving the tip a nice polished feel.

Not all species of fungi look different, though: "Cryptic" species are those whose appearance is very similar to another species.  And some species produce multiple morphotypes.  That's why we take samples of different morphotypes.  We'll extract their DNA and sequence a short region of it, which allows us to identify the species of fungus colonizing each root tip.

Plates of root tip samples.  At the bottom of each tube is a bit
of tree root with a fungal infection that looks like the above
examples.  It's floating in an extraction solution, that breaks up
the tissue and lets the DNA out of the cells.

Here, I'm processing a plate of samples by pipetting
different buffers into each well.  The goal is to process all
the seedlings and perform all the DNA extractions before
heading back to the States.  DNA is very stable in freezers,
so once it's been extracted, I can store it for future work.

After the seedling's root system has been examined, I separate out the roots from the shoot (that is: I cut the seedling in half), and save each portion to be dried and weighed.  Later, I'll be able to use these data to do things like estimate growth rate (by measuring the distance between trunk scars) and calculate fungal infection per unit tree biomass.

So far, I've accumulated 17 such plates of DNA samples in the freezer, and I've still got two-and-a-half coolers-full of seedlings to process.  On with the race!