Friends, this past week, I did a fair amount of lab work, but it was all in aid of keeping things going, rather than learning something. This is a common lot of the experimental biologist.

For the cell wall project, described in the last two posts (here  and here), I jousted with Xena, warrior princess. No indeed, she and I did not run at each other bearing lances. This particular warrior is a sputter coated named Xenasput. A sputter coater is a device to lay down a thin coat of metal on samples bound for the scanning electron microscope. The coat is created through a process called sputtering, in which a cloud of metal atoms is created that then lets the atoms fall on the sample like so much snow. Because Xenasput is a feisty thing and often refuses to sputter, I liken these sample-coating sessions to a joust with her namesake. Happily, Xena sputtered to life without coming to blows. I coated two of the stubs I prepared the week before, enough to keep me busy on the ‘scope for hours and I’ll coat up the next batch in due time. Its not that I enjoy jousting with Xena but rather that fresher coats seem to image better.

Anyway, why do my samples need a coat of metal? In scanning electron microscopy, the image arises from electrons scattered by the sample but scattering is incomplete. This is a problem for both left and right hands. On the left hand, there might not be enough scattered electrons to form a decent image; On the right hand, the n0t-scattered electrons hang around on the sample, forming a cloud of negative charge that interferes with the electron beam. A metal coat solves both problems because metal scatters electrons more effectively than do organic compounds and because a metal coat is conductive, allowing the non-scattered electrons to flow off of the sample (through the stub and instrument to ground).

Also for the cell wall project, I dried another set of sections (the third and last set). I truly hope I don’t need them, and I might have left them soaking in the ethanol in which they were mailed. But, allowing for a little bit of superstition to intrude into this otherwise flawlessly rational discourse, by spending the time to dry them, I make it less likely that I will need them. And allowing for intuition to intrude, I think storing the samples dry will be better for them than storing them in ethanol.

Another job needing my attention was passaging tobacco BY-2 cells. I grow these cells because they are cute. Here is a picture:

They grow as elongated filaments, which is not that different from how a cell in a root grows but is certainly different from the isotropic cell clusters formed by almost all other plant tissue culture lines. These cells are advantageous for imaging and I have several projects in mind where the cells would be perfect. The cells are a bit like pets though, they need to be passaged once a week, without fail. This is not difficult – put 0.5 mL of the old culture into 70 mL of fresh medium, although it has to be done using ‘sterile technique’ to prevent anything else from growing in the culture medium. I have three different lines that I am maintaining and of course one is passaged on Tuesday and the other two on Wednesday. At some point, I need to get them all together on to a Tuesday schedule.

There is one more project that kept me busy. This one also has to do with cell walls but not with imaging them, rather with understanding how they are put together. Dexian Luo, who worked as a postdoc in the lab until the end of December, isolated some genes from the grass Brachypodium distachyon and put them into the eudicot, Arabidopsis thaliana. The genes in question are those implicated in synthesizing cellulose (called CESA) and the arabidopsis lines in question harbor mutations in one or another of them. The main idea is to learn to what extent the cognate gene from the grass will fill in for the one in the eudicot.

Dexian obtained some striking results when she analyzed the primary transformants. We are excited about this but to do this job properly we need to assay two generations later. In that way, we have a homozygous line expressing the introduced gene in a definable way. That is perhaps a glib and not terribly informative explanation. But suffice to say, we want those ‘second generation’ youngsters. Based on where they all are now, I estimate we will have these lines in November.

Dexian left a bunch of plants growing and my job is to water them and when they are ready, harvest the seeds. For getting the first and second generations, I harvest seed from each plant individually. The flats of plants are in the greenhouse and I sit out there surrounded by carefully labeled bags, holding a pair of tweezers in one hand and scissors in the other. I grab a dry pod (a silique is the botanical term), snip it off the stem, being careful not to let the pod shatter and release its seed in the process, and place it in the bag. The bags will then sit for a month allowing the seed to dry. Then I’ll have to thrash it out (to remove the pods and other chaff) and plate for another round.

The inflorescence stems of arabidopsis are long and develop from the bottom up, so that pods at the base are dry while pods at the top are green. If I were to wait for them all to be dry, then the ones at the bottom would have shattered and lost their seed. To get as many seeds as possible, I do this sit-and-snip business over a couple of weeks, a few times per flat.

I also have a few flats of newly transformed plants. We haven’t analyzed these at all yet but Dexian set up the transformations because they will serve as important controls for the rest of the material. Harvesting these is faster because seed from all the plants can be pooled. I bring a flat to a bench in my lab where I have a dozen pieces of plain white paper spread in a large array, with the center of the array having an extra sheet, creased sharply down the middle. I pick up a pot containing four or five plants, and holding it sideways over the creased paper, I run the dried shoots and seed pods through my fingers, gently kneading them. This causes the ripe pods to shatter and also to detach. The green ones stay put, for now. After going through the whole flat, I pour the seed and detached pods into a bag.

I did both kinds of harvesting this week. Seeds in the bag, as it were. But plenty of more seed and more bags until all this is done.

So no discoveries or even data this week. But the boulder rolls.