My last post was months ago, not because I got tired of writing but because I was out of my lab and stuck in my office. I suppose I could blog about office work, writing and reading, things I am thinking about. But for some reason I want to focus on things that I do. Experiments.
On Monday I started the way experiments always start: making solutions. I discovered that the 10 x PBS was supporting a flourishing crop of mold. I made fresh 10 x PBS and then 1x PBS, PBS supplemented with 0.5 M NaCl, and PBS supplemented with 0.2% Triton X-100. The latter is a detergent much used in biology. And oh yes, by PBS, I mean phosphate-buffered saline, a more or less universal solution for manipulating bits of organisms in the lab, especially when the bits in question are not required to do anything beyond keep their structure. I also needed to remove flotsam had washed up around the Vibratome, the thing not having been used in an age. I made sure that despite its long slumber the Vibratome still vibrated, and I found super glue, applicator sticks, 6-well dishes, and small transfer pipettes. I laid out a red dining hall tray next to the Vibratome and soaked a small pile of paper towels on the tray to serve as a moist cutting surface.
Then I got the seedlings from the dark room. Carefully unrolling the jelly roll far enough to expose a few seedlings, I chose one with a straight root and placed it on the wet towels. I put a thin layer of gel-type superglue on the Vibratome stub, cut the root about 7 mm from the tip, and placed the tip segment on the glue, nudging the segment so it aligned perpendicular to the blade. I waited 20 seconds and then covered the now solidly glued-down root segment with a mL or so of PBS. At that point, I aligned the blade and started slicing. I transferred the slices (which are about 0.1 mm thick) to two wells of PBS. I got about 8 sections per root, so I sliced 3 roots, for 12 sections per well.
I moved sections in one well to 0.5 M NaCl for 1 h and then into 0.2% Triton X-1oo for another hour, followed by three rinses in PBS. The sections in the other well were just moved from PBS to PBS. Over the next few days, I dehydrated the sections, moved them from the 6-well plate to small glass vials, infiltrated them in tertiary butanol, and then freeze dried them. I had been worried about the pump for the freeze drier but this time the vacuum was almost as good as new. The sections are bone dry in the vials, waiting for me to mount them for scanning electron microscopy. Next week.
What I am doing here is seeing whether the salt and detergent soaks will rid the sections of the cytoplasmic debris that likes to obscure the cell walls in maize roots. For the experiment I plan, I need to observe the cell wall. If the debris shrugs at my salt and triton double whammy then I will have to try more intense (and expensive) things, such as protease K. Results to follow next weekend!
Full disclosure: This spurt of labbing follows on from what I was doing, and blogging about, almost two years ago and before. In the fall of 2017 and spring of 2018, I figured out to do an experiment on maize roots. This was a new system for me and required ironing out a kink or ten. I blogged about this in perhaps excessive detail. I got the system sorted out and started to accumulate actual data and I ought to have continued right on through that summer (of 2018). But I didn’t. There was one thing, and then there was another; the time seems to have been consumed like an ice cream cone — gone before it could even melt. I am happy, and a little nervous, to be getting back at last.