Today, Birmingham was definitely a bight zone. Tremendously sunny, not even a passing shower. I seized the day-bright and took my bike north along the canal towpath. I was not dissuaded even when the towpath became a narrow muddy rut. The sun kept shining. I passed under the mighty Galton bridge (Figure 1; hopefully no connection to the eugenicist).
Figure 1. The Galton Bridge from the Birmingham Canal towpath.
But there is a dark zone at work. As I explained in the last post, I have been trying to get good staining of roots with fast scarlet. By good, I had been focusing on ‘bright’. The simplest method, namely dissolving the dye in water, gave weak staining, more or less everywhere in the root. Last week (and the week before) I have been trying protocol permutations (!) to brighten things up.
The brightening has been working. This past week, I compared pH 6 and pH 7 in the same experiment and found much improved brightness compared to water. And although the difference was not great, roots stained at pH 7 were perceptibly brighter than pH 6. For grins, at both pee-haiches, I compared the standard in-the-literature concentration of 0.01% dye to 0.03%. I could see no difference, suggesting that the dye amount is not limiting for staining. And actually that is consistent with an earlier trial where I got similar staining from soaking the roots for 45 min vs 5 hours.
But the brightening is uneven. Roots are bright in the meristem and the adjacent part of the elongation zone. They are also bright in the mature zone. But the region of the elongation zone next to the mature zone, what one might call the shootward portion, was not bright. The dimness extended into the mature zone too. These new staining permutations, despite brightening things up in general, still have a zone of darkness. Unfortunately, this zone is crucial for my twisting project; I need to stain the cellulose there too.
The zone in question should contain cellulose at more or less the same concentration as the adjacent zones; therefore, I believe the zone stains weakly because the dye’s access to cellulose is blocked by something in the cell wall. I tried spiking the pH 7 dye solution with 25 mM KCl or with 0.01% triton X-100 (detergent) but the dead zone was brightened up by neither.
I also examined the plants that had grown on the dye since gemination. At the ‘standard’ concentration (0.01%), root elongation was slammed; elongation was likewise affected at 0.003%, albeit less severely. However, at 0.001%, roots grew the same as controls, at least on day 7 when I measured them (11.9 ± 0.2 mm vs 12.3 ± 0.4 mm; these are means ± SEM). Through the fluorescence microscope, these roots looked a lot like roots stained at pH 7. Generally nice and bright but—low and behold—the dead zone was sucking up light like a black hole.
As an aside, the roots grown on 0.001% fast scarlet had root hairs every one of which had stopped after forming a bulge. Evidently, the dye inhibits a specific step in root hair emergence. Someone should study this! And dang it, I am chagrined by having no photo to share. I need to work on solving the no-camera-on-the-microscope; for you and me both!