Look at this glop!

This image comes from material that had been neither fixed nor incubated in cryoprotectant for more than a few minutes (for background to this story, please see previous post). It shows the cell wall in a fiber cell. In the wild type, the cell wall would look something like a textile, filled with fibrils running straight from one side to the other. But the cell walls of the fiber cells in the sections I looked at this week, whether fixed or not, there was little but amorphous material, aka glop. Thus, we can exclude the possibility that the glop was some kind of artifact caused by the fixative or by the cryoprotectant. The latter is itself a kind of glop, as viscous as honey (which btw would probably serve as an adequate cryoprotectant), and there could have been some kind of strange interaction between it and the cell wall. It is true that to cut the sections, Joseph needed to incubate the segments in that stuff, but only for a few moments before freezing. In those few moments, little if any of the viscous cryoprotectant would have been able to penetrate through the cuticle and reach the interior of the section. Happily, the material handled like this was nevertheless able to be sectioned. Probably, the cryoprotectant simply needed to surround a segment to be able to provide a strong enough matrix for successful sectioning when frozen.

It remains a formal possibility that the glop in the images (as in Fig. 1) represents cytoplasmic debris that sticks like bubble gum to the cell walls. This is what I thought when I first saw it. But now I don’t think so. For one thing, it doesn’t look like the cytoplasmic debris that I have seen previously. For another, there are no gaps, and one would expect even the most tenacious glop once in a while to lose its grip. Finally, Joseph treated half the sections with bleach, the other half he left alone. In both, the glop looked more or less the same. To be sure, in this round of material, even the non-bleached samples were pretty clean, as judged for example by parenchyma cells, which sometimes are rather clotted with leftover cytoplasm. That means I don’t have verification that the bleach was good and bleachy. Maybe it had gone off? Maybe, but bleach doesn’t usually go off. It is a simple chemical and there is not much that can happen to it while it sits in the bottle.

However, I plan to look at more bleached versus un-bleached sections to see whether better evidence of bleaching can be found. I have heaps of unexamined sections at my disposal, just need to mount and coat. That might happen this week. Among other things, we need to decide whether to use bleach for the final protocol. If the samples are really clean enough without it, then best to skip because, along with removing cytoplasm, bleach can eat away at the structures we want to examine. In some protocols in the literature, the cell walls are covered by cytoplasm and bleach has proven essential to reveal cell walls. I need to find out to what extent that is true with this material. Next week…