This week, among my pursuits, academic and trivial, was just a little bit of lab work. Again, I used a microscope to check out what happens to our BY2 cells when we grind them. The week before last, I looked at cells that had been ground using a ball mill. This machine is like a paint mixer but on a smaller scale. It has two arms onto which you clamp a tube with your sample (in my case, BY2 cells; but it also works on yeast cells or even hunks of stems or leaves). The arms vibrate, in the neighborhood of 10 to 100 times a second. Along with the cells, the tube contains a steel ball-bearing, which bangs back and forth against the sample and the tube wall, breaking the cells apart. To make this operation more successful, tube containing sample and ball bearing is frozen, because in the frozen state, things are ever so much more brittle.
But as we found out week-before-last, the cell walls survive this and generally are not broken open. When I looked at this preparation and saw few if any signs of broken open cells (the tell tale flap of single cell wall), I also saw that each cell retained its nucleus and much cytoplasm. Besides breaking open the cells, I’d like to use this material to study “the cell wall”, I was disappointed to see all that contamination.
For last week’s test, instead of grinding cells with the ball mill, Eri used a good old mortar and pestle. Old is right: according to Wikipedia, humans have been pestling for 35 thousand years. And certainly mortars and pestles have been standard lab equipment for as long as I can remember. The problem with them of course is that, being manual, the conditions are difficult to standardize; also when there are loads of samples to grind, one’s arms hurt. The mortar and pestle has its good points: the action is slow and therefore avoids heating the sample and also the forces brought to bear are large and effective. But in any case, here we just wanted to see what would happen.
When grinding tissue for cell walls, the standard protocol calls for placing the ground up cell power directly in ethanol. But ethanol might cause the cytoplasm to precipitate and essentially become stuck to the cell walls. Eri ground cells and placed them in ethanol in the ordinary way, but for a second sample, after grinding, she put the powder in an aqueous buffer containing a concentration of salt that more or less matches that of the cell. In that condition, the cytoplasm might not precipitate and would instead be free to float away through any holes that were opened up through the cell wall. For this sample, after a time in aqueous buffer, the material was treated with alcohol so that the samples would have undergone similar steps.
The results were promising. The mortar and pestle opened up some cells. I could see single cell wall flaps, at a reasonable frequency. What was remarkable though was the difference between alcohol and aqueous buffer. With the alcohol, there were nuclei in most of the cells and lots of clotted cytoplasm. By contrast, the material washed in buffer was far cleaner. Few nuclei, scant cytoplasm, the cell walls looked pretty clean. Improved purification for sure.
But the ground up cells were mainly stuck together, into large clumps, in both conditions. How to separate them? Separation will help us find the single cell wall flaps. To separate cells, we will try a water bath sonicator. Not as old as mortar and pestle, a sonicator is a venerable method for breaking up clumps. In fact, I found an old sonicator in the lab, made by Koh-i-noor, and designed to unclog their Rapidograph pen tips. I have no idea where I scrounged that from but it runs. Now to see if those sound waves will un-clump the ground cells.