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Monday, 21 November 2011

Thin sections and a microtome

One way to beat the depth-of-field problem is to cut thin sections of things so all of the parts of your specimen are at the same level, just one cell thick. You can do this using the sort of razor-blade that has a strong backing edge. These are very sharp and dangerous, so get adult advice before you start. Thin sections can be mounted in water under a cover slip, and they will let you see cells, though without stains (we'll come to those later) you won't see much internal detail.

The basic microtome. I inserted a plug of carrot into the wing
nut, which is fitted back to front onto the bolt. You need a
dish of water to put the sections in, and something to pick them up.
Before long, you will realise that these 'thin sections' are usually wedge-shaped, and you can see better detail at the thin side of the wedge. If you want a thin section that is even all over, you need a microtome. Microtomes are expensive, but there is a way to make one almost for zilch—well, mine cost just $2! All I needed was a razor blade and a matching half-inch Whitworth wing nut and bolt (some hardware is still sold in "old" units).

ADULT SUPERVISION is essential for this one—risks include cuts and possibly broken blades hitting the eye. You will need a bolt with a wing nut to match, safety goggles, a safe very sharp blade and an old cutting board.

The equipment you will need. Note the paintbrush. the dish, the
chopping board. the $2 microtome and the backed razor blade in
particular. You will need the needle later, to mount a section.
For your first attempt, cram a short piece of carrot (or celery) into the wing nut's threaded hole, then slip the bolt into the wing nut back-to-front, with the 'wings' at the end nearest the bolt.

When you go to slice a section of carrot, you will see why the nut has to be this way around. Once the bolt has a grip on the nut, put on the safety goggles, get the cutting board and use the blade to trim off all the carrot that is sticking out of the nut.

As the bolt slowly moves into the nut, the carrot in the threaded hole is slowly pushed out on the other side, and if you slide a sharp blade across the flat surface of the wing nut, you will cut that tiny bit off, producing a thin section that can be mounted on a slide. Trim off any bits that are sticking out of the nut.

Now you can start. Turn the nut slightly, so a tiny amount is pushed out of the threaded hole in the nut, and slice downwards.

Hold the apparatus as shown on the left: this shot was posed to let you see a section coming off. Don't use it as a guide!

This is the way to hold the equipment.

Notice in two of these shots the Petri dish with water in it, that you drop the sections into as you cut them. A saucer or even a jar lid will do just as well.

 You lift the section with the brush . . .
 Carry it across to the dish . . .

And put it in the water. While you are learning, put the slices in a dish of water repeat the operation until you master the method and throw away your first attempts. Examine the sections in a wet mount if you wish, but now you are ready to section difficult stuff like leaves.

For small items like leaves or stems, you will need some scrap polystyrene foam to wrap around what you are sectioning. You can also use cork or a piece of carrot or potato for this, anything that grips tightly on the leaf or stem.

If you read up on professional microtomes, you may see references to using wax instead of foam, but I recommend against this. It is hard to make a water mount of a waxy section, because wax and water don't mix. Most professional reference books recommend using very toxic chemicals to dissolve the wax, so polystyrene foam is safer than wax. Do some experimenting first: and remember that a piece of foam larger than the hole can always be squeezed and 'screwed' into the nut, once it is wrapped around the leaf or other object.

Put the wing nut on the bolt again, with about one full turn of the nut on the bolt, then fill the empty portion of the nut with whatever you want to section. If you really want to section a leaf in wax, prop the bolt upright in an old jar or can, poke the leaf in, and then drip candle wax in, until the leaf is surrounded with wax and leave it to set.

The sections will usually be wedge-shaped, but if you choose to look at the thinnest part, you will be able to see something like this shot, which is one of the sections you see being cut above. It isn't perfect, but it's a good start.

Of course, I have left out a big step: actually getting the section on the slide and putting a cover slip on top without any bubbles. That is called Making a wet mount, and I will deal with that next.

For comparison, here is a small portion of the first-ever drawing of cells, done from a section of cork, cut by Robert Hooke with a pen-knife in 1665 (this link gives you the whole thing in Wikipedia).

1 comment:

  1. I read Your post which was really Good waiting for next post