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Introduction to Hand-sectioning & Wax-embedding
Preamble
I've had a few complaints about me (whoops... sorry - 'my') style of writing. It
seems that my use of slang and colloquialisms (unrefined speech in local
dialect) has not been to everybody's liking. Well, if those who complained
would like to step up and take the time to share their knowledge here with me, I could offer - not only a valuable
course on microscopy... but an English lesson as well.
I never said I woz (sorry, 'was') an English teacher. What I am is an ordinary bloke who knows his way 'round amateur
microscopy - better than a lot of folks. However, as I really want to try and get folk up and able to do microscopy,
and I'm not easily hurt by remarks about my standard of English, I've asked my mate Mol of Mic-UK to translate
and modify my pages here so they are easier to understand in terms of dialect and grammar.
At the end of the day - even criticism is important: there's no point in me writing all this stuff if no-one understands
what I'm saying, now is there?
Well, I've had my moan, so lets get down to the project.
Hand-Sectioning Wax-embedded Sections - what is it?
To study microscopic processes in both plant and animal tissue, it is necessary to have very thin (transparent)
slices of the material mounted on a glass slide. The problem is that it is quite difficult to just simply slice
off a piece of anything in such a way that it is thin, of uniform thickness, and remains structurally intact during
the process of cutting it through to mounting it onto a glass slide.
Preserving the structure
By far the main problem is 'preserving the structure'. Imagine for a moment - using your mind as a microscope -
a simple plant stem... still living! Here is a structure predominantly made up of a set of vertical tubes, with
even smaller tubes joining them together horizontally, periodically along the plant stem. Inside each tube, liquids
flow carrying nutrients and gasses up from the roots to be dispersed throughout the plant.
If by a stroke of magic, we could suddenly slice away a thin section of the stem and look at it under a microscope
in a .0001th of a second, we would be able to see - momentarily - the precise structure of the material and the
living cells of the stem and nutrients. I say 'momentarily' because as soon as we start to look, the stem itself
would start to change shape and distort as water within the structure dries out, and the cells making up the structure
collapse and die. The structure and nature of living things are only truly preserved by their action and state
of 'being alive'!
Fixing
We don't have 'magic' but we do have science. It is possible to take a sample of a living thing and 'fix' it using
chemicals to halt the living processes and hold them in as close a state as possible to when they were living and
active. We can also retain most of the original structure by ensuring our chemicals replace the liquid (mostly
water) that was in the sample when it was alive.
The problems come when we remove the sample from our 'preservative' and expose it to the air ( thus inadvertently
initiating a 'drying out' process) in order to cut sections from it and mount them on glass slides.
What do we need to do to preserve the structure of a sample, from the moment it is removed from a preservative
solution, to the final moment it is mounted in a suitable resin or compound on a glass slide?
We need to ensure the structure is 'supported' !
A clumsy analogy... but it is a bit like using wooden 'shuttering' on wet concrete - where the wooden planks define
and retain a desired structure until the concrete dries.
In microscopy, we can use molten wax to permeate and infiltrate the sample - replacing any of the liquids present
so their removal does not cause the structure to collapse. We can then allow the wax to go hard - possibly flexing
the structure slightly - but then enabling us to handle the sample for sectioning (cutting into slices) and for
sticking the sections onto glass slides before removing the supportive wax.
This process is called wax-embedding!
This Project
The process of creating a finished slide, complete with a stained specimen section, is a complex one. It involves
the application of refined knowledge gathered over many years not only by myself, but from many enthusiast microscopists
- some now sadly no-longer with us. The process involves preparing a specimen embedded in a supportive wax block;
preparing a cutting edge so sharp - and yet still so strong - that it will slice through hard microscopic structures
without tearing them; taking a thin slice of wax carrying the specimen and fixing it onto a glass slide so that
it lays flat and adheres to the glass; and finally clearing the wax from the slide and the specimen so that the
final preparation can receive a mountant material and cover slip.
It is the most daunting process an enthusiast can ever undertake.
Most enthusiasts, even with the seemingly simple process of cutting a section from a wax block, will find their
endeavors frustrated and will believe it impossible ever to cut a neat clean section thin enough to mount. So let
me just say that the only reason they will have any problems is not because it is a difficult process: it is because they do not know the few basic secrets
that will ensure success. I do... and if you stay with me on this project - you will know these things to.
To ensure you don't have to take in a lot of information all in one hit, I have divided our project up into parts.
Their order may not follow the logical sequence of the entire process from start to finish but there is a high
degree of logic involved with how I am going to present this.
In practice, the process order of preparing a wax-embedded specimen and then deriving a set of finished mounted
slides from it would go something like this:-
- Prepare Specimen (clean and preserve it)
- Prepare wax solution
- Embed specimen in solution (can take several days for wax to
infiltrate)
- Prepare and hone a cutting edge
- Mount the wax block in a microtome and cut sections
- Fix sections onto glass slides
- Bake in oven (first clearance of wax)
- Solvent-clean the wax from the slides
- Stain the fixed specimens on the slides
- Apply mountant and cover slips.
This list alone is enough to put you off. Don't let it! This project is one of the most important ones you are
ever likely to encounter on practical microscopy. It offers a unique reward. If you follow through and become able
to create mounted slides from your own wax-embedded specimens, you will have achieved a special status. Very few
enthusiasts have this knowledge and capability... yet it is one of the most important skills to possess. When you
create your first finished slide, you will have achieved something quite incredible - because from that moment
on you will be keeping the knowledge and capability of microscopists before you alive and you will have become
a critical asset towards keeping enthusiast microscopy alive this century. I kid you not!
What I propose to do is work through these stages in a slightly different way - starting with the following two
:-
- Prepare and hone a cutting edge
- Mount the wax block in a microtome and cut sections
It is important to note that a microtome (a tool for holding a sample of material
in such a way that it can be offered to a cutting edge in a precise manner) can be a bench microtome or a rocking
microtome.
The former is a microtome which can be clamped to a bench. It is used to hold samples that are going to be cut
by hand. The latter is a more expensive device which not only holds the sample - but will perform the cutting of
sections as well. It is a more precise tool - yet still requires a lot of skill in its set-up and operation.
We will be using the bench microtome. In fact - in this project, I will be using the Brunel Bench Microtome - obtainable
from the Onview On-line shop!
(continue...)
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