Hello, I'm Geoff Meyer.
In this lecture, I'm going to briefly describe
how tissue is prepared for microscoping viewing
and secondly, a little bit about histological
These are 2 areas of histology that are highly
They require very highly-trained professional
technologists, so I'm only going to briefly
cover the basics of these 2 processes, enough
for you to understand to enable you to then,
when you study histology in more detail and
all the tissues in the practical classes and
in these lectures, you'll have an understanding
of what stains really show you and you'll
have an understanding of the technology behind
looking at slides and even some of the artifacts
that occur during preparation of histological
So it's really a lecture or a demonstration
more in 2 parts.
Firstly, to briefly describe the process of
preparing tissue for microscopic examination
and I'm going to confine it just to light
using paraffin embedded sections.
They're the most common slides you're going
to examine in your histology classes.
And then lastly, I'm just going to briefly
describe and show you
some of the common histological stains you'll come across in
your study of histology.
Here is a technologist with what we call a
It's a machine used to cut histological sections.
If you look at the technologist's left hand
just above his fingers, you can see a bit
of tissue stained a browny color embedded
in a highly-stained paraffin block,
and the technologist is collecting sections
as he cuts them on this microtome.
He's turning the wheel with his right hand
and that wheel lifts and then brings down the paraffin
block of tissue onto the knife and then as
the tissue section is cut from the knife,
he will remove it and then
float it on a water bath to flatten it out
and then he'll put it on the slide.
So let me just go through some of the basic
Firstly, what you need to do is you get the piece of tissue
from either the animal or the human that you're going to
You don't need a large block of the tissue.
And then you have to fix it for at least 24
hours or more in what we use as a fixative.
Fixation, to fix something, means to stabilize
all its components.
A fixative is a compound such as formalin
or paraformaldehyde or glutaraldehyde
that is used to stabilize and fix components of
cells such as proteins, phospholipids, etc.
Some fixatives won't fix all the components
of a cell.
Other fixatives are better to use but more
expensive to use and they're often reserved
for more detailed preparation of tissue, for
instance, for examining with an electron microscope.
You can fix tissue in 2 ways.
Normally, you cut a piece of tissue such in
taking a biopsy from a person or you might
collect the tissue by cutting it from an animal
and dropping it or immersing it in the fixative,
and the gently shaking that fixative
around the specimen
to allow the fixative to penetrate into the specimen.
That's the common way of doing it.
In a lot of animal studies, animal tissue,
sometimes we perfuse the animal.
The animal is anesthetized and then a needle
is put into a major vessel and saline is passed
through to wash all the blood out and then
fixative follows, and that allows very efficient,
rapid penetration of fixative into all the body tissues,
and then the tissue is removed and then
examined after processing histologically.
After fixation, you then pass the tissue through
a series of alcohols to dehydrate the specimen,
to take all the water out of it, and that's
important because what you want to do then
is you want to replace that alcohol with a
substance such as xylol that
is miscible with paraffin because you then
want to infiltrate all the spaces between
the cell tissue that you have preserved or
fixed with paraffin and carried through in
this vehicle of xylol or chloroform, and that
paraffin is then set or you imbed the block
in the paraffin, you leave it for a while
to set just like you would other components
you might make that require a setting process
like cement, and then what you do is you put
it on the microtome you see here or you saw
previously in more detail,
and you cut very thin sections off that paraffin block.
Sections for light microscopic examination
are normally about 5 µm to 10 µm thick.
This enables you using normal light microscopy
and staining to get almost the limit of resolution
with the light microscope to look at details
of the tissue.
Once you cut that section, you then float it on a glass bath
and pick it up on a glass slide, and then you have to go
through the process
of getting rid of that embedding medium which is going to
stand in the way
of the viewing of the individual components of the tissue.
So to remove that, you again pass it through
alcohols to dissolve it all away
and then you stain this section with whatever staining you
wish to use
to show details or the components of the tissue.
The normal stains we use are going to be hematoxylin
That's the common stain used in histology
and it's the common stain used for all the
sections you're going to look at, or most
of them anyway, you're going to look at in
your histology classes.
But certainly, there are some very special
stains that are used to show up very specific
components of tissue that I'll show you in
the latter part of this lecture.
After you stain, you've got to re-dehydrate
the sections once again in alcohol
and then you again clear it all away with the xylol.
Make sure all the paraffin and all the debris
or all the non-tissue material is removed,
and then you put a cover slip on that slide,
you don't forget to label the slide so you
know what the tissue is of, and then you're
ready to view the slide.