The information on THIS page is also available as an online PowerPoint, with pictures of some equipment used in our own preparation laboratory. The PowerPoint gives a more detailed, step-by-step description.
To view this PowerPoint, Click Here. It opens in a new window.
The following links lead to the respective sections on this page.
1. General Remarks
Histology uses many tools : from a simple magnifying glass through light microscope, microscopes using fluorescent techniques, polarised light to the high resolution electron miscroscopes. To serve our specific needs we shall concentrate on the light microscopic features of tissues with a few selected techniques. Also note that the technical details of preparation of tissues for study is outside the scope of this unit. The introduction to histological techniques given here suffices for us to understand the implications of the processes for our study.
Most of the slides you will see have sections or thin slices of tissue on them. The thickness of sections for light microscopy is usually between 5 and 8 microns (or micrometres; one micrometre is one-thousandth of a millimetre or one-millionth of a metre). This is rather thin by ordinary standards, but electron microscopy requires sections even thinner than this! Obviously, the human hand cannot cut sections of this thickness, it is done by a machine called microtome.
2. The Material and How We Obtain It
Body tissues (with the exception of tendons, cartilage and bone) are rather soft in the fresh state and being organic matter, are prone to decomposition. Before a section is cut the tissue must be preserved and hardened. This is done by chemical treatment with "fixatives". Formaldehyde in solution (called formalin) is one of the common fixatives. Still, a formalin-fixed piece of tissue needs to be supported by something before it is attached to the microtome for cutting. The supporting material commonly used is paraffin wax. Since the tissues are water-based wax cannot penetrate them. The tissue is first dehydrated with ethyl alcohol and then the alcohol is replaced by a paraffin solvent like toluene. This process is done with an automated machine which immerses the tissue serially in alcohol and then xylene. It is then immersed in a bath of molten paraffin. After the tissue is infiltrated with paraffin it is allowed to solidify, yielding a "block" of paraffin with the tissue in it. Next, it is cut into sections by the microtome. The sections are floated on warm water to straighten them out and fixed to the slides that we see.
This process is called paraffin processing. For light microscopy we may also use other methods of hardening tissues. One such method is freezing the tissue and sectioning it. “Frozen sections” retain some tissue components which may be lost in paraffin processing and thus have an advantage, but frozen sections are thicker, and do not last long. We may also actually use chemical reactions to demonstrate some cellular components especially enzymes or certain chemical elements. These are very specialized methods and have their limitations.
Most of the tissue components are colourless. For identifying different components (cytoplasm, nuclei, extracellular material) we need to colour them with "stains". This involves dissolving the paraffin, restoring water in the sections, staining and once again "dehydrating" them. Finally, a very thin "coverglass" or coverslip is used to to protect the section. The coverslip is attached by a synthetic adhesive.
3. Effects of Processing
We need to understand some of the effects this entire process has on the tissue.
1. We are looking at two-dimensional pictures of body structures. This is like studying an engineer’s drawing of the plan of the house and imagining what the house will look like. The drawing of a plan is like a section passing through as many features (windows and doors, for example) of the house as possible. Realise that a section of the house close to the floor level will show most of the doors but no windows. We therefore need to learn to interpret the sections. This happens with practice and one always has to ask the question : “what would this be like in three dimensions?”.
2. Everything in a histological section appears as it was at the moment the tissue was taken from the body and fixed. In other words, it is a picture at a moment in time. The same cell/s might have appeared different at a different moment in time. Cells grow, divide, change their features as they enter different phases of functioning, even change their location and die. Fortunately we shall be seeing so many cells at a time that we may expect to see them various cells in different stages in their life.
3. The tissue we see in the section has been subjected to a lot of chemical and physical treatment as outlined above. Overall we may say that the dead, sectioned, physically and chemically treated (including staining!) cells and tissues are unlikely to have the appearance they had in life. Other than staining, all such changes are called artifacts (some may spell the word “artefacts”, it does not matter!). We are not expected to analyse these artifacts in detail or know all of them at this stage, but it helps to know some common ones. At this stage there are no illustrations of these artefacts. In the pages that follow, artefacts will be commented on as and when they are seen.
3a. Artefacts of chemical treatment.
The most common artifact of this kind is shrinkage. The process of dehydration by alcohol causes this. All types of tissue components may appear smaller than they are and clear spaces may appear around cells and between layers of tissues. As we see more of them we shall learn to distinguish them from normal cavities in the body. very often shrinkage is actually helpful as it allows us to see some cells and tissue components more clearly!
Loss of fat is another important artifact. The toluene used to replace alcohol prior to the paraffin bath is also a solvent for fats. Fat containing cells therefore appear “empty”.
3b. Artefacts introduced by the microtome.
Tissues and tissue components differ in their hardness. In general connective tissues tend to be harder, while nervous system structures are very delicate. An ideally processed tissue cut with the ideal, sharp microtome knife should not have any artefacts.
A defective knife can leave nicks, tears or parallel scratch marks on the sections. With harder tissues they may be unavoidable. A very soft tissue may be compressed. Sometimes cells or chunks of tissue may just be "knocked off", leaving an empty space.
3c. Artefacts of staining and mounting
Occasionally stain particles may be seen in sections. This happens when the staining chemicals are not dissolved properly or the stain is not fresh, leading to precipitation. This is an avoidable artefact!
More important, air bubbles may be trapped when the coverslip is attached over the stained section. These are obvious as tiny spherical features.
Folds in sections. Sometimes wrinkles on the wax sections cannot be straightened and one can see folds or pleats in the section. This is frequent artifact in sections with a hard component, difficult to avoid even with the greatest care.
Faulty dehydration is another common artefact, giving rise to a "cloudy" appearance of the section.
Some stains tend to fade over time (just as colour photographic prints do!).
remember that a slide may just be dirty or dusty. Usually dirt on the slide is
"out of focus" when the section is in sharp focus, because
the thickness of the coverslip separates the two.
4. A Note on Staining Methods.
As has been mentioned above, we need to stain most of the sections to enhance contrast between components of cells and tissues. The most common method of staining paraffin sections uses two stains, called Haematoxylin and Eosin. The process is named by the acronym H & E. Haematoxylin is a reddish purple dye which turns a shade of blue during the staining process. Eosin is pink or orange (even yellowish orange). Haematoxylin is described as a "basic" dye (as against acidic) and eosin as an acidic dye. This description is chemically not entirely accurate, but, without going into the theoretical aspects, serves a useful purpose.
Acidic components of tissues have affinity for the basic haematoxylin. These are called "basophilic" components and appear blue in H & E sections. Examples are nuclei of cells (they contain DNA), ribosomes and rough endoplasmic reticulum (containing RNA). Most other components are stained non-specifically by eosin though some have strong affinity to eosin. Such components are stained pink and are said to be acidophilic or eosinophilic. H & E stained sections can yield a huge lot of information about the components of most tissues.
H & E staining does have limitations. For example, muscle cell cytoplasm and connective tissue fibres are both eosinophilic. If we wish to distinguish between them we must use stains which show them up in different colours. A number of such methods are available, collectively called Masson techniques or trichrome stains. They stain collagen blue or green, muscle cytoplasm red, and nuclei are stained bluish black by a variant of haematoxylin.
Similar "differential" staining can be used to show collagen and/or elastic fibres separately in a connective tissue.
Nervous tissue is stained by a large variety of techniques used to show cell membranes, nuclei, healthy or degenerated myelin sheaths.
A complete discussion of all these techniques is beyond the scope of this unit, but examples will be commented on as we come across them.
Key Points :
Fixation : preservation and hardening.
Dehydration : removal of water; can lead to shrinkage.
Clearing : "filling" the tissue with a paraffin solvent - dissolves natural fats as well.
Paraffin embedding and cutting sections. Sections may "wrinkle" while cutting.
Attaching sections to glass slides.
Staining : Routine stains : haematoxylin and eosin.
This information is also available
in an online PowerPoint, with pictures of some equipment used in the preparation
To view this PowerPoint, Click Here. It opens in yet another new window, so that you do not lose the thread of these pages.