How to Prepare Histology Slides
How to prepare histology slides is not usually knowledge required for first-level courses in Anatomy & Physiology (e.g. ITEC) and Human Biology (e.g. A-Level in UK). It is, however, useful to have a general awareness of the steps involved in preparing histology slides.
It might be useful for school and college students to know a bit about how to prepare histology slides as part of general appreciation of laboratory biology and techniques used in medical research. Familiarity with terminology used in the discipline of histology is also useful when reading around the subject or communicating with other professionals working in various fields within health sciences e.g. during work experience.
The five main stages in the preparation of histology slides are:
Notes about each of these stages:
Samples of biological tissue are 'fixed' to preserve the cells/tissue in as natural a state as possible and prevent postmortem decay (autolysis and putrefaction). Chemical fixatives are very carefully selected substances whose properties must meet many criteria. Even the most careful fixation alters the sample to a certain extent and may potentially introduce artifacts that can interfere with interpretation of images of the fine detail of cells, incl. all their organelles, that can only be observed using an electron microscope (such fine detail of cells that can only be seen using electron microscopes is called 'cellular ultrastructure').
Tissue processing is done to remove water from the biological tissues, replacing such water with a medium that solidifies, setting very hard and so allowing extremely thin sections to be sliced. This is important because biological tissue must be supported in an extremely hard solid matrix to enable sufficiently thin sections to be cut. Some typical values are:
Removal of water is 'dehydration'.
After tissues have been dehydrated and before they can be "sectioned" i.e. sliced very thinly (see the thicknesses mentioned above) they must be secured in a very hard solid block in such a way that the hardened material used to secure all parts of the biological tissues in place is transparent to the optical method used for viewing the finished samples.
Different types of embedding techniques and materials are used depending on the sample being prepared and the other types of processing involved in preparing that particular sample.
In general, tissue samples are placed in molds together with liquid embedding material which is then hardened. The result of this stage in the preparation of histology slides is hardened blocks containing the original biological samples together with other substances used so far in the preparation process.
Sectioning an embedded tissue sample is the step necessary to produce sufficiently thin slices of sample that the detail of the microstructure of the cells/tissue can be clearly observed using microscopy techniques (either light microscopy or electron microscopy).
Possible orientations at which tissue samples may be sectioned include:
The method used to actually cut sections from the hardened block of tissue depends on the type of microscopy that will be used to observe it and hence the thickness of sample required. In the case of samples to be studied using light microscopy, a steel knife mounted in a microtome may be used to cut 10μm tissue sections which are then mounted on a glass microscope slide. In the case of samples to be studied using transmission electron microscopy, a diamond knife mounted in an ultramicrotome may be used to cut 50 nm tissue sections which are then mounted on a 3-millimeter-diameter copper grid.
Finally, the mounted sections are treated with an appropriate histology stain.
Why are histology samples stained ?
Put another way, what is the purpose of histology stains ?
Biological tissue has very little variation in colours/shades when viewed using either an ordinary light (optical) microscope or an electron microscope. Staining biological tissues is done to both increase the contrast of the tissue and also highlight some specific features of interest, depending on the type of tissue and stain used.
There are many histology stains. Histology stains are normally selected according to the type of tissue to be observed. Some stains are more widely used than others while some are only used to study very specific types of biological tissue.
For more information see What is Histology, Histology Stains, Histopathology and the Structure of a Cell. The report Differential Staining With Acid Dyes* may also be of interest. *Copyright Bryan D. Llewellyn. Made available for download from this website for educational purposes within conditions of use stipulating that the text is unchanged and no charge is made. Accessed by us from http://stainsfile.info/StainsFile/downloads.htm, no-longer online.