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Introduction to microscopy
Introduction to microscopy

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3 Histochemical techniques

Since most cell structures are transparent, very little detail of the structure can be seen, unless the cells are stained. The same is true of components of the extracellular matrix. Because different parts of the cell are biochemically different, they take up specific stains to varying degrees. For example, haematoxylin binds strongly to acids and consequently binds to nuclear DNA and stains nuclei blue. Histologists have developed many stains which are suited to particular purposes, allowing cell structures to be differentiated. It is important to remember that the colours of stains are not the real colour of a particular tissue, and that a structure that appears as one colour using one stain, may be a quite different colour using another stain.

The great majority of routine histology is done with haematoxylin and eosin (H&E) staining, because it is quick, cheap and informative. Staining with H&E is very reliable although it does show some variation depending on the exact formulation of the stain, and the stain density is considerably affected by the thickness of the sections - thicker sections take up more stain. It is also generally done before any additional staining techniques, because histology with H&E can confirm the basic tissue type and help to localise the lesion. (The term lesion is used by pathologists to indicate any area of damage, infection, inflammation, tumour, necrosis or otherwise abnormal tissue.) For example, to diagnose a lymphoma within a lymph node, one would initially carry out H&E staining to confirm the basic diagnosis and localise the affected cells, before doing immunohistochemistry to identify exactly which type of lymphocyte was present.

A wide variety of other histochemical stains are also available, each of which can help identify particular structures. Some are relatively simple to perform, merely requiring that the section is dipped in the stain for a set time. Others require a number of sequential steps, and in some cases the results can be surprisingly variable or unpredictable. For example, the silver staining technique originally developed by Camillo Golgi (he of the Golgi apparatus) is notably temperamental (Figure 7). Some of the more commonly used techniques are outlined below:

  • Giemsa stain consists of a mixture of methylene-blue and eosin. It is mostly used on methanol-fixed blood films, where it stains erythrocytes pink and the different types of leukocyte, allowing their identification according to size and shape of their nucleii. It also binds to some pathogens, including spirochaetes (syphilis), trypanosomes (sleeping sickness and Chagas disease) and plasmodium (malarial parasites). In addition it can also be used to stain some bacteria in tissue sections pink, and it is therefore particularly useful if infection is suspected.
  • Gram stain is used to identify and differentiate bacteria. For example, staphylococci, streptococci and pneumocci are Gram-positive and stain a deep blue, whereas coliforms and neisseria are Gram-negative and stain pink.
  • Ziehl Nielsen stain is used to identify 'acid-fast' bacilli, including mycobacteria (tuberculosis and leprosy), which appear as black rods.
Figure 7 Axons and dendrites of a neuron - Bielchowsky silver stain, a development of the Golgi silver staining method.

Note that identification of the bacteria requires that the shape of the bacteria can be distinguished as well as their ability to take up stain, so sections have to be observed using the highest magnification possible.

Many stains are useful because they can differentiate elements in the tissue. They include:

  • Luxol fast blue/Cresyl violet is used to identify myelin which stains blue, while other elements of the nervous system stain pink or violet.
  • Oil red O is a dye that is more soluble in fat than in water or alcohols, hence it is used as a stain for neutral lipids. For example when myelin is broken down in the CNS, in diseases such as multiple sclerosis, macrophages take up the lipid-rich debris and stain strongly with this dye.
  • Masson's trichrome stain, covers a variety of different techniques that developed from Masson's original formulation, each of which uses three dyes to stain different structures. It is valuable for distinguishing elements of connective tissue. Typically the cell cytoplasm, muscle and keratin are stained red, nucleii are black and collagen is blue. This stain benefits from having tissue fixed using Bouin's fixative, although formalin-fixation is still workable.
  • Periodic acid Schiff (PAS) stains carbohydrates magenta, including components of the basal lamina, surface glycoproteins on cells and intracellular carbohydrates such as glycogen in hepatocytes. Cells that secrete mucus are also strongly stained.
  • Alcian blue is often combined with PAS, as it stains acidic mucins blue, whereas PAS stains neutral mucins red, hence it can be used to distinguish elements of the extracellular matrix. It also stains some fungi and parasites.
  • Congo red is used to identify deposits of protein in tissue called amyloid.
  • Silver staining methods have a long history; they deposit silver, which appears black onto structures that reduce silver nitrate. They can be particularly valuable for identifying individual cells, such as a single nerve cell within a group of cells, because the methods do not uniformly stain every cell of a type within the tissue. The image of a single cell within a complex tissue can be very informative, but getting the precise conditions to produce this partial staining can be difficult.
  • Toluidine blue is a particularly versatile dye that stains nuclei blue, and can be used to differentiate different types of granules (e.g. within mast cells). Because it can permeate the resins that are used to embed sections for electron microscopy, it is often used as a preliminary stain, to identify sections that will later be examined by EM.
  • Van Gieson stain binds to collagen in the extracellular matrix, staining it pink. Often it is combined with a stain for elastic fibres (elastic van Gieson) which stain black, allowing the two major elements of connective tissue to be differentiated.

The methods described above are only a small proportion of those that are available. As can be seen in the examples of trichrome stains, dyes may be used in combination to obtain additional information from the sections. The precise methodology and timings for the staining procedure may also vary slightly between laboratories. Some laboratories regularly use high temperatures in their staining procedures (microwaving) during particular steps, either to enhance the staining or reduce the time taken. Some stains are also more labile than others, and need to be remade regularly to maintain consistent results.