HISTOLOGIST encyclopedia topics

Technical procedure

Fixation

Fixatives are used to preserve tissue from degradation, and to maintain the structure of the cells inclusive of sub-cellular components such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria). The most common fixative is 10% neutral buffered formalin (4% formaldehyde in phosphate buffered saline). Formaldehyde-based fixatives preserve tissues or cells by irreversibly cross-linking primary amino groups in proteins with other nearby nitrogen atoms in protein through a CH₂ (methylene) linkage, thereby "fixing" proteins in the shape that they're in. This process destroys the biological functionality of proteins, and can also denature them to a certain extent. This can be detrimental to certain histological techniques.

Processing

Tissues must be impregnated with the material that will be used for embedding so that the tissue samples adhere to the embedding material, therefore the type of processing depends on the kind of embedding material that will be used. The most common histological technique is paraffin wax embedding, and many larger facilities utilize automated tissue processing machines. During this process samples are immersed in multiple baths of progressively more concentrated ethanol to remove the water from (dehydrate) the tissues, followed by a clearing agent, such as xylene, to remove the alcohol, and finally hot molten paraffin wax which replaces the xylene.

Embedding

After the tissues have been dehydrated and impregnated with the embedding material they are ready for embedding. During this process the tissue samples are placed into moulds along with liquid embedding material which is then allowed to harden. The hardened blocks containing the tissue samples are then ready to be sectioned.

Embedding can also be accomplished using frozen, non-fixed tissue in a water-based medium. Pre-frozen tissues are placed into moulds with the liquid embedding material, usually a water-based glycol or resin, which is then frozen to form hardened blocks.

Sectioning

The tissue is then sliced into very thin (0.5–10 microns; 1000 microns = 1 mm) sections using a machine called a microtome. These slices, usually thinner than the average cell, are then placed on a glass slide for staining.

Frozen tissue embedded in a freezing medium is cut on a microtome in a cooled machine called a cryostat.

Staining

Routine staining: This is done to give contrast to the tissue being examined, as without staining it is very difficult to see differences in cell morphology. Hematoxylin and eosin (H&E) are the most commonly used stains in histology and histopathology. Hematoxylin colors nuclei blue; eosin colors the cytoplasm pink. To see the tissue under a microscope, the sections are stained with one or more pigments.

Special staining: There are hundreds of various other techniques that have been used to selectively stain cells and cellular components. Other compounds used to color tissue sections include safranin, oil red o, Congo red, fast green FCF, silver salts, and numerous natural and artificial dyes that were usually originated from the development dyes for the textile industry.

Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis.

Histology samples have often been examined by radioactive techniques. In historadiography a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used to visualize the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy.

Recently, antibodies are used to specifically visualize proteins, carbohydrates, and lipids: this is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail. Digital cameras are increasingly used to capture histological and histopathological image

Common laboratory stains

Fungi—black

Stain	Common use	Nucleus	Cytoplasm	Red blood cell (RBC)	Collagen fibers	Specifically stains
Haematoxylin	General staining when paired with eosin	Blue	N/A	N/A	N/A	Nucleic acids—blue Blue eER (ergastoplasm)—blue
Eosin	General staining when paired with haematoxylin	N/A	Pink	Orange/red	Pink	Elastic fibers—pink Reticular fibers—pink
Toluidine blue	General staining	Blue	Blue	Blue	Blue	Mast cells granules—purple
Masson's trichrome stain	Connective tissue	Black	Red/pink	Red	Blue/green	Cartilage—blue/green Muscle fibers—red
Mallory's trichrome stain	Connective tissue	Red	Pale red	Orange	Deep blue	Keratin—orange Cartilage—blue Bone matrix—deep blue Muscle fibers—red
Weigert's elastic stain	Elastic fibers	Blue/black	N/A	N/A	N/A	Elastic fibers—blue/black
Heidenhains'azan trichrome stain	Distinguishing cells from extracellular components	Red/purple	Pink	Red	Blue	Muscle fibers—red Cartilage—blue Bone matrix—blue
Silver stain	Reticular fibers, nerve fibers, fungi	N/A	N/A	N/A	Reticular fibers—brown/black Nerve fibers—brown/black
Wright's stain	Blood cells	Bluish/purple	Bluish/gray	Red/pink	N/A	Neutrophil granules—purple/pink Eosinophil granules—bright red/orange Basophil granules—deep purple/violet Platelet granules—red/purple
Orcein stain	Elastic fibres	Deep blue [or crazy red]	N/A	Bright red	Pink	Elastic fibres—dark brown Mast cells granules—purple Smooth muscle—light blue
Periodic acid-Schiff stain (PAS)	Basement membrane, localizing carbohydrates	Blue	N/A	N/A	Pink	Glycogen and other carbohydrates—magenta

Table sourced fromMichael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins.

The Nissl method and Golgi's method are useful in identifying neurons.

Alternative techniques

Alternative techniques include cryosection. The tissue is frozen and cut using a cryostat. Tissue staining methods are similar to those of wax sections. Plastic embedding is commonly used in the preparation of material for electron microscopy. Tissues are embedded in epoxy resin. Very thin sections (less than 0.1 micrometer) are cut using diamond or glass knives. The sections are stained with electron dense stains (uranium and lead) so that they can be seen with the electron microscope.

History

In the 19th century, histology was an academic discipline in its own right. The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had dueling interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible.

Histological classification of animal tissues

There are four basic types of tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. All tissue types are subtypes of these four basic tissue types (for example, blood cells are classified as connective tissue, since they generally originate inside bone marrow).

Epithelium: the lining of glands, bowel, skin and some organs like the liver, lung, kidney
Endothelium: the lining of blood and lymphatic vessels
Mesothelium: the lining of pleural and pericardial spaces
Mesenchyme: the cells filling the spaces between the organs, including fat, muscle, bone, cartilage, and tendon cells
Blood cells: the red and white blood cells, including those found in lymph nodes and spleen
Neurons: any of the conducting cells of the nervous system
Germ cells: reproductive cells (spermatozoa in men, oocytes in women)
Placenta: an organ characteristic of true mammals during pregnancy, joining mother and offspring, providing endocrine secretion and selective exchange of soluble, but not particulate, blood-borne substances through an apposition of uterine and trophoblastic vascularised parts
Stem cells: cells able to turn into one or several of the above types

Note that tissues from plants, fungi, and microorganisms can also be examined histologically. Their structure is very different from animal tissues.

Related sciences

Cell biology is the study of living cells, their DNA and RNA and the proteins they express.
Anatomy is the study of organs visible by the naked eye.
Morphology studies entire organisms.

Artifacts

Artifacts are structures or features in tissue that interfere with normal histological examination. These are not always present in normal tissue and can come from outside sources. Artifacts interfere with histology by changing the tissues appearance and hiding structures. These can be divided into two categories:

Pre-histology

These are features and structures that have being introduced prior to the collection of the tissues. A common example of these include: ink from tattoos and freckles (melanin) in skin samples.

Post-histology

Artifacts can result from tissue processing. Processing commonly lead to changes like shrinkage, color changes in different tissues types and alterations of the structures in the tissue. Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered. A common example is mercury pigment left behind after using Bouin's fixative to fix a section.