Classification principles
To study tissues under a microscope, they must be contrasted. All staining agents used in histology are divided into exactly four groups. The main criterion for this division is the chemical nature of the stain itself, as well as its affinity for specific structures inside the cell or in the extracellular matrix. Understanding this classification is the key to deciphering any histological specimen.
Acidic stains and oxyphilia
By their chemical nature, these substances are acids and their acidic salts. The key property of tissue structures due to which they bind to such substances is called oxyphilia.
Typical representatives of this group are bright pink eosin and acid fuchsin. Their main "targets" are oxyphilic structures rich in basic chemical groups:
- Cytoplasmic proteins of most cells (including hemoglobin contained in erythrocytes).
- Non-cellular structures, primarily collagen fibers of the intercellular substance.
Basic stains and basophilia
This group is represented by basic salts. The ability of tissues to bind to them is called basophilia. Among the most well-known representatives are carmine, azure II, and hematoxylin (furthermore, the active coloring agent in the latter is the product of its oxidation — hematein).
Basic stains selectively bind to structures that are rich in organic acids. Such basophilic targets include:
- Cell nuclei and ribosomes. Staining occurs due to the high content of nucleic acids (DNA and RNA) in them.
- Amorphous component of the intercellular substance. Here, the reaction is due to the presence of hyaluronic acid and other similar compounds.
Neutral and indifferent stains
Neutral stains are a complex mixture of basic and acidic coloring agents. Their mechanism of action consists in simultaneously staining various tissue components in their specific colors. Classical targets for them (neutrophilic structures) are the specific granularity of neutrophil leukocytes and the cytoplasm of polychromatophilic erythroblasts.
Indifferent stains differ radically from the rest. By nature, they are hydrophobic and lipophilic substances that are completely insoluble in water. The most famous representatives are Sudan III and Sudan IV.
Their staining mechanism is based not on a chemical reaction with tissue components, but on the purely physical dissolution of the stain itself in fats and lipids. As a result of this process, lipid droplets acquire a rich bright orange color, making them easy to identify on the specimen.
Briefly: answers to topical questions
How do metachromatic stains differ from orthochromatic ones?
Metachromatic stains are distinguished by their ability to change their original color upon contact with chromotropic substances, while information about orthochromatic stains is absent in these materials. The phenomenon of metachromasia is due to the high concentration of glycosaminoglycans in the tissue. Metachromatic stains include:
- Toluidine blue — stains the substance not blue, but another (violet-red) color.
- Methylene blue — also causes the phenomenon of metachromasia.
Which histological stains are used to detect glycogen?
Schiff's reagent (as part of the PAS reaction) and Best's carmine are used to detect glycogen. The following methods are used in histological practice:
- Schiff's reagent — used in the PAS reaction after oxidation of the specimen with periodic acid. Glycogen clumps are stained purple, dark red, or dark crimson.
- Best's carmine — used for specific differentiation of glycogen and gives a red coloration.
Enzymatic control (treatment of sections with amylase) is used to confirm the presence of glycogen.
What stains are used to detect elastic fibers?
Elastic fibers are detected using orcein staining or a combination of picrofuchsin and hematoxylin, since they are not contrasting under standard methods. Staining methods:
- Orcein — a selective method in which elastic fibers and membranes acquire a cherry (or cherry-red) color against a pale pink background of other tissues.
- Picrofuchsin with hematoxylin — a differential method that stains elastic fibers yellow under the action of picric acid, collagen fibers red, and cell nuclei violet.
Basophilia is easy to remember: BASIC stains bind to acids (DNA, RNA in nuclei). Oxyphilia is the opposite: ACIDIC stains color proteins and cytoplasm.
Class and exam questions
What is the difference between oxyphilia and basophilia?
Oxyphilia is the ability of structures to stain with acidic dyes (cytoplasmic proteins and collagen stain this way). Basophilia is the affinity for basic dyes, typical of acid-rich structures (nuclei, ribosomes, amorphous ground substance).
What is the mechanism of action of indifferent stains?
Unlike other groups, they do not enter into chemical reactions with tissue. Indifferent stains (e.g., Sudan III) physically dissolve in lipids and fats, staining them bright orange.
What do neutral stains color?
They consist of a mixture of acidic and basic substances, staining neutrophilic structures: the specific granularity of neutrophil leukocytes and the cytoplasm of polychromatophilic erythroblasts.
What else to study on this topic
- The process of oxidation of hematoxylin to hematein
- Staining of erythrocyte hemoglobin with eosin
- Detection of collagen fibers with acid fuchsin
- Chemical nature of hyaluronic acid and its basophilia
- Physicochemical properties of Sudan III and Sudan IV
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