The reaction of the anionic groups varies with pH. Thus
• At a high pH (about 10), all three groups are ionized and available for reaction by electrostatic linkages with the basic dye.
• At a slightly acidic to neutral pH (5 to 7), sulfate and phosphate groups are ionized and available for reaction with the basic dye by electrostatic linkages.
• At low pH (below 4), only sulfate groups remain ionized and react with basic dyes.
Therefore, staining with basic dyes at a specific pH can be used to focus on specific anionic groups, and because the specific anionic groups are found predominantly on certain macromolecules, the staining serves as an indicator of these macromolecules.
As mentioned, hematoxylin is not, strictly speaking, a basic dye. It is used with a mordant (i.e., an intermediate link between the tissue component and the dye). The mordant causes the stain to resemble a basic dye. The linkage in the tissue-mordant-hematoxylin complex is not a simple electrostatic linkage, and when sections are placed in water, hematoxylin does not dissociate from the tissue. Hematoxylin lends itself to those staining sequences in which it is followed by aqueous solutions of acidic dyes. True basic dyes, as distinguished from hematoxylin, are not generally used in sequences wherein the basic dye is followed by an acidic dye. The basic dye then tends to dissociate from the tissue during the aqueous solution washes between the two dye solutions.
Acidic dyes react with cationic groups in cells and tissues, particularly with the ionized amino groups of proteins
The reaction of cationic groups with an acidic dye is called acidophilic![Gr., acid-loving]. Reactions of cell and tissue components with acidic dyes are neither as specific nor as precise as reactions with basic dyes.
Although electrostatic linkage is the major factor in the primary binding of an acidic dye to the tissue, it is not the only one; because of this, acidic dyes are sometimes used in combinations to color different tissue constituents selectively. For example, three acidic dyes are used in the Mai-lory staining technique: aniline blue, acid fuchsin, and orange G. These dyes selectively stain collagen, ordinary cytoplasm, and red blood cells, respectively. Acid fuchsin also stains nuclei.
In other multiple acidic dye techniques, hematoxylin is used to stain nuclei first, then acidic dyes are used to stain cytoplasm and extracellular fibers selectively. The selective staining of tissue components by acidic dyes is due to relative factors, such as size and degree of aggregation of the dye molecules and permeability and "compactness" of the tissue.
Basic dyes can also be used in combination or sequentially (e.g., methyl green and pyronin to study protein synthesis and secretion), but these combinations are not as widely used as acidic dye combinations.
A limited number of substances within cells and the extracellular matrix display basophilia
These substances include
• Heterochromatin and nucleoli of the nucleus (chiefly because of ionized phosphate groups in nucleic acids of both)
• Cytoplasmic components such as the ergastoplasm (also because of ionized phosphate groups in ribosomal RNA)
• Extracellular materials such as the complex carbohydrates of the matrix of cartilage (because of ionized sulfate groups)
Staining with acidic dyes is less specific, but more substances within cells and the extracellular matrix exhibit acidophilia
These substances include
• Most cytoplasmic filaments, especially those of muscle cells
• Most intracellular membranous components and much of the otherwise unspecialized cytoplasm
• Most extracellular fibers (primarily because of ionized amino groups)
Certain basic dyes react with tissue components that shift their normal color from blue to red or purple; this absorbance change is called metachromasia
The underlying mechanism for metachromasia is the presence of polyanions within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the dye molecules are sufficiently close to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dye molecules.
Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups, such as the ground substance of cartilage, heparin-containing granules of mast cells, and rough endoplasmic reticulum of plasma cells, exhibit metachromasia. Therefore, toluidine blue will appear purple to red when it stains these components.
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