Theory of histological staining

Reagent-tissue interactions

Coulombic attractions have been termed salt links or electrostatic bonds, and have been the most widely discussed reagent-tissue interactions. They arise from electrical attractions of unlike ions, e.g. the colored cations of basic dyes and tissue structures rich in polyanions such as phosphated DNA or sulfated mucosubstances ( ). However, binding of dye ions to an ionic tissue substrate also depends on charge magnitude, the amount of non-dye electrolyte in the dyebath, electrical repulsions between ions of similar charge, and swelling or shrinking of tissue substrates ( ). These phenomena are relevant for all ionic reagents, not just dyestuffs. For example when using periodate as the oxidant in the PAS procedure, the periodate anions do not readily react with anionic polysaccharides, such as chondroitin sulfate ( ). Moreover, even uncharged tissue substrates acquire an ionic character after binding ionic reagents, e.g. during staining of glycogen by the PAS procedure.

Reagent-tissue attractions not depending on isolated electric charges include dipole-dipole, dipole-induced dipole and dispersion forces; collectively described as van der Waals’ forces . These occur between all reagents and tissue substrates. However, as extensively delocalized electronic systems favor larger dipoles and greater polarizability, van der Waals’ forces are more significant when tissues or stains contain such moieties. Consequently, proteins rich in tyrosine and tryptophan residues, and nucleic acids containing heterocyclic bases, favor van der Waals’ attractions. This is also true for the large aromatic systems of bisazo dyes and bistetrazolium salts, halogenated dyes such as rose Bengal and phloxine, and indoxyl and naphthyl enzyme substrates ( ). For instance, van der Waals’ attractions contribute substantially to stain-tissue affinity when staining elastic fibers which are rich in aromatic desmosine and isodesmosine residues with polyaromatic acid and basic dyes such as Congo red and orcein ( ).

Hydrogen bonding is occasionally discussed in the biological staining context. This attractive interaction arises when a hydrogen atom lies between two electronegative atoms e.g. oxygen or nitrogen, though being covalently bonded only to one. Water is hydrogen bonded extensively to itself, forming the clusters important for the hydrophobic effect discussed below. This effect also applies to molecules carrying hydrogen bonding groups present in many dyes and tissue components. As water molecules vastly outnumber dye molecules, hydrogen bonding is not usually important for stain-tissue affinity when aqueous solvents are used. Exceptions arise when hydrogen bonding is favored by the substrate, as with collagen tissue fibers ( ). A related attractive phenomenon, halogen bonding ( ), may also contribute to staining affinity. This could explain strong staining seen with dyes such as eosin Y (4 arylbromo substituents), phloxine (4 bromo plus 4 chloro), and other halogenated fluoresceins.

Covalent bonding between tissue and stain is yet another source of stain-tissue affinity. This process underpins the commonly used PAS reactive stains, as well as the historic Feulgen nuclear stains. The polar covalent bonds between metal ions in dyes such as hematein and tissue substrates are another possible example. Dye-tissue binding considered to involve such polar bonds is termed mordanting. However, this is of uncertain status since the characteristic staining properties of mordant dyes may have other, or additional causes. Unlike most cationic dyes used as biological stains, common cationic metal-complex dyes are strongly hydrophilic ( ) and resist extraction into alcoholic dehydration fluids ( ).

Solvent-solvent interactions

A major contribution to stain-tissue affinity when using organic reagents or dyes in aqueous solution is the hydrophobic effect . This involves no stain-tissue attractions, but is the tendency of hydrophobic groupings (e.g. leucine and valine side chains of proteins; biphenyl and naphthyl groupings of enzyme substrates and dyes) in an aqueous milieu to come together, even though they were initially dispersed. This interaction occurs because water molecules are linked together by hydrogen bonds into transient clusters whose presence is favored by hydrophobic groups. Any process breaking clusters into individual water molecules occurs spontaneously, as this increases system entropy (cf. the second law of thermodynamics). Consequently, removing cluster-stabilizing hydrophobic groups from contact with water by placing them in contact with each other, is thermodynamically favored. Accounts of the hydrophobic effect are provided by biochemists, amongst others ( ). The effect becomes more important as substrate and reagent become more hydrophobic. Thus, when staining fats with Sudan dyes applied from substantially aqueous solutions, the hydrophobic effect provides major contributions to affinity. Although the phenomenon is sometimes termed hydrophobic bonding, no dye-tissue hydrogen bonds are involved.

Staining using Sudan dyes in non-aqueous solvents does not involve the hydrophobic effect. However, as described in chemical thermodynamics texts (e.g. ), the tendency of a system to change spontaneously to maximize its disorder, and for entropy to increase, provides an explanation. Presence of dye in solvent and lipid constitutes a more disordered system than dye restricted to the solution. So dye disperses, and staining occurs. Such increases in entropy involving substrate and stain occur in all types of histological staining.

Stain-stain interactions

Dye-dye interactions can also contribute to affinity. Even in dilute solutions, dye molecules can attract each other, forming aggregates. In aqueous solutions this may be driven by the hydrophobic effect, but in both aqueous and non-aqueous solutions van der Waals’ attractions between planar dye molecules occur. Dye aggregation increases with concentration, e.g. when high dye concentrations build up on tissue sections. With basic (cationic) dyes, such as toluidine blue, this occurs on substrates of high negative charge density e.g. sulfated polysaccharides in mast cell granules giving metachromatic staining . This color effect arises because dye aggregates have spectral properties different from the monomeric dye. That dye-dye interactions contribute to affinity in tissue sections was demonstrated quantitatively by .

Other cases where stain-stain interactions contribute to affinity include metallic nano and micro-crystals generated by gold or silver impregnation ( ), ionic metal sulfide precipitates formed in Gomori-type enzyme histochemistry, and the purple azure-eosin charge transfer complex produced during Romanowsky-Giemsa staining of cell nuclei ( ).

A minor anomaly

Some stains are not taken up by their tissue targets. In negative staining the shapes of structures are disclosed by filling or outlining them with a stain. Examples include demonstrating canaliculi in bone matrix using Schmorl’s picro-thionine stain and visualizing individual microorganisms using nigrosine.

Solubility, an unacknowledged factor

The solubility of stains and staining reagents is a key practical property. Thus, when staining fat with a Sudan dye, the upper limit of staining intensity is set by dye solubility in the lipid, and is further influenced by dye solubility in the staining bath solvent. Solubility is also critical for dye retention after staining, as discussed below. Solubility has complex causes but, in general, the stronger the reagent-reagent interactions, the lower the solubility. Physicochemical texts provide overviews of solubility e.g. .