Degrees affinity of the dye to specific tissue sites rather than other tissue components relates to intensity of staining (number of binding sties) or type and intensity of attractive forces acting. Dye to tissue binding includes both chemical and physical forces eg: hydrophobic bonding, Van der Waals’s forces, Coulombic attraction, hydrogen bonds and covalent bonds. Where solvent-solvent, dye-solvent, dye-dye and dye-tissue interactions are all occurring simultaneously and during the staining process.
Dye to tissue binding also relies on other factors: rate of penetration (tissue lattice structure and molecular size of dye), rates of uptake/reaction and rates of loss especially during differentiation Orthochromasia: tissue staining the same colour and shade as the one dye but of varying intensity eg. Methylene blue Metachromasia: tissue staining the same & different shades and colours as one dye. Eg. Toluidine blue (binds to a highly charged tissue elements which alters the absorbed wavelength of visible light, altering the colour of the bound dye).
Polychromasia: tissue staining many colours and shades as the staining solution is made up from compound of dyes eg. Any of the romanowsky type dyes used for staining of blood films or bond marrow smears (Leishman’s, Giemsa, Wright’s stains) most dyes are prepared as salts, auxochromes on a dye can be classed as either acidic or basic
Haematoxylin staining can be either progressive (does not need differentiation, as is not overstained) eg. Gill’s, Mayer’s recipes or regressive (needs differentiation as there is overstaining). Eg. Mostly aluminium mordanted, harris’s, Heidenhain’s recipes. It all depends on haematoxylin formulation or recipe.
Differentiation is the removal of excess dye from over-stained tissue; it’s done to see details of cellular structure. The bond between the basic haematoxylin dye and the mordant is acidified with acid alcohol. This detaches the dye so that excess can be washed away and haematein is amphoteric when acidified it has a red bias
Blued with Scott’s bluing solution.
Strengthens the haematein/mordant bond precipitates the dye and prevents further loss Haematein when alkaline it has a blue bias Eosin dyes is an xanthene dye, there are at least 3 common types Y, B & S. eosin Y is the most widely used Used as an acid (negatively charged) counterstain a dye Contrasts well with the primary staining dye Highlights and discriminates between the primary staining and different tissue elements. Binds to positively charged (cationic) tissue site such as cytoplasmic proteins.
Often made up as an alcoholic solution Solubility in water is great and so would remain in solution not preferentially bind to tissue sites Staining intensity is a matter of preference Water solubility is of importance. Avoid rinsing off stained slides with water or even low concentration alcohols; it will be washed out of the tissue section. Therefore water differentiates eosin.