Quarterly Journal of Microscopical Science, Vol s3-104, 413-439, Copyright © 1963 by Company of Biologists

An Approach to the Thermodynamics of Histological Dyeing, Illustrated by Experiments with Azure A

¹ Department of Anatomy, University of the Witwatersrand Medical School, Hospital Street, Johannesburg

Methods are proposed for the estimation of the rate, activation energy, heat, and affinity of staining of histological sections, and approximate results are given for the staining of mucin, mast-cell granules, chromatin, cytoplasmic ribonucleic acid (RNA), cartilage matrix, and other structures by azure A.

The half-staining time t is the time taken by a substrate under given staining conditions to achieve half the intensity of staining it would reach at equilibrium, and is approximately equal to the time taken to stain in a given, fairly dilute dyebath to the same intensity as at equilibrium in a dyebath of half the given concentration. The activation energy E of staining is given by E = ln t₍₁₎/t₍₂₎ x RT₁T₂/(T₂-T₁), where t₍₁₎ and t₍₂₎ are the half-staining times at absolute temperatures T₁ and T₂ respectively, and R is the gas constant. The activation energy of staining reflects the effect of temperature on rate of staining, and may be regarded as an index of substrate permeability. Half-staining times and activation energies of staining with azure A increase in the order mucin, mast-cell granules, chromatin, RNA, and interstitial cartilage matrix. Times of half-destaining and activation energies of destaining also are probably largely determined by substrate permeability. Differential staining dependent on differences in rate of staining may be enhanced by the use of chilled and stirred dyebaths, and by the use of dyes of large particle size.

The heat of dyeing H, sometimes regarded as the sum of the heats of formation of the various dye-substrate bonds, approximately equals RT₁T₂/(T₂-T₁)x ln [D]₁/[D]₂, where [D]₁ and [D]₂ are the concentrations of dyebath giving equal intensity of staining of the substrate at equilibrium at temperatures T₁ and T₂. Approximate figures for H in kcal/mole for staining with dilute azure A are: mucin, -8; chromatin and cartilage matrix, -7; cytoplasmic RNA, -5.5; mast-cell granules, - 2 to - 4. The higher the value of -H the more is staining inhibited by a rise in temperature of the dyebath.

The affinity of a dye for a substrate may be regarded as the standard free energy change accompanying the staining process, which under certain conditions is given approximately by F° = - RT ln /(1-)[D], where is the fraction of available staining sites in the substrate occupied by dye when the substrate is at equilibrium with a dyebath of concentration [D]. Differential staining of substrates with a high affinity for the dye is facilitated by the use of dilute dye solutions. Approximate values of F° for staining with azure A at 4° C and pH 4.0, in kcal/mole, are: cartilage matrix, -3.8 (orthochromasia) and - 5.3 (metachromasia); mast-cell granules, -4 (orthochromasia) and -4.4 (metachromasia); RNA, -3.1; mucin, between - 2.7 and -3.4; chromatin, -3.1; thyroid colloid, -2.3; Xenopus poison gland secretion, -2.3 It is suggested that part of the high affinity of sulphate groups for basic dyes is due to an increase in entropy during staining, resulting from dispersion of a large hydration shell surrounding the sulphate groups before attachment of the dye.