Quantitative observations on the kinetics and mechanisms of binding of electron stains to thin sections through hen erythrocytes
Authors: Richardson W. D. , Davies H. G. 
Published: April 2021

ABSTRACT
The kinetic studies described in this paper have enabled us to define the staining conditions which lead to occupation of all available binding sites in sections through biological material. In a hypothetical section containing stainable regions the concentration, c, of bound stain in any thin layer is a function of its distance, x, from the surface in contact with the staining solution and the staining time t. Theoretical considerations indicate that there are 2 extreme types of kinetics depending on the relative values of 2 diffusion or migration rates : r1 that of the staining solution into the depth of the section, and r1 that of the solution into the stainable regions. When r1&gt; &gt;r2, called type A kinetics, penetration of stain throughout the depth of the section is ‘instantaneous’ and binding to stainable regions is slow. Two families of curves can be constructed, each member of which has a particular value of t: first c-vs-x curves and second, derived from them by integration, E-vs-d curves, where E is the electron-scattering density of a stained region relative to clear resin and d is section thickness. When r1&lt; &lt;r2 called type B kinetics, the staining solution diffuses relatively slowly into the section, and all the binding sites in each stainable region are occupied ‘instantaneously’. Similarly there are 2 families of curves, c-vs-x and E-vs-d, but of different shapes. When r1∼r2 there is an intermediate type AB kinetics. At any point on an E-vs-d curve the slope is proportional to the concentration of bound stain. The penetration time, tp, is defined as the time taken for the concentration of the staining solution to reach the same value at the bottom of a 100-nm section as at the top surface in contact with the solution.
Models for the cytoplasm and chromatin bodies of erythrocytes are proposed, based on their physical structure and on interferometric determinations of the fractional volumes occupied by resin, biological material and free space. Staining mechanisms can be explained in terms of diffusion or migration through a system of interconnecting spaces comprising the resin, as well as a second, hydrophilic, pathway connecting the 2 surfaces of the section, arising from long threads of DNA-histone.
Experimentally, E-vs-d curves are derived by treating single grids, each carrying a series of sections of increasing thickness, for increasing times. E is measured directly in the microscope with a Faraday cage. Sections have been treated at 20° and at 60 °C with aqueous and, or, ethanolic solutions of (a) magnesium uranyl acetate or uranyl acetate, either alone or followed by Pb-citrate; (b) PTA; and (c) KMnO,. From the E-vs-d curves upper and lower limits for tp have been tabulated. Also tabulated are values of E for sections too nm in thickness, at equilibrium when all available sites are filled. E/100 nm is approximately proportional to the concentration of bound stain per unit volume. The kinetics of stain uptake are discussed in terms of the models. The anomalous binding of KMnO4 and ethanolic PTA previously reported have beeh explained and the significance of our data for the interpretation of electron micrographs is also discussed.