One-bath trichrome staining: investigation of a general mechanism based on a structure-staining correlation analysis

Dye pairs of contrasting colours were selected from acid dyes of varied chemical characteristics. The 44 dye pairs were investigated in a one-bath trichrome staining system in which the dye-baths were strongly acid. Dye concentrations, concentration ratios and staining times were varied for each dye pair. Thirty dye pairs stained collagen fibres distinctly different colours to muscle cytoplasm, while 14 dye pairs gave muddy, non-selective staining. Comparison of dye structures showed that in selective pairs the larger dye always stained the collagen fibres, with cytoplasm being coloured by the smaller species. With 28/30 of the selective dye pairs the differences in anionic weights of the dyes was greater than 200. However, in dye pairs giving non-selective staining, the anionic weights of the members of 13/14 of the dye pairs differed by less than 200. As no other structural feature correlated so clearly with selectivity, it was concluded that the selectivity of one-bath trichromes is diffusion-rate controlled, involving the interaction of differentially permeable tissue sites (collagen being more permeable than muscle cytoplasm) with dyes diffusing at different rates (large dyes slower than small). In keeping with this, lengthening staining times reduced staining selectivity. The rate control mechanism suggested a rational trouble-shooting guide for one-bath trichromes, encompassing such practical factors as dye concentration, embedding medium, fixative, dye-bath pH, section thickness and staining time.