Mechanism of connective tissue techniques. I: The effect of dye concentration and staining time on anionic dye procedures

Anionic dye connective tissue procedures were performed by staining for 5 min and 24 h with (a) 0.00018 M and 0.0018 M solutions of 28 dyes, and 0.018 M solutions of 21 dyes in saturated picric acid (SPA), and (b) 0.0018 M and 0.018 M solutions of 20 dyes in 1% (w/v) phosphomolybdic acid (PMA). The staining obtained with dyes in SPA was classified as selective (no cytoplasmic staining), moderately selective (traces of cytoplasmic staining) and non-selective (all other staining patterns). The staining of collagen and cytoplasm with dyes in PMA was separately classified on a scale of 1-5 (1 = no staining, 5 = maximum staining). The selectivity of the staining obtained with SPA with solutions of dyes at concentrations of 0.00018 M and 0.0018 M, and both staining times, was correlated (p < 0.001) with an empirical sulphonic acid constant (SAC) defined as the (number of dye sulphonic acid groups/dye molecular weight) x 10(3). Correlation with molecular weight was poor and was significant only when staining was performed with 0.00018 M dye solutions for 24 h. The dyes were divisible into three groups: group 1 (selectivity independent, or almost independent of staining time), group 2 (selective to moderately selective when staining was performed for 5 min), and group 3 (non-selective). The SAC of the group 1 dyes differed significantly from those of the group 2 and 3 dyes. Selectivity was essentially lost at dye concentrations of 0.018 M. The staining with acidic dyes (no amines or substituted amines) in PMA differed significantly (p < 0.001) from that obtained with amphoteric dyes (containing basic substituents).