MECHANISM OF CONNECTIVE-TISSUE TECHNIQUES .1. THE EFFECT OF DYE CONCENTRATION AND STAINING TIME ON ANIONIC DYE PROCEDURES

Anionic dye connective tissue procedures were performed by staining fo r 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) phosphomolyb dic acid (PMA). The staining obtained with dyes in SPA was classified as selective (no cytoplasmic staining), moderately selective (traces o f cytoplasmic staining) and non-selective (all other staining patterns ). The staining of collagen and cytoplasm with dyes in PMA was separat ely classified on a scale of 1-5 (1 = no staining, 5 = maximum stainin g). The selectivity of the staining obtained with SPA with solutions o f 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 con stant (SAC) defined as the (number of dye sulphonic acid groups/dye mo lecular weight) x 10(3). Correlation with molecular weight was poor an d 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), gr oup 2 (selective to moderately selective when staining was performed f or 5 min), and group 3 (non-selective). The SAC of the group 1 dyes di ffered significantly from those of the group 2 and 3 dyes. Selectivity was essentially lost at dye concentrations of 0.018 M. The staining w ith acidic dyes (no amines or substituted amines) in PMA differed sign ificantly (p < 0.001) from that obtained with amphoteric dyes (contain ing basic substituents). In general, acidic dyes stained cytoplasm. Am photeric dyes with the exception of indigocarmine stained collagen. Ho wever, most of these dyes also stained cytoplasm. In contrast to the r esults obtained with dyes in SPA, selectivity correlated strongly with molecular weight and only poorly with the SAC. Staining time and dye concentration affected selectivity only when the acidic dyes were used for 5 min at concentrations of 0.0018 M and 0.018 M. The data obtaine d do not permit a clear distinction between the rate control and chemi cal affinity models for the mechanism of staining with anionic dyes. H owever, it seems possible that different groups of dyes stain by diffe rent mechanisms.