Phosphotungstic Acid-Hematoxylin; Spectropho-Tometry of the Lake in Solution and in Stained Tissue

Job's method of continuous variation (Ann. Chim., ser. 10; 9, 113-203, 1928) was applied to tungstate-hematein and molybdate-hematein lakes. For phosphotungstic acid-hematein (PTA-Hn), sodium tungstate-hematein, or molybdic acid-hematein, it demonstrates that each forms a single complex in solution, with ratios of composition (PTA to Hn) 1:2, 1:2 and 1:1 respectively. PTA-Hn was the best stain, as judged by its ability to differentiate collagenous connective tissues from muscle, and to delineate nuclear and cytoplasmic detail. Hematein is used in place of hematoxylin because a reproducible, potent staining solution can be prepared without lengthy aging periods or uncertain oxidants. With this lake, staining is independent of fixation and obviates the necessity of mordanting procedures prior to staining. Microspectrophotometric evidence indicates that the blue component of the well-recognized polychromasia of these lakes is a true example of bathochromic metachromasia. Staining by the lake at various pH values and by acid and basic dyes after PTA treatment of tissues indicate that PTA-Hn is an acid lake dye and therefore different from the iron and aluminum hematoxylin lakes. Observations on the distribution of metachromasia suggest that it is topographically related to tissue components having a high content of basic protein.