Observations on histological methods involving the use of phosphotungstic and phosphomolybdie acids, with particular reference to staining with phosphotungstic acid /haematoxylin

An attempt has been made to elucidate some of the factors involved in differential staining of tissues with Mallory’s phosphotungstic acid /haematoxylin, and to find out how far these factors are applicable to other histological methods in which phosphotungstic or phosphomolybdic acid is used. Sections have been subjected to various pretreatments to find whether specific chemical groupings are responsible for any of the staining reactions, but the results obtained are not always easy to interpret. The most important single factor in determining the staining reaction of a tissue material with PTAH appears to be its relative permeability to the two colour-complexes of the PTAH mixture. The red complex, of larger molecular size, penetrates collagen; muscle-fibres are penetrated mainly by the smaller blue complex, while red bloodcorpuscles fixed with formaldehyde are not penetrated by either. The staining reactions of muscle and red blood-corpuscles can be altered by a methylation procedure, by treatment with performic acid or formic acid, or by mild alkaline hydrolysis; but this appears to be due to alteration of permeability rather than to chemical alteration of any specific dye-binding groups. The effects of blocking reactions indicate that the binding of both complexes is to basic groups in the tissues, though it is possible that hydroxyl groups and carboxyl groups may also be involved. It appears that similar factors control the differential staining with other techniques which involve the use of complex acids (Baker, 1958), and that the chemical specificities which have been claimed for some of them are not well founded.