We have devised methods for the separation and isolation of the important indicator constituents of litmus, azolitmin, and erythrolitmin, with a view to employing them as vital stains. Analysis of the color intensities of these dyes shows slight differences in them, azolitmin being the weaker pigment, weight for weight. Study of a third coloring matter, erythrolein, which exists in litmus has shown it to be an unsatisfactory indicator, and toxic for animals.

Analyses with the spectrophotometer of the absorption of light by erythrolitmin and azolitmin, prepared by our methods, and tested over a wide acid-alkali range, show them to be pure substances, comparable in this respect with synthetic indicators.

The errors in the interpretation of the indicator phenomena on vital staining, which are incident to changes in the concentration of the dyes, are so slight as to be negligible. The salt and protein errors on the other hand are large. The factors responsible for the Donnan equilibrium fail to influence the distribution of the indicators between fluid and gelatin.

Erythrolein was found useless when employed for vital staining, and azolitmin proved unsatisfactory since it colors poorly and is toxic. But erythrolitmin can be used to great advantage. It is readily absorbed, and in non-toxic doses stains intensely. The range of pH at which it changes from red to blue fits it for the demonstration of changes in the reaction of living tissues. By reason, however, of the salt and protein errors to which it is liable, the pH cannot be accurately ascertained.

Intravital staining with erythrolitmin yields results similar to those following injection of purified "whole litmus."

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