Does Methylene Blue Penetrate Into Living Cells?

Abstract

Experiments with dyes tend to favor the theory that the penetration of a basic dye into living cells depends on the fact that the dye has an apparent dissociation constant, and that in the form of free base, which predominates at higher pH values, it penetrates very readily, while in the form of salt it penetrates so slightly that its entrance may be neglected.

According to this theory, methylene blue, which behaves like a strong base, 1 should not penetrate, since it exists in the form of a salt. But the literature is full of statements that living cells are stained by methylene blue. No one, however, has heretofore tested the dye which has actually entered a living cell from a solution of methylene blue. Until this is done, we are in no position to conclude that the dye found inside a living cell is methylene blue, rather than one of its lower homologues (such as trimethyl thionin or “Azure B”, which is less basic), since it is very difficult 2 to obtain methylene blue absolutely free from such dyes (especially at higher pH values where some of it changes very readily to the other forms).

Previously, the writer 3 has found that unless there was injury or contamination of the sap from the deeply stained cell wall, the vacuole of the living cell of Valonia took up practicatlly no dye 4 from methylene blue dissolved in sea water at pH 5.4, while it took up more at pH 9. Chloroform behaved similarly, in that the higher the pH value of the aqueous solution, the more dye was taken up by the chloroform.