Histology FAQ

Staining, Histochemistry and Histotechnology

(Frequently Asked Questions)


Dr. John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada



FAQ Home > Staining Methods, Histochemistry


Methyl blue and methylene blue


A method calls for methyl blue, in a mixture with eosin Y. The nearest name I can find on a bottle is methylene blue. Will it be OK to use it instead?


No! The only thing these two dyes have in common is a blue color. Otherwise they have opposite staining properties.

Methyl blue, an acid triphenylmethane dye, is one of the components of aniline blue. Aniline blue is a generic name that includes methyl blue (C.I. 42780; Acid blue 93) and water blue or ink blue (C.I. 42755; Acid blue 22). Most dyes that are sold under these names are mixtures of both dyes, but some are mostly methyl blue. A contaminant known as sirofluor is also present in these dyes, and is exploited in fluorescent stains for callose in plants. In staining applications any dyes sold as aniline blue, methyl blue and water blue are interchangeable, provided that the batch meets the Biological Stain Commission's standards in respect of content of reducible blue dye and performance in standardized staining procedures.

Methyl blue (aniline blue) is used in Mann's eosin-methyl blue method and in various trichrome stains such as Mallory's, Gomori's, Cason's and Heidenhain's AZAN. It colors collagen fibers and a few other materials.

Methylene blue (C.I. 52015; Basic blue 9) is a basic thiazine dye. It may have more scientific uses than any other dye. As a simple stain, applied from a mildly acidic solution (pH 3 to 4) it colors nucleic acids and acidic carbohydrates. At neutral or alkaline pH is colors everything. Methylene blue is used in conjunction with eosin and other dyes in stains for blood cells and parasites, and it is also extensively used in bacteriology. Products of degradation (demethylation or "polychroming") of methylene blue are essential components of the commonly used Romanowsky-Giemsa stains for blood cells. The purple coloration of leukocyte nuclei and magenta color of malaria parasites seen with Wright's and Giemsa's stains, are due to one of these products, the dye known as azure B (C.I. 52010).

Methylene blue (and some other thiazine dyes) can provide beautiful and selective staining of the living neurons and their cytoplasmic extensions, and has been much used to demonstrate the innervation of peripheral tissues. Methyl (aniline) blue cannot be used in this way.

Reference:  Conn's Biological Stains. Entries under the various named dyes.

John Kiernan
London, Canada