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

Mayer's and Gill's hematoxylins


I would like to know the differences between two types of hematoxylin: Mayer's and Gill's.

Answer 1.

Haematoxylin dye concentration for Mayer is 1 gm/L compared with 2 gm/L for Gill-I. The preservative for Mayer's is chloral hydrate and for Gill it is ethylene glycol. The acidifying agent for Mayer's is citric acid, whereas for Gill it is acetic acid.

Both have very good shelf lives of two years or more under correct storage conditions. They both are used mainly as progressive stains, and are well suited to use as counterstains as well. Gill-I has some some strong adherents for progressive cytology staining.

It is possible to make either of these in a non-toxic formulation without compromising performance or shelf life.

Mike Rentsch

Answer 2.

Both stains are hemalums: they are solutions containing hematein (from oxidized hematoxylin), an aluminium salt (the "mordant," which forms dye-metal complexes with hematein), an organic acid to adjust the pH, and a hydrophilic compound (glycerol, ethylene glycol or chloral hydrate). The last ingredient is variously said to modify the solubilities of other ingredients, retard the oxidation of hematoxylin, "preserve" the solution or do nothing at all. In most hemalums the hematein is generated by adding enough of an oxidizing agent (most often the iodate ion) to oxidize about half the hematoxylin. The unoxidized hematoxylin provides a reservoir from which more hematein is slowly produced by atmospheric oxidation. This compensates for the atmospheric over-oxidation of hematein to trioxyhematein (which is useless), thereby prolonging the life of the solution.

The compositions of Mayer's and Gill's hematoxylins are set out below. Mayer's recipe was published in 1863, that of Gill, Frost and Miller in 1974. Gill's hematoxylin closely resembles "haematal-16," a mixture published by J. R. Baker in 1962 that contained ethylene glycol but no organic acid.

   MAYER                                          GILL

   Hematoxylin        1 g                     Hematoxylin       2 g
   Potassium alum   50 g (0.09M )      Al sulfate           17.6 g (0.03M)
   Sodium iodate     0.2 g                  Sodium iodate    0.2 g
   Citric acid           1 g                     Acetic acid         40 ml
   Chloral hydrate    50 g                   Ethylene glycol    250 ml
   Water to make   1000 ml               Water to make   1000 ml

   Molar ratio of Al ions to haematein molecules in the freshly made solution:
                                 Mayer: 32
                                 Gill:     11

A high ratio of aluminium:dye slows down staining and increases the selectivity for nuclei. Both these hemalums are used progressively; in principle, Gill's should stain more quickly than Mayer's. The effect of excess aluminium is seen most strikingly with Ehrlich's hematoxylin, which is saturated with alum and relies on atmospheric oxidation (slow) to provide a  low concentration of hematein from an initially large (6 to 7 g/L) reservoir of hematoxylin. Ehrlich's hematoxylin is the slowest of the progressive hemalum stains (up to 30 minutes, compared with 3 to 10 minutes for Mayer's or Gill's). Hemalums for regressive nuclear staining (e.g. Delafield's, Harris's) have lower aluminium:dye ratios than the progressive stains. Acid-alcohol extracts the dye-metal complex more slowly from nuclei than from other components of tissues.

Some references. These are for practical, rather than chemical or theoretical (i.e. speculative) aspects of hemalum staining.

Baker, J.R. (1962). Experiments on the action of mordants. 2. Aluminium-haematein. Quarterly Journal of Microscopical Science 103: 493-517.
Bancroft, J.D. & Cook, H.C. (1984). Manual of Histological Techniques. Edinburgh: Churchill-Livingstone.
Bancroft, J.D. & Stevens, A., eds. (1996). Theory and Practice of Histological Techniques, 4th ed. London: Churchill-Livingstone.
Ehrlich, P. (1886). Die von mir herruhrende Hamatoxylinlosung. Zeitschrift fr wissenschaftliche Mikroskopie 3: 150.
Kiernan, J.A. (1999). Histological and Histochemical Methods: Theory and Practice, 3rd ed. Oxford: Butterworth-Heinemann.
Llewellyn, Bryan. Stains File.

J. A. Kiernan
Department of Anatomy,
The University of Western Ontario,
LONDON, Canada  N6A 5C1