Hematoxylin Harris Stain

Hematoxylin stains are commonly employed for histologic studies, often employed to color the nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or colored complexes (dye-mordant-tissue complexes), the color of which will depend on the salt used. Aluminum salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.

The three main alum hematoxylin solutions employed are Ehrlich’s hematoxylin, Harris’s hematoxylin and Mayer’s hematoxylin. The name “haemalum” is preferable to “hematoxylin” for these solutions because haematein, a product of oxidation of Hematoxylin, is the compound that combines with aluminum ions to form the active dye-metal complex. Alum hematoxylin solutions impart to the nuclei of cells a light transparent red stain which rapidly turns blue on exposure to any neutral or alkaline liquid.

Alum or potassium aluminum sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH of water to form insoluble aluminum hydroxide. In the presence of excess acid, aluminum hydroxide cannot be formed thus failure of aluminum hematoxylin dye-lake to form, due to lack of OH ions. Hence, acid solutions of alum hematoxylin become red. During staining alum hematoxylin stained sections are usually passed on to a neutral or alkaline solution (e.g. hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminum haematin complex. This procedure is known as “blueing”.

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing hematoxylin, a tap water substitute consisting of 3.5 g NaHCO3 and 20g MgSO4X7H2O in one liter of water with thymol (to inhibit formation of molds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10 degrees Celsius for blueing sections may even produce pink artifact discolorations in the tissue.

The straining of nuclei by hemalum does not require the presence of DNA and is probably due to binding of the dye-metal complex to arginine-rich basic nucleoproteins such as histones. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes is prevented by chemical enzymatic extraction of nucleic acids. Such extractions do not prevent staining of nuclei by hemalum.