Question.

Many stains are acidified, but some are adjusted to a neutral or even an alkaline pH. Why? Are different dyes differently affected by pH changes?

Answer.

For a full answer to your question you will need to refer to a textbook of histological techniques. Here is a simplified answer. It applies to basic (cationic) and acid (anionic) dyes with fairly small molecules. Attraction of opposite electric charges plays a major part in staining by such dyes.

The structural macromolecules in a section of a tissue have numerous side-chains that can form either positive or negative ions.

Acid dyes (attracted to positive sites in tissue).

The positive ions are associated mainly with proteins.

The side chain of the amino acid arginine (a guanidino group) is a strong base. That means it always carries a positive charge, even at a high pH. It can therefore always attract a negatively charged dye ion. At pH 9 or above, all staining by a simple basic dye (biebrich scarlet is commonly used) is due to arginine.

The other organic group that can form positive ions is the amino group, which occurs at the N-terminus of every chain of amino acids and on the end of the side-chain of lysine. Amino groups are weak acids: at high pH they are not ionized, but at low pH an amino group collects a hydrogen in (proton) from the solvent and becomes positively charged. The amino group of lysine can collect a proton even when there are not many around, as in a neutral or slightly alkaline medium. Consequently, lysine behaves as a cation and binds acid dyes at pH about 8 or below. N-terminal amino groups are weaker acids: they cannot be protonated much above pH 6, so they are not stained by neutral or alkaline solutions of acid dyes. More and more amino groups become protonated (ionized) as the pH is lowered. Staining with an acid dye therefore occurs more rapidly and more strongly from the more acid solutions. At a pH around 2, these dyes stain everything.

The foregoing remarks apply to a "typical" acid dye with sulfonic acid side-chains. Sulfonic acids are strong acids; they exist in solution only as sulfonate anions. (Eosin is not "typical" in this way because it is a salt of a weak acid. Moreover eosin solutions must not be acidified too much or insoluble unionized eosin will be precipitated, leaving a colorless solution.)

Basic dyes (attracted to negative sites in tissue).

The three negatively charged chemical groups present in a section are:

1. Sulfate (actually half-sulfate) of many carbohydrate components (glycoproteins in some mucus, heparin in mast cell granules, chondroitin sulfates in cartilage matrix, etc.) These are strong acids: ionized even at low pH. Sulphate groups therefore bind cationic (basic) dyes at any pH. They are the only things stained at pH 1.

2. Phosphate groups, associated with DNA and RNA. These are weak acids, so they become protonated (not ionized) if the concentration of protons (hydrogen ions) is high enough. Typically this occurs below about pH 2.5. The phosphates of nucleic acids are fully ionized at pH 3.5 to 4. A basic dye at pH 3 to 4 stains nuclei, cytoplasm that is rich in RNA and. of course, all the sites of half-sulfate esters.

3. Carboxyl groups. These occur as parts of amino acids (C-terminal and the side-chains of glutamic and aspartic acid), sialic acids (mucus and other glycoproteins), glycosaminoglycans of extracellular matrix carbohydrates (hyaluronic acid, chondroitin sulfates etc) and free fatty acids (frozen sections only). Carboxyl groups ionize over quite a wide range of pH, from 5 up to about 8. The higher the pH, the stronger and more rapid the staining by a basic dye. At or above pH 8 it stains everything.

Alkaline solutions of basic dyes are used for staining semi-thin plastic sections. With anything thicker the color is too dark to show structural details. For more selective staining, basic dyes are applied as acidic solutions. At pH 1 only the sulfated materials are displayed. As the pH rises from 2.5 to 4.5, nuclei and RNA stain with increasing speed and intensity.

Remember that these simplified arguments do not apply to all dyes, or even to those most commonly used in routine work.

Further reading.

Horobin, R.W. (1982). Histochemistry: An Explanatory Outline of Histochemistry and Biophysical Staining. Stuttgart: Gustav Fischer.
Kiernan, J.A. (1999). Histological and Histochemical Methods: Theory and Practice, 3rd ed. Oxford: Butterworth-Heinemann.
Horobin, R.W. (1988). Understanding Histochemistry: Selection, Evaluation and Design of Biological Stains. Chichester: Ellis Horwood.
Lyon, H. (1991). Theory and Strategy in Histochemistry. A Guide to the Selection and Understanding of Techniques. Berlin: Springer-Verlag.

John A. Kiernan
Department of Anatomy & Cell Biology
University of Western Ontario
London, Canada.
(kiernan[AT]uwo.ca)