Quarterly Journal of Microscopical Science, Vol s3-103, 477-492, Copyright © 1962 by Company of Biologists

Ionic and Non-Ionic Bonds in Staining, with Special Reference to the Action of Urea and Sodium Chloride on the Staining of Elastic Fibres and Glycogen

¹ Medical School, University of the Witwatersrand, Hospital St., Johannesburg, S. Africa

1. A powerful hydrogen bonding agent such as urea may affect staining in several ways:

(a) by competing for hydrogen bonding sites in the tissue or on the dye particle, it may inhibit staining in which the dye-substrate link is a hydrogen bond;

(b) because it is a dipole, urea may have some affinity for charged sites in the tissue, and thus tend to reduce electrostatic staining;

(c) urea may in some cases decrease staining by increasing the solubility of the dye;

(d) by inhibiting association of dye particles in solution, where this is due to hydrogen bonding, urea may sometimes facilitate penetration and staining of dense substrates.

2. Sodium chloride may inhibit staining due to electrostatic forces by ionic competition, but may also decrease staining in some cases by aggregating and precipitating the dye.

3. Prolonged exposure to hot acidic methanol may not only block acidic groups, but also block or destroy non-acidic groups, including the 1,2-glycol group.

4. Urea inhibits the staining of all substrates by Best's carmine. The staining of glycogen is inhibited also by methylation and by periodic acid, but not by deamination or sodium chloride. Staining of mucin and nuclei is inhibited by methylation and deamination, while that of mucin is also inhibited by periodic acid and sodium chloride. It is suggested that the staining of glycogen and mucin by Best's carmine is by hydrogen bonding on to 1, 2-glycol groups, the attachment requiring in the case of mucin stabilization by an ionic linkage with some strongly basic group. The staining of nuclei may also be by hydrogen bonding, on to an unknown group.

5. The staining of elastic fibres by aldehyde-fuchsin, resorcin-fuchsin, orcein, chlorazol black E, and luxol fast blue is inhibited by urea. This is consistent with the role of hydrogen bonding in elastic staining suggested by previous workers.

6. The staining of mast-cell granules and cartilage capsules by aldehyde-fuchsin is inhibited by salt, while that of elastic fibres and interstitial matrix is inhibited by urea. It is suggested that aldehyde-fuchsin contains:

(a) large dye cations which selectively stain negatively charged substrates of high permeability, such as mast-cell granules and cartilage capsules, and

(b) smaller dye particles which stain elastic fibres and the dense interstitial cartilage matrix, by hydrogen bonding.

7. Sodium chloride inhibits the staining of mast-cell granules and cartilage matrix by orcein, but not that of nuclei or elastic fibres. In the presence of urea, dilute orcein stains nuclei selectively, apparently by electrostatic attraction reinforced by a linkage of unknown nature.

8. Urea and phthalate both decrease the staining of mucin by a number of basic dyes, but increase the staining of certain substrates, especially by alcian blue. It is suggested that both substances inhibit staining in which hydrogen bonding is important, and increase the staining of dense substrates by large-particled dyes by inhibiting aggregation of the dye particles and thus facilitating penetration.