Differential Staining of Aborted and Nonaborted Pollen

TLDR

Acidifying the staining solution with glacial acetic acid is essential for distinguishing green pollen walls from red protoplasm. The authors prepared a single staining solution containing ethanol, malachite green, water, glycerol, phenol, chloral hydrate, acid fuchsin, orange G, and glacial acetic acid, adjusting the acid volume according to pollen wall thickness and accelerating staining by flaming or heating, resulting in green aborted pollen and red nonaborted pollen. The stain proved durable, lasting about a month, and was effective for small, lightly colored nondehiscent anthers in both angiosperms and gymnosperms, distinguishing aborted (green) from nonaborted (red) pollen.

Abstract

A single staining solution was made by compounding it in the following order (dyes were from British Drug Houses): ethanol, 10 ml; 1% malachite green in 95% ethanol, 1 ml; distilled water, 50 ml; glycerol 25 ml; phenol, 5 gm; chloral hydrate, 5 gm; acid fuchsin 1% in water, 5 ml; orange G, 1% in water 0.5 ml; and glacial acetic acid, 1–4 ml. For best results in differentiation to give green pollen walls and red protoplasm, the staining solution should be acidified with glacial acetic acid. The amount of acid to be added depends upon thickness of the pollen walls: for very thin-walled pollen, 1 ml; for moderately thin walls, 2 ml; and for thick-walled or spiny-walled pollen, 3 ml of acid. For pollen inside non-dehiscent anthers, 4 ml of acid should be used. Staining is hastened by flaming the slide (for loose thin-walled pollen) or by immersing thick-walled pollen or anthers for 24–48 hr at 50 C. In the typical stain, aborted pollen grains are green; nonaborted, red. The method is useful for pollen inside nondehiscent anthers if these are small and not too deeply coloured naturally. The stain is very durable, especially if the coverslips are sealed with param wax. The staining solution will keep well for about a month. It is useful both for angiosperms and gymnosperm microgametes.