Orcein is extracted from two species of lichens, Rocella tinctoria and Lecanora parella. Orcein also is available in a synthetic form, but the natural form is preferred for chromosome analysis, because it gives better contrast. Orcein is used in form of a 1% solution in 45% acetic acid. This solution is prepared by pouring 55 mL boiling glacial acetic acid over 1 g orcein powder. The solution is cooled, 45 mL of distilled water added, and filtered. This solution is unstable and should be prepared fresh before use.
Aceto-orcein staining does not require the addition of iron ions. The staining prodedure is similar to the aceto-carmine method. Fixed material is transferred for an approprioate time to 1% aceto-orcein and then analyzed by the squash technique.
Drain off the fixative and place the roots in 1% acetocarmine for 1 to 3 h. Heat until the acetocarmine begins to boil. Cut off the root cap with a razor blade and squeeze the meristematic tissue out with a lancet needle. Add a drop of acetocarmine or 45% acetic acid. Place a razor blade (double-edged) to one side and add a cover slip. Tap the cover slip gently with the needle end of a probe. Slide the razor blade out and heat to a point just below boiling (steam will form beneath the slide). Then, quickly squash with thumb or forefinger between two layers of filter paper. Be careful to not move the cover slip at this point.