Autoclaving as an aid in the clearing of plant specimens.

Numerous recipes exist for the clearing of plant tissues prior to examination as whole mounts (see annotated bibliography by Lersten 1967). Broadly speaking, these methods may be classified into 3 groups; mild, drastic and very drastic procedures, depending upon the severity with which the tissue is extracted by the clearing procedure. For some purposes mild procedures are all that are needed for high-quality microscopy of the cleared specimen. Solvent extraction of pigments with aqueous ethanol, followed by infiltration with a liquid of appropriate refractive index (e.g. chloral hydrate, xylene, Farrant’s medium, methyl benzoate, methyl salicylate, or glycerine) would be an example. In other cases, slightly more drastic action (e.g. lactic acid or lacto-phenol at 60 C or 1% KOH at room temperature) is required, especially if the tissue is rich in starch. In many cases, very drastic procedures (5% KOH for several days, followed by chlorine water bleaches to remove phenols) are necessary. In all cases, specimens rarely clear adequately in less than 48 hours, and difficult specimens may take several weeks. In 1968, Bevege introduced the use of the autoclave to clear roots infected with vesicular-arbuscular mycorrhizas, reducing the time required to 30 min. We have found that autoclave treatment of leaves produces similar results, and suggest the following treatments.