Sectioning and Staining Pathological Phloem

Abstract

A technique is described for simultaneously staining numerous batches of nonembedded sections of pathological material. Staining baskets containing sections are placed in a Büchner funnel which is agitated by a motor, and stains and rinses are quickly changed by draining the funnel. Three staining techniques are described for pathological phloem. For studying sections, the most useful of the three is a combination of hematoxylin and lacmoid. Material is fixed 24-48 hr in Randolph's “Craf”, a modified Navashin's solution. Sections are mordanted in 4% ferric ammonium sulfate, washed several times in water, stained progressively with 0.006% aqueous hematoxylin, washed in tap water, stained 15-72 hr in 0.1% lacmoid, rinsed in tap water, washed 10 min in 1% NaHCO₃, dehydrated in 30%, 70%, and anhydrous tertiary butyl alcohol, cleared in clove oil and mounted in Canada balsam. Sieve plate callose and lignin stain blue, cellulose walls of living cells stain purplish black, and walls of nonliving cells stain from brown to purplish black. The vacuoles of some ray cells stain red. For colored photomicrographs, sections stained with a mixture of Congo red and lacmoid, and then dehydrated and mounted as indicated above are excellent for demonstrating sieve plate callose. Lacmoid alone is very useful for readily detecting callose distribution.