Identification of Cauose by its Diachrome and Fluorochrome Reactions

Abstract

Methods for callose deposits can be applied to both living and fixed plant tissue, because the stains required are relatively nontoxic and may be added to incubating media. For fixed preparations, killing must be rapid. Tissue is placed in alcohol-acetic acid (3:1 v/v) cooled to —74 C in a Pyrex beaker by solid CO2. After natural warming to room temperature, the fixative is removed by washing in water. Sections are prepared freehand or with a cryostat microtome. Paraffin embedding is permissible if sections are dewaxed and hydrated before staining. Staining is done either with resorcinol blue (light microscopy) or aniline blue (fluorescence microscopy). Each staining method is usually specific but callose is more confidently identified when shown by both methods. The resorcinol blue is specially prepared, thus: Dissolve 3 gm of resorcinol in 200 ml of distilled water, add 3 ml of conc. NH4OH, and heat 10 min on a steam bath. Stopper the flask with a plug of cotton. When a dark blue color appears, in about 6 hr, reheat for 30 min, filter into an evaporating dish, and heat again until NH3 cannot be detected. For staining, dilute 3 drops of this solution with 10 ml of tap water or with a neutral buffer solution. Callose becomes cobalt blue in 1-2 min. For fluorescence, stain sections 5-10 min in 0.005-0.01% water-soluble aniline blue (National Aniline 161, Cert. No. NK-10) dissolved in M/15 K2HPO4. Callose fluoresces bright yellow in ultraviolet or blue light. Sections stained by either method are mounted in a medium made as follows: Add 150 gm of powdered gum arabic and a few crystals of thymol to 100 ml of 35% aqueous K-acetate. Let stand 2-3 days, then dilute with enough K-acetate solution to obtain a honey-like syrup, and add a large crystal of thymol. Check the pH, as the medium must be slightly alkaline.