The estimation of sugar concentration in individual sieve-tube elements by negative staining

The presence of water-soluble compounds in sectioned plant tissue can be visualized in negative contrast by freeze-substitution in acetone, followed by embedment in Epon containing 6% Sudan B. The contents of mature sieve tubes and companion cells of bean (Phaseolus vulgaris L.) showed strong, and mostly uniform, negative staining. The degree of negative staining was measured by microspectrophotometry. Since, in sieve tubes, virtually all of the solute is the translocated sugar, the sugar concentration can be estimated by comparison with similar measurements made on sections from pith blocks which were infiltrated with sugar solutions and processed by the same procedures. Sieve tubes contained a solution of about 11.2% (w/v) sucrose; companion cells contained a similar concentration of sucrose. Negative staining, and therefore the sucrose concentration, in immature sieve-tube elements and companion cells was much less than in their mature counterparts.