Leaf Clearing Protocol to Observe Stomata

Figure 1. Leaf clearing and fixing. The ethanol:acetic acid solution fixes the tissue and the tissue becomes transparent.

Leaf Clearing Protocol to Observe Stomata and Other Cells on Leaf Surface

by Sharma N. (2017)

In bio-101 2017-09-05 – DOI: 0.21769/BioProtoc.2538

https://bio-protocol.org/bio101/e2538

Figure 2. Washing the tissue. Cleared tissue (A, B) is washed in 1 N KOH (C, D). Tissue is washed in water (E, top slide) and placed in Hoyer’s solution (E, bottom slide).

Abstract

In this protocol, leaves are cleared and fixed in an ethanol and acetic acid solution, and mounted in Hoyer’s solution.

Figure 3. Slide preparation. Individual leaves form a precipitate when they are placed in Hoyer’s solution.

The cleared leaves are imaged under differential interference contrast (DIC) microscope. This protocol is beneficial for studying stomata, hair cells, and other epidermal cells in plants.

Figure 4. Same leaf imaged with Brightfield or DIC filters. DIC image (B) gives better resolution than Brightfield (A) to observe different cell types. Arrows: stomata (young and mature).
Figure 5. Quantification of stomata. Number of stomata (X) and pavement cells (Y) are counted to calculate stomatal index.

Published by

Willem Van Cotthem

Honorary Professor of Botany, University of Ghent (Belgium). Scientific Consultant for Desertification and Sustainable Development.

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