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.2013 May 7;1(5):apps.1300016.
doi: 10.3732/apps.1300016. eCollection 2013 May.

An improved clearing and mounting solution to replace chloral hydrate in microscopic applications

Affiliations

An improved clearing and mounting solution to replace chloral hydrate in microscopic applications

Thomas S Villani et al. Appl Plant Sci..

Abstract

Premise of the study: This study presents Visikol™, a new proprietary formulation that can be used as an efficient replacement for chloral hydrate as a clearing agent for microscopic examination. In the United States, chloral hydrate is regulated and therefore difficult to acquire. •

Methods and results: Fresh and dry samples of the following plants: ginger (Zingiber officinale), maté (Ilex paraguariensis), lime basil (Ocimum americanum), oregano (Origanum vulgare), and mouse-ear cress (Arabidopsis thaliana), were cleared using Visikol or chloral hydrate solution and compared using a light microscope. •

Conclusions: This new method can be used successfully to clear specimens, allowing identification of diagnostic characteristics for the identification of plant materials. Visikol is as effective as chloral hydrate in providing clarity and resolution of all tissues examined. Tissues become transparent, allowing observation of deeper layers of cells and making it effective in research, botanical and quality control, and for educational applications.

Keywords: Visikol; acidified chloral hydrate; botanical quality control; clearing; histology; microscopy.

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Figures

Fig. 1.
Fig. 1.
Light micrographs of dry and powdered botanical specimens cleared with chloral hydrate (left column) and with Visikol (right column). (A–F) Ginger rhizome. Annular vessel element and fibers (A, B); abundant starch grains in rhizome (C, D); thin-walled parenchyma cells (E, F). (G–J) Maté leaves. Leaves, upper epidermis with underlying palisade cells, large and closely packed (G, H); lower epidermis surface showing anomocytic stomata and circular cuticular striations (I, J).
Fig. 2.
Fig. 2.
Light micrographs of fresh, whole-mounted specimens cleared with Visikol. (A, B) Basil leaf. Epidermis with diacytic stomata, capitate and peltate glands (A); mesophyll cells with chloroplasts (B). (C–F) Oregano leaf. Covering trichomes with thick cell walls over the vein and capitate glands (C); close-up capitate glands (arrow) (D); epidermis and peltate oil gland (E); mesophyll cells (F). (G, H)Arabidopsis thaliana root. Root tip cellular differentiation (G); xylem differentiation in root (arrow) (H).
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References

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    1. Gardner R. O. 1975. An overview of botanical clearing technique. Stain Technology 50: 99–105 - PubMed
    1. Haseloff J. 2003. Old botanical techniques for new microscopes. BioTechniques 34: 1174–1182 - PubMed

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