Plant microtechnique

The microscopic analysis of herbal drugs is necessary for the characterization and identification of species, with major implications in quality control, forensic investigation and identification of archaeological materials, among other uses.

Preparation of specimens

Sectioning

The safety razor blade can be used to produce sections which are thin enough for study under magnifications of 100-400x. As each section is cut it will slide onto the knife blade, lubricated by 50% alcohol solution. The section can be examined temporarily in water (a 50% glycerin solution will be needed if the section is meant to be kept for several months). The sections can be transverse, longitudinal and tangential.

Peeling

The epidermis of most leaves can be readily removed by the scraping method. First place a square of leaf on the slide (keeping the leaf irrigated with a few drops of sodium hypochlorite). Then hold the leaf with a cork and scrape gently with a razor blade. Finally trim off the thin part, transfer to a dish with tap water and brush off loose cells. Then it will be ready to be mounted on a slide. With some materials it is unnecessary to scrape the leaf. In these cases the peeling is done by folding the leaf to break the surface, and either stripping directly, pulling one part of the leaf downwards relative to the other, or by holding as thin a layer of the surface as possible between forceps and peeling it back.

Clearing

When the study of sections with their inclusions is finished, another observation will be conducted, with "cleared" sections. Sections can be cleared by transferring them from a dish of 50% alcohol solution to a dish of water with a brush or fine forceps. Then they are placed in a petri dish containing undiluted sodium hypochlorite (household bleach).The time needed for cell contents to dissolve away is usually of 5 minutes, and can be determined by visual inspection. After immersion in the bleach, the sections are thoroughly washed in water (it is recomended to use fine forceps instead of a brush because its bristles will dissolve).

Staining

There are several staining combinations that may be used to enhance details within sections. Sections of freshly cut material should be washed gently, to remove cell debris which will obscure details once the section has been stained. Specific stains are prescribed for particular characters:

Reagents used for the microscopic examination:

1% aqueous methylene blue: All cell walls turn blue, except cutin or cutinized walls which remain unstained.

Phloroglucin/HCl: The phloroglucin is added to the section, and then the diluted HCl. Lignin turns red.

Sudan III: is a reagent for triglycerides detection, which turns them red.

Lugol’s solution: is a reagent for starch detection, which turns them blue or violet.

Rutenium red: Mucilage and some gums turn pink. Sections can be mounted directly in the stain.

Identification of microscopic botanical characters

The following are some particular features and the families where they can be found. Not all members of the families listed will necessarily show the features mentioned, but the features regularly occur where indicated.

Leaf

Anisocytic stomata: Brassicaceae, Plumbaginaceae.

Anomocytic stomata: Berberidaceae, Capparaceae, Liliaceae, Polygonaceae, Ranunculaceae

Diacytic stomata: Acanthaceae, Caryophyllaceae.

Latex-containing cells: Apocynaceae, Convolvulaceae, Papaveraceae.

Laticifers, articulated: Papaveraceae, Euphorbiaceae.

Laticifers, non-articulated: Apocynaceae, Asclepiadaceae.

Paracytic stomata: Juncaceae, Magnoliaceae, Poaceae, Rubiaceae.

Peltate hairs: Bombacaceae, Elaeagnaceae, Oleaceae

Sclereids: Margraviaceae, Oleaceae, Theaceae, Trochodendraceae.

Silicified hairs: Poaceae.

Stinging hairs: Euphorbiaceae, Loasaceae, Urticaceae.

Stem

Cluster crystals: Bombacaceae, Cactaceae, Chenopodiaceae, Malvaceae, Rutaceae, Tiliaceae, Urticaceae.

Cystoliths: Cannabinaceae, Moraceae, Urticaceae.

Raphides: Balsaminaceae, Dilleniaceae, Liliaceae, Margraviaceae, Rubiaceae.

Solitary crystals: Flacourtiaceae, Mimosaceae, Papilionaceae, Rutaceae, Tamaricaceae

Superficial cork: Apiaceae, Asteraceae, Corylaceae, Fagaceae, Labiatae, Meliaceaae, Proteaceae.

Softwood

Resin ducts (axial): Picea, Pinus (Gymnosperms)

Use of monographs and micrographs

Monographs may contain schematic drawings of the main microscopic features of powdered drugs, which are useful in the comparison and identification of specimens.

Further reading

Ruzin, S. E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York.

References

Ray, F. E., 2006. Esau’s Plant Anatomy. Wiley & Sons, New Jersey.