Common Staining Techniques and Laboratory Exercises Cell shape and orientation of certain structures within a cell can be helpful

scaly bark (e.g., Pinus, Pyrus). Other genera (e.g., Vitis, Clematis, Lonicera) form continuous cylinders of periderm called “ring bark.” Still other plant species are intermediate between the scaly and ring barks (e.g., Arbutus, Platanus, and Eucalyptus).

Commercial cork is produced from many trees, in particular, Quercus suber. Cork is valued because it is impenetrable to gas and liquid and has strength and elasticity and is lightweight. Stripping the periderm from a 20-year-old tree is the first step in forming commercial cork. The exposed cells of the phelloderm and cork die, and a new phellogen is rapidly formed as a wound response. Within 10 years, this second phellogen is ready for harvest.

This process continues at 10-year intervals until the tree is approximately 150 years old. The dark brown spots that may be noted within commercial cork (e.g., wine bottle corks) are lenticels, isolated areas in the periderm consisting of suberized and nonsuberized cells.

3.3 Common Staining Techniques and Laboratory Exercises Cell shape and orientation of certain structures within a cell can be helpful in classification of a species. In order to learn about plant anatomy and specific plant structures within the plant body plan, it is important to take a practical approach. In the following sections we describe examples of inexpensive laboratory exercises that can be performed as training for forensic botanists.

3.3.1 Epidermis

This is a surface tissue of young plants and herbaceous plants that lack lateral meristems. The epidermis may only be one cell layer in thickness.

1. Obtain a leaf from Tradescantia (Spiderwort) or Zebrina (Wandering Jew).

2. Tear the leaf at a 90° angle to the veins. Look for a small region of the lower (purple) epidermis at the edge of the tear.

3. Using forceps and a clean razor blade, make a wet mount of this purple tissue with a drop of water on a microscope slide. Place a coverslip on top of your sample. A drop of methylene blue may be added to increase the contrast between the cell types.

4. First examine the slide under low magnification (4X or 10X). Two types of cells should be present: epidermal cells and guard cells. Guard cells occur in pairs and surround the stomata. The epidermal cells should form an interlocking layer or sheet of cells with the guard cells interspersed in pairs throughout the layer.

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3.3.2 Trichomes

Specialized epidermal cells (trichomes) appear as “hairs” on the surface of the plant epidermis. They can be one cell or multicellular, and are useful features for species classification. Some trichomes reflect light as an optical illusion for insects or contain stinging chemicals for protection from predators.

1. Examine leaves of a Coleus plant and a tomato (Lycopersicon) plant under a dissecting microscope. Locate the trichomes. Carefully record their structure, noting the number and shape of the cells as well as the spacing on the leaf surface.

3.3.3 Periderm

In 1665, Robert Hooke described the cellular nature of cork, which comprises the most abundant part of the periderm in woody plants.

1. Prepare a dry mount of a very thin section of cork tissue. Cork samples may be obtained from supermarket wine bottles. The section does not have to be very large, in fact, smaller than a millimeter is sufficient. Do not add water or a coverslip.

2. Observe the tissue with both low (4X) and then high (100X) magni-fication. Note the uniform size and general appearance of the cork cells. These are dead cells with no cytoplasm. What are observed under the microscope are components of a waxy cell wall.

3.3.4 Collenchyma

Collenchyma consists of living elongated cells that have an uneven thickness to their primary cell walls. They are usually arranged in strands in stems and petioles. Collenchyma cells are classified as either “angular” or “lamellar” by their patterns of cell-wall thickening. Angular collenchyma has cell walls in which the corners are extra thick (e.g., celery). Lamellar collenchyma has thick-enings on both the inner and outer cell walls (e.g., tomato and sunflower).

1. Cut a thin section of a celery petiole and place on a microscope slide without a coverslip. Observe under the dissecting microscope. What different cell types are distinguishable?

2. Stain the section with toulidine blue for one minute. Rinse the excess stain and observe again under the dissecting microscope. Describe what you see.

3. Under low magnification (10X), locate the small clusters of cells on each of the ridges along the outer portion of the petiole. These are the strands of collenchyma tissue (e.g., strings). Do not misinterpret collen-chyma for the vascular bundles located in the interior of the section.

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4. Peel a strand of collenchyma from the petiole. How much tension is required to break the strand?

3.3.5 Sclerenchyma

Sclerenchyma serves as structural support of stems, leaves, and other plant parts that are no longer elongating. Sclerenchyma cells are generally dead at maturity, and there are two common types of sclerenchyma cells: sclereids and fibers.

1. Prepare a wet mount of a small amount of the fleshy part of a pear fruit. The small, slightly hard cells that you notice when you eat a pear are clusters of sclereids (groups of a few cells or perhaps 30 cells).

2. Prepare a second wet mount of pear tissue and stain for one minute with phloroglucinol, a highly acidic stain that reacts with lignin.

Observe the stained specimen for sclereid cells again. What is their general shape and thickness?

3.4 Summary

The accurate forensic identification of different plant species is often depen-dent on knowledge of plant anatomy and different plant cell types. Botanical evidence may be significant, as in a large wood chip that is readily identifiable by wood grain and texture. However, fragments of plant material may be extremely small and may require thorough microscopic examination to identify, first, what plant part it is. Second, species identification may be possible if an appropriate reference guide is available for comparison against the evidentiary sample. Simple staining procedures and a basic recognition of cellular patterns could be the key step for solving the case. In addition, further forensic research studies can be performed to aid in establishing good pictorial reference guides for local plant material for the different quadrants of the U.S.

General References

Bruce, D.M., Mathematical modelling of the cellular mechanics of plants, Philos.

Trans. R. Soc. Lond. B. Biol. Sci., 358, 1437, 2003.

Fahn, A.H., Plant Anatomy, 3rd edition, Pergamon Press, New York, 1982.

Flurer, C.L., Crowe, J.B., Wolnik, K.A., Detection of adulteration of locust bean gum with guar gum by capillary electrophoresis and polarized light microscopy, Food Addit. Contam., 17, 3, 2000.

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Friedman, W.E., Cook, M.E., The origin and early evolution of tracheids in vascular plants: integration of palaeobotanical and neobotanical data, Philos. Trans.

R. Soc. Lond. B. Biol. Sci., 355, 857, 2000.

Genard, M., Fishman, S., Vercambre, G., Huguet, J.G., Bussi, C., Besset, J., Habib, R., A biophysical analysis of stem and root diameter variations in woody plants, Plant Physiol., 126, 188, 2001.

Jagels, R., Visscher, G.E., Lucas, J., Goodell, B., Palaeo-adaptive properties of the xylem of Metasequoia: mechanical/hydraulic compromises, Ann. Bot., 92, 79, 2003.

Niklas, K.J., The influence of gravity and wind on land plant evolution, Rev. Palaeobot.

Palynol., 102, 1, 1998.

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