Different Sections Different Views

Anatomical characteristics that are used for identification of plant parts are located either on the surface or in the inner tissues of the plant part. If the structures are on the surface, as is typical of delicate leaves and floral structures, and the material is thin enough to allow for light to pass through, no sectioning is required and the sample can be cleared and viewed (see "Surface View" section). Materials rich in thermolabile compounds that can be destroyed when exposed to heat, such as mucilage and starch, are most often viewed as powders, for which

FIGURE 10.2 Softening and clearing of samples with chloral hydrate and boiling. (a) Use of chloral hydrate to soften root for sectioning; (b) section placed on a slide and passed over a flame for clearing; (c) softening relatively large pieces by boiling in chloral hydrate. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.2 Softening and clearing of samples with chloral hydrate and boiling. (a) Use of chloral hydrate to soften root for sectioning; (b) section placed on a slide and passed over a flame for clearing; (c) softening relatively large pieces by boiling in chloral hydrate. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

softening and sectioning are not necessary. For analyzing powders, a small amount of the powdered sample is applied to a slide and thoroughly mixed with water. The sample can then be viewed. In the case of thicker materials such as stems, rhizomes, roots, barks, fruits, seeds, and some leaves, sectioning is required. Figure 10.3 provides a schematic overview of how sections are prepared.

Three primary types of sections are used in microscopy: transverse, radial longitudinal, and tangential longitudinal sections (Figure 10.4). The types of sections and their applications to various plant parts are presented in Table 10.2. Transverse sections (also known as cross sections) are taken perpendicular to the long axis of the plant part—usually roots, barks, and stems. Radial

Plant parts

Whole or cut plant organs

Powder

Cellular structure

Surface view

Leaves, petals, sepals

Thick leaves, fruits, seeds, stems, cork

Inner structure

Leaves

Stems, rhizomes, roots, fruits, seeds

Thermolabile Cell contents (e.g. Starch, mucilage)

Roots, rhizomes, barks, fruits, seeds

Sectioning

Paradermal section

Cut Upper side

Cross section

Cross section, longitudinal section

Staining

Documentation

Storage

Drawings, photos *

Permanent mounting Glycerine-gelatin

Powder, any section, scraped fragments

Sectioning

Paradermal section

Cut Upper side

Cross section

Cross section, longitudinal section

Staining

Drawings, photos *

Permanent mounting Glycerine-gelatin

Water

Iodine, methylene blue

Drawings, photos

FIGURE 10.3 Schematic overview of the preparation, sectioning, mounting, staining, and storage of botanical microscopy samples. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

sections are taken parallel to the long axis of the plant part directly through the center of the stem (on radii). Tangential sections are also taken parallel to the long axis of the sample but are cut off the centerline (along a tangent) of the sample.

Each type of section affords the analyst a different anatomical view of the internal arrangement of tissues in a plant organ. Depending on the species and plant part, some views will be of more value in terms of providing diagnostic characters. After sectioning, most materials require sp

Transverse section

Radial longitudinal section

Tangential longitudinal section

Paradermal section f^VXfl_A_f^/V/L___-_A^-v/i" iV»_^_fl^M^

FIGURE 10.4 Cutting plane for preparing different sections; example of root showing cork (ck), cortex (c), secondary phloem (sp), and xylem (x). (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

Table 10.2 Types of Sections and Their Applications

Sample Preparation

Description of Preparation

Plant Organ

Surface view

A view of the surface of a whole, unsectioned plant organ. The object is thin enough for light to pass through and can be placed directly on the slide so that the surface may be viewed. Prepare with chloral hydrate solution.

Most useful for delicate plant parts such as thin leaves and flowers

Paradermal section

A view of the surface of a plant organ that has been sectioned parallel to the surface. The section is placed on a slide with the surface side up. Prepare with chloral hydrate solution for leaves; prepare with water for preservation of starch.

Most useful for thick leaves, fruits, and seeds, organs that often have diagnostic features on their surfaces, but are too thick to be viewed without sectioning

Transverse section (cross section)

The plant part is sectioned perpendicular to its main axis. Prepare with chloral hydrate solution and water for preservation of starch.

Most useful for bark, stems, rhizomes, roots, and fruits. Cross sections may also be important for some leaves. This view uniquely allows for the differentiation between stems, stolons, or rhizomes, on the one hand, and roots on the other hand, based upon the arrangement of the wood.

Longitudinal radial section

The plant part is sectioned directly through its center and parallel to its main axis. Prepare with chloral hydrate solution and water for preservation of starch.

Most useful for bark, stems, rhizomes, and roots. These sections reveal the details of elongated structures, such as vessels, tracheids, fibers, and secretory ducts, and illustrate the way that medullary ray cells cross these structures at right angles. They uniquely allow for the identification of the types of wall thickenings of tracheary elements and the differentiation between fibers and sclereids.

Longitudinal tangential section

The plant part is sectioned perpendicular to the longitudinal radial section and not through the center of the organ (i.e., tangential to its outer rounded surface). Prepare with chloral hydrate solution.

Most useful for examining the ray structure of the wood in roots because the characteristic lens-shaped medullary rays are well illustrated

clearing of pigments and cell contents (e.g., chlorophyll) with chloral hydrate solution in order for the structures to be viewed (see "Clearing and Mounting" section). Some materials also require the use of specific stains or reagents to be viewed (Table 10.3).

Surface Views If the plant organ is thin enough for light to pass through after preparation with chloral hydrate solution, and the diagnostic characteristics are present on the surface, then the sample can be viewed from the surface and no sectioning is necessary (Figure 10.5a). In such cases, the sample is laid flat directly on a glass slide and covered with chloral hydrate solution and a cover slip; the solution is heated to a boil (cleared). Different characteristics may be present on either side of the organ.

In order for both surfaces to be observed in a single viewing, the sample can be cut in half before or after boiling and one half turned over on the slide and placed next to the other. It is important to mark on the slide which half shows the upper surface and which shows the lower because it can be difficult to distinguish which surface is which with the naked eye. With leaves, the relative prominence of the vein can aid in this determination. In most leaves, the vein is more prominent on the lower side than the upper. Additionally, when the focus is directly beneath the upper epidermis of a leaf, the regular arrangement of the circular palisade cells will be discernible; when one is looking beneath the lower epidermis, the irregular spongy parenchyma will be apparent.

Paradermal sectioning: This form of sectioning is predominantly used to view the surface characteristic of thick leaves, fruits, and seeds. Most leaves and flowers are sufficiently thin that these characters can be viewed adequately

Table 10.3 Common Reagent Preparations

Reagent

Preparation

Purpose

Chloral hydrate solution

60 g chloral hydrate and 40 mL water are mixed (60% solution). Heating and stirring under a fume hood are required for dissolution (alternate solution: 50 g chloral hydrate in 20 mL water).

For clearing soft cell contents and making temporary mounts

Ether-ethanol

Equal parts ether and ethanol (96%)

Defatting and clearing (removal of fixed oils, fats, resins, volatile oils, tannins, chlorophyll)

Sodium hypochlorite

Dilute to strong aqueous solution of sodium hypochlorite (0.9-8.0%)

Bleaching of chlorophyll and dark-colored secretions

Glycerin gelatin

30 g of gelatin (Gelatina alba) is added to 120 mL cold water and stirred immediately with a glass rod. Let the mixture rest for at least 15 minutes without stirring to allow the gelatin to swell, then warm carefully (without stirring) in a water bath until the solution is clear. Intense heating and formation of air bubbles should be avoided. To the warm solution, add 150 g of 85% glycerin and 300 mg of a parabene mixture (three parts p-hydroxybenzoic methyl ester and two parts p-hydroxybenzoic acid propyl ester, dissolved in a sufficient amount of hot water). The solution should be clear. While it is still warm, pour into a glass vial for solidification. After the mixture cools, cap the vial for storage.

Permanent mountant

without further preparation. Some species, such as eucalyptus (Eucalyptus globulus) and senna (Senna alexandrina), have leaves that are too thick and coriaceous (leathery) to be viewed in surface view and therefore are best viewed by preparing a paradermal section (Figure 10.5b).

Paradermal sections of leaves are prepared by bending a piece of the sample around the tip of the forefinger or a round object, such as a pencil or pen, and securing it

a b with the thumb and middle finger. With a razor blade in the other hand, the epidermis of the sample is carefully and thinly sliced, with care taken not to cut into the finger (Figure 10.6). This procedure can be repeated so that both the upper and lower surfaces are sectioned and viewed by placing them next to one another on the same slide. Fruits and seeds are usually rigid enough that paradermal sections can be made by holding them in one hand while

FIGURE 10.5 Surface views of leaves with and without sectioning. (a) Surface view of Senna alexandrina leaf without sectioning; (b) surface view of Senna alexandrina leaf paradermal section. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.6 Preparation and view of paradermal sections. 1. Paradermal sections are used for samples too thick for surface view. 2. Bend sample around finger or pencil with epidermis up and secure with the thumb and middle finger (a and b). 3. With a blade in the other hand and taking care not to cut the finger, carefully and thinly slice the surface layer of the sample parallel to the surface of the epidermis (c-e). (a) Preparing paradermal section by wrapping a leaf around a finger; (b) preparing paradermal section by wrapping a leaf around a cylindrical object (e.g., a pencil); (c) preparing a paradermal section of Ginkgo biloba leaf; (d) preparing paradermal section of Achillea millefolium stem. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.6 Preparation and view of paradermal sections. 1. Paradermal sections are used for samples too thick for surface view. 2. Bend sample around finger or pencil with epidermis up and secure with the thumb and middle finger (a and b). 3. With a blade in the other hand and taking care not to cut the finger, carefully and thinly slice the surface layer of the sample parallel to the surface of the epidermis (c-e). (a) Preparing paradermal section by wrapping a leaf around a finger; (b) preparing paradermal section by wrapping a leaf around a cylindrical object (e.g., a pencil); (c) preparing a paradermal section of Ginkgo biloba leaf; (d) preparing paradermal section of Achillea millefolium stem. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

slicing parallel to the surface with the other hand. If the test sample is brittle, it should be softened with chloral hydrate solution prior to sectioning.

Methodologies for Preparing Sections There are three primary means of preparing transverse or longitudinal sections: (1) by hand with the naked eye, with or without a mount; (2) by hand using a stereomicroscope, with or without a mount; or (3) with a microtome. For quality control purposes and confirmation of identity and purity, handmade sections are typically of sufficient quality and much more time efficient than those produced using a microtome. All of the descriptions, drawings, and photomicrographs in the Atlas section of this text are of handmade sections. The skills necessary for proper sectioning come with practice. One of the most important considerations for preparing adequate sections is to ensure that the

e

FIGURE 10.6 (continued.) Preparation and view of paradermal sections. 1. Paradermal sections are used for samples too thick for surface view. 2. Bend sample around finger or pencil with epidermis up and secure with the thumb and middle finger (a and b). 3. With a blade in the other hand and taking care not to cut the finger, carefully and thinly slice the surface layer of the sample parallel to the surface of the epidermis (c-e). (e) Preparing paradermal section of Crataegus monogyna fruit; (f) surface view of Ephedra stem; (g) view of paradermal section of Ephedra stem. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.6 (continued.) Preparation and view of paradermal sections. 1. Paradermal sections are used for samples too thick for surface view. 2. Bend sample around finger or pencil with epidermis up and secure with the thumb and middle finger (a and b). 3. With a blade in the other hand and taking care not to cut the finger, carefully and thinly slice the surface layer of the sample parallel to the surface of the epidermis (c-e). (e) Preparing paradermal section of Crataegus monogyna fruit; (f) surface view of Ephedra stem; (g) view of paradermal section of Ephedra stem. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

section includes the entire range of structural elements from the surface of the material to its center.

Sectioning by Hand Hand sections are made simply by holding the moistened or softened plant part to be sectioned between the thumb and forefinger of one hand and, using the forefinger as a guide, cutting with the razor in the other hand. Hand sectioning is primarily used for materials that are of a relative hardness and size that allows for manipulating and cutting the sample by hand.

More delicate materials that are difficult to handle require a mount. It is important for the cutting edge to be sharp and to be drawn across the material to cut it—in contrast to pulling the razor through the sample—because this can distort the structures. When sections are prepared by hand, use of the stereomicroscope is optimal (Figure 10.7). The enhanced magnification enables the microscopist to produce a section that is sufficiently thin and of uniform thickness—attributes that are extremely important for clearly viewing anatomical characteristics.

FIGURE 10.7 Hand sectioning with a stereomicroscope. (Image courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

Sectioning Using a Mount The second method for preparing slides is by use of a mounting material or platform designed to hold the plant material steady and allow for a thin, uniformly sliced section to be made. Historically, pithy materials such as cork or the stems of elder (Sambucus spp.) or sunflower (Helianthus spp.) were used as mounts for sectioning of delicate leaves and flowers, and harder plant parts required the use of harder wood. Today, a commonly used mount can be made from a piece of polystyrene (described in detail in Figure 10.8). For sectioning, it is best to use protective single-edged razor blades (Figure 10.9). Brittle objects must be softened prior to sectioning. The optimal preparation of each sample will vary depending upon genus, species, and plant part and will be learned over time.

Sectioning Using the Microtome As an alternative to the manual sectioning techniques described, sections can be prepared using a microtome, which is a mechanical apparatus specifically designed for preparation of microscopy sections (Figure 10.10). There are both handheld and tabletop microtomes. The most important advantage of sections prepared with a microtome is that they are of well-defined and uniform thickness. The thickness of a section prepared in a microtome can vary between 1 and 10 ^m and is completely homogeneous. The preparation of such highly precise sections is important when well-defined and absolutely consistent photomicrographs are needed. However, this is not generally required for most practical purposes and the ability to develop sufficiently good sections by hand comes with practice.

The primary disadvantage with microtome sectioning is the time required, which can be from several days to even a few weeks. In this multistep process, the sample requires softening and clearing in various solutions (water, ethanol/ n-butanol, liquid and solid paraffin, etc.) and remains in them for several hours, followed by another multistep process for creating a permanent slide.

"Handheld" microtomes consist of a platform on which a sample can be fastened so that it protrudes above two flat surfaces at varying heights. A long blade is then slid across the even plane of the mounting surface to create a uniform section as thin as 0.005 mm. This type of microtome sectioning is very similar to use of a polystyrene mount; the former gives more uniform sections and the latter is more time efficient and sufficient in most cases.

Regardless of the technique used for sectioning, after sectioning, the sections can be stored in a dish of water to maintain their moistness and suppleness. They can then be transferred to a slide for staining, clearing, and viewing.

FIGURE 10.8 Sample preparation using a polystyrene mount. (a) Prepare a polystyrene mount as illustrated. The central vertical cut must be exactly at a right angle to the top plane; (b) the top plane must be narrow for slicing and the central vertical cut must be deep enough to spread open; (c) insert the sample (fresh parts or organs which were softened by boiling with chloral hydrate solution) to be sectioned into the mounting platform, allowing part of the tissue to protrude. The sample should be inserted exactly vertically in order to get a high-quality section of a vascular bundle; (d) if the sample is inserted at an angle, the examination of vascular bundles is impossible. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.8 Sample preparation using a polystyrene mount. (a) Prepare a polystyrene mount as illustrated. The central vertical cut must be exactly at a right angle to the top plane; (b) the top plane must be narrow for slicing and the central vertical cut must be deep enough to spread open; (c) insert the sample (fresh parts or organs which were softened by boiling with chloral hydrate solution) to be sectioned into the mounting platform, allowing part of the tissue to protrude. The sample should be inserted exactly vertically in order to get a high-quality section of a vascular bundle; (d) if the sample is inserted at an angle, the examination of vascular bundles is impossible. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

Clearing and Mounting the Test Sample After the plant part has been softened and prepared for surface view or sectioned, it must be mounted onto a microscope slide for clearing and viewing. Cells of plant parts contain air, which will cause the cell to appear dark and nontransparent, as well as a variety of contents including light-absorbing compounds such as chlorophyll, starch, mucilage, proteins, chloroplasts, resins, and volatile oils, to name a few. In order for the structure of the tissues to be visible, these contents must be cleared by mounting the sample in a fluid medium and then boiling, usually with chloral hydrate solution.

Similarly, dry objects cannot be viewed using a typical microscope with transillumination for the same reasons b a

FIGURE 10.8 (continued.) Sample preparation using a polystyrene mount. (e) Using a very sharp razor blade and squeezing the mount to stabilize the sample, carefully slice the sample by moving the blade evenly along the top flat surface of the mount. This portion of the sample is discarded. The sample is now ready to be sectioned; (f) make sure the top plane is at right angles to the sample at all times. If during sectioning the plane inclines, restore the correct angle; otherwise, a proper transverse section of even thickness cannot be made. If the sample is brittle or requires more softening, a drop of chloral hydrate solution can be placed on the cut edge; (g-j) holding the blade parallel to the surface of the mount, slice the sample through the mount with a continuous pulling movement parallel to the central slit. Taking care not to squeeze the object by avoiding movement of the blade directly toward the sample will provide the best sections. The preparation of thin sections requires sharp razor blades. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.8 (continued.) Sample preparation using a polystyrene mount. (e) Using a very sharp razor blade and squeezing the mount to stabilize the sample, carefully slice the sample by moving the blade evenly along the top flat surface of the mount. This portion of the sample is discarded. The sample is now ready to be sectioned; (f) make sure the top plane is at right angles to the sample at all times. If during sectioning the plane inclines, restore the correct angle; otherwise, a proper transverse section of even thickness cannot be made. If the sample is brittle or requires more softening, a drop of chloral hydrate solution can be placed on the cut edge; (g-j) holding the blade parallel to the surface of the mount, slice the sample through the mount with a continuous pulling movement parallel to the central slit. Taking care not to squeeze the object by avoiding movement of the blade directly toward the sample will provide the best sections. The preparation of thin sections requires sharp razor blades. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.8 (continued.) Sample preparation using a polystyrene mount. (g-j) Holding the blade parallel to the surface of the mount, slice the sample through the mount with a continuous pulling movement parallel to the central slit. Taking care not to squeeze the object by avoiding movement of the blade directly toward the sample will provide the best sections. The preparation of thin sections requires sharp razor blades; (k) apply the sliced section to a glass slide and push aside and discard all portions of the mounting platform that have also been sliced. To ensure that a high-quality section is obtained, prepare duplicate or triplicate samples. These can be laid side by side and compared. Turn half of the sample over so that both sides are viewed. The sample is now ready for clearing; (l) the clearing of the sections utilizes the same procedure as for clearing unsectioned plant parts. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.8 (continued.) Sample preparation using a polystyrene mount. (g-j) Holding the blade parallel to the surface of the mount, slice the sample through the mount with a continuous pulling movement parallel to the central slit. Taking care not to squeeze the object by avoiding movement of the blade directly toward the sample will provide the best sections. The preparation of thin sections requires sharp razor blades; (k) apply the sliced section to a glass slide and push aside and discard all portions of the mounting platform that have also been sliced. To ensure that a high-quality section is obtained, prepare duplicate or triplicate samples. These can be laid side by side and compared. Turn half of the sample over so that both sides are viewed. The sample is now ready for clearing; (l) the clearing of the sections utilizes the same procedure as for clearing unsectioned plant parts. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.9 Making transverse and longitudinal sections of stems, barks, rhizomes, or roots. (a) Preparing a transverse section of a thin, weak, herbaceous stem using a polystyrene mount; (b) preparing a transverse section of a rigid and woody root or rhizome by hand; (c and d) radial longitudinal section of a rigid and woody root or rhizome. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

and also because the cells are shrunken in a way that distorts their natural size and shape. Therefore, all specimens made from dry plants must be placed on the slide in a fluid mountant that will fill the air spaces and expand the cells to their normal size and shape. Once the cells have been moistened and their contents have been cleared, the cell structures and remaining characteristics (e.g., crystals) may be clearly viewed. Chloral hydrate solution serves as a reagent for clearing the object and as a mountant (Table 10.3).

Other mountants must be used in order to view ther-molabile cell contents that are destroyed by heating, such as starch (found in most roots), mucilage (found in some roots, leaves, barks, and seaweeds), or water-soluble particles such as sugars; all of these can serve as important diagnostic characters for some species and organs. The choice of mountant can have a great influence on how clearly defined the cells and tissues appear. The greatest definition is provided by a fluid with a refractive index different from the object being viewed. A lower refractive index is best because this puts the outline of the structure

FIGURE 10.9 (continued.) Making transverse and longitudinal sections of stems, barks, rhizomes, or roots. (e) Tangential longitudinal section of bark. Preparation using a mount may be preferable for such small pieces of bark (Caution: when sectioning, take care not to cut finger!); (f) transverse section of nonwoody root. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.9 (continued.) Making transverse and longitudinal sections of stems, barks, rhizomes, or roots. (e) Tangential longitudinal section of bark. Preparation using a mount may be preferable for such small pieces of bark (Caution: when sectioning, take care not to cut finger!); (f) transverse section of nonwoody root. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

being viewed on the side away from the object. The ratio of the refractive index of the object to the refractive index of the mountant should be approximately 1.06. Examples of this ratio in common mountants are chloral hydrate solution = 1.08 and glycerol = 1.06.

Clearing and mounting plant material by boiling with choral hydrate solution is the method most commonly used for viewing cell structure and the arrangement of cells in a sample (Figure 10.10). The prepared sample is placed on the microscope slide and a few drops of chloral hydrate solution are added. After it is covered with a glass cover slip, the slide is held over a flame or placed on a hotplate until the solution bubbles slightly. The procedure should be repeated until the sample appears cleared.

It is important that the object does not become dry. If necessary, additional drops of chloral hydrate solution can be placed under the cover slip (tends to introduce air bubbles) or along one edge (will be drawn under the cover slip by capillary action). The addition of a drop of glycerol (50-85%) to one edge of the cover slip on the cleared and

FIGURE 10.10 Microtome with test sample mounted in paraffin wax. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.11 Clearing and mounting a test sample by boiling with chloral hydrate solution. (a) Place a part of the plant organ (approximately 2-3 mm; in this example, a part of a leaf) or a section on a slide; (b) add a few drops of chloral hydrate solution to the sample; (c) place a cover slip onto the sample; (d) take care that the whole space under the cover slip is filled with chloral hydrate solution. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.11 Clearing and mounting a test sample by boiling with chloral hydrate solution. (a) Place a part of the plant organ (approximately 2-3 mm; in this example, a part of a leaf) or a section on a slide; (b) add a few drops of chloral hydrate solution to the sample; (c) place a cover slip onto the sample; (d) take care that the whole space under the cover slip is filled with chloral hydrate solution. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

cooled slide will prevent the crystallization of the chloral hydrate solution for several days (Figure 10.11). In the United States, chloral hydrate is a class IV controlled substance and its use requires a drug registration unit number from the Drug Enforcement Administration (DEA).

Mounting for the Detection of Thermolabile Cell Contents For the microscopic analysis of botanicals rich in thermolabile compounds such as starch or mucilage, at least two sets of slides need to be prepared: one to view the structures and the other to view the starch or mucilage. For viewing the structures, sections are prepared by boiling the sample in chloral hydrate solution as described. This will destroy the starch and mucilage, which are important characteristics of many species but interfere with the viewing of structures. For viewing starch, mucilage, or inulin, a separate set of plates must be made by preparing a powder of the sample in unheated water (Figure 10.12). If powder is not readily available or there is not enough material to make a powder, test material of sufficient thinness may be obtained by scraping or grating the dry plant parts.

e

FIGURE 10.11 (continued.) Clearing and mounting a test sample by boiling with chloral hydrate solution. (e) Hold the slide over the flame of a microburner until the chloral hydrate solution bubbles slightly; (f) take the slide away from the flame; after a few moments of cooling, repeat boiling until the bubbles and color are cleared; (g) if the chloral hydrate solution evaporates and air appears around the test sample, add more chloral hydrate along one edge of the cover slip and let it seep under until the whole area under the cover slip is filled. The sample must remain moist; (h) repeat boiling and cooling until the test sample appears translucent and free from air under the microscope (Caution: excessive boiling can cause the cover slip to fly off.). (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.11 (continued.) Clearing and mounting a test sample by boiling with chloral hydrate solution. (e) Hold the slide over the flame of a microburner until the chloral hydrate solution bubbles slightly; (f) take the slide away from the flame; after a few moments of cooling, repeat boiling until the bubbles and color are cleared; (g) if the chloral hydrate solution evaporates and air appears around the test sample, add more chloral hydrate along one edge of the cover slip and let it seep under until the whole area under the cover slip is filled. The sample must remain moist; (h) repeat boiling and cooling until the test sample appears translucent and free from air under the microscope (Caution: excessive boiling can cause the cover slip to fly off.). (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

If fresh material or soft plant parts that cannot be powdered require examination, then sections must be prepared. Sections are useful because they show the location of the starch (also mucilage or inulin) in the plant tissues. For many species, this can be diagnostic. The powder or scraped material is placed on a microscope slide, a few drops of water are added without heating, and the mixture is stirred to free a greater amount of starch granules or mucilage from the cells. After placing the cover slip, the slide is ready for staining, which is required for the observation of starch and mucilage. For example, iodine solution stains starch dark blue, often making it visible to the naked eye, and a solution of methylene blue stains mucilage dark blue against a pale blue background (Figure 10.13). Table 10.3 lists other reagents that are used.

Defatting The powders of oily seeds such as flax may need to be defatted prior to microscopic examination

FIGURE 10.11 (continued.) Clearing and mounting a test sample by boiling with chloral hydrate solution. (i) After clearing, remove the cover slip and cut the sample in half if a surface view is being done. Turn one half over so that both surfaces can be viewed on a single slide (note: the leaf can also be cut prior to clearing); (j) replace the cover slip over the test sample; (k) add chloral hydrate solution along one edge of the cover slip until the whole area under the cover slip is filled (Caution: excess chloral hydrate solution should be cleaned from the slide to prevent it from coming into contact with the microscope objectives because it can damage them over time.); (l) to prevent crystallization of the chloral hydrate, a few drops of a glycerin-water solution (50-85%) can be applied at one edge of the cover slip. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.11 (continued.) Clearing and mounting a test sample by boiling with chloral hydrate solution. (i) After clearing, remove the cover slip and cut the sample in half if a surface view is being done. Turn one half over so that both surfaces can be viewed on a single slide (note: the leaf can also be cut prior to clearing); (j) replace the cover slip over the test sample; (k) add chloral hydrate solution along one edge of the cover slip until the whole area under the cover slip is filled (Caution: excess chloral hydrate solution should be cleaned from the slide to prevent it from coming into contact with the microscope objectives because it can damage them over time.); (l) to prevent crystallization of the chloral hydrate, a few drops of a glycerin-water solution (50-85%) can be applied at one edge of the cover slip. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

in order to view their cell structure better. Materials with high concentrations of oil can cause the droplets to form a layer that obstructs viewing of the structures. Similarly, it is sometimes best to defat oily materials prior to powdering because fatty powders are difficult to powder and sieve. A mixture of equal parts ether and ethanol (96%) can be used to remove fixed oils, fats, resins, volatile oils, tannins, or chlorophyll (Table 10.3). Material that is difficult to powder can be scraped or grated in lieu of being powdered.

Bleaching Some herbaceous stems, barks, and woods (as well as chlorophyll in leaves) contain dark-colored secretions that restrict visibility of cell structures. These sometimes require bleaching in addition to clearing. For bleaching, a solution of sodium hypochlorite is added to the

FIGURE 10.12 Preparation of powdered plant parts. (a) On a slide, add a small amount of powder to a few drops of chloral hydrate solution or water, depending on what structures are being viewed; (b) stir to make a moist powder; (c) place a cover slip over the sample, then carefully clear the slide in the flame of a microburner. To view starch and mucilage in water, do not heat. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

FIGURE 10.12 Preparation of powdered plant parts. (a) On a slide, add a small amount of powder to a few drops of chloral hydrate solution or water, depending on what structures are being viewed; (b) stir to make a moist powder; (c) place a cover slip over the sample, then carefully clear the slide in the flame of a microburner. To view starch and mucilage in water, do not heat. (Images courtesy of Prof. Dr. Reinhard Länger, AGES PharmMed, Vienna, Austria.)

section until bleaching is completed, after which the reagent is removed and the section is washed. Prolonged bleaching in the reagent can cause the removal of starch and lignin, which is not desirable. If chloral hydrate solution is used for clearing and mounting, bleaching is not necessary.

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Responses

  • Darcey Jamieson
    Why is it that chloral hydrate solution is mount to a prepared radial longitudinal section?
    11 months ago
  • jacopo dellucci
    Can trichomes be found in stem barks?
    19 days ago

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