Materials Included In Kit

Additional Materials Required

Prelab Preparation

Plants

1. Plant red bean seeds at least two weeks in advance. You may also use the seedlings from the Transpiration Laboratory Kit, AP^® Biology Lab 9A, or purchase plants, such as vinca, chrysanthemum or coleus.
2. Remove leaves and stem sections from the plants just before they will be used in this activity.

Melted Paraffin (just prior to lab)

1. Pour 50 mL of distilled water into a 250-mL beaker.
2. Place the 250-mL beaker on the hot plate.
3. Pour the paraffin into the 100-mL beaker.
4. Place the 100-mL beaker into the 250-mL beaker.
5. Heat the hot plate at a low setting to melt the paraffin—do not boil the wax.
6. Place the beaker of melted paraffin in a common area for student use.

Safety Precautions

Ethyl alcohol is toxic by ingestion due to the presence of a denaturing agent. Glycerin may cause an allergic irritation reaction to skin and eyes. Contact with strong oxidant may cause an explosion. Toluidine Blue O Solution is moderately toxic by ingestion. Hot paraffin wax may cause minor skin burns. Razor blades are very sharp—use extreme caution when handling the razor blade. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Follow all normal safety precautions. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Ethyl alcohol and glycerin may be disposed of according to Flinn Suggested Disposal Method #26b. Toluidine Blue O Stain and paraffin may be disposed of according to Flinn Suggested Disposal Method #26a. Razor blades may be disposed of according to Biological Waste Disposal method Type V.

Lab Hints

• Enough materials are provided in this kit for 8 groups of students. The laboratory activities can reasonably be completed in one 50-minute class period. The students should read the Background section and the entire Procedure before coming to lab, and the data compilation and calculations can be completed after the lab.
• Ensure that students label all materials with their group number.
• Plants that work well in this lab include vinca, chrysanthemum, coleus and beans.
• Students may use the bean plants from the Transpiration Laboratory Kit—AP^® Biology Lab 9A. Ensure the leaf and stem sections are free of petroleum jelly residue.
• To ensure that clean pipets are not confused with used pipets, tape a test tube to the side of each solution bottle. Place a clean pipet into each test tube for student use.
• Toluidine blue O (TBO) is a blue aqueous dye that binds to negatively charged groups on organic compounds. However, different anionic groups form different bonds with the dye creating different colors. For example, pectin will stain red or reddish purple; lignin stains blue; phenolic compounds stain green to blue-green.
• Staining can be done on the microscope slide if the stem cross-sections are very fragile. Place the slide on paper towel to prevent damage to the benchtop.

Teacher Tips

• The structure of the stem can be studied by placing stalks of celery in water containing red food dye. The food dye moves quickly up the stalk and leaves a red-colored trace of its movement. The celery stalks are firm enough that free-hand razor sections can be made for microscopic analysis without further preparation or staining. The stalks can also be split lengthwise to show the pathway of xylem up the stalk.
• Extend the lab by having students compare their slide preparations to the Roots and Stems—Compariset Prepared Microscope Slides (Flinn No. ML1419).
• Extend the lab by having students prepare cross-sections of monocot stems, such as corn seedlings.
• Extend the lab by having students prepare leaf impressions of several types of leaves including the tops of plants that float on top of water.

Sample Data

Answers to Questions

1. Were most of the stomata open or closed in your preparation? Propose a likely reason why the stomata are either open or closed.

   If the stomata are mostly closed the plant may have been exposed to a strong light source, a breeze, or it may be dehydrated. Open stomata indicates that conditions are favorable for photosynthesis.

2. What is the function of the guard cells? How does the function of the guard cells differ from that of epidermis cells?

   The guard cells open and close the stomata regulating transpiration and photosynthesis. Epidermis cells function in plant protection from infection and from evaporation through the majority of the plant’s outer surfaces.

3. How does the structure of each type of cell relate to its function?
   

   Epidermal cells secrete a waxy cuticle to prevent water loss. Guard cells swell and deflate to open and close pores for transpiration. Parenchyma cells contain chloroplasts for photosynthesis. Sclerenchyma cells are dead and hollow to allow water to be pulled upward to the leaves. Collenchyma cells have perforated end walls to transport sugars, amino acids and small molecules throughout the plant.

4. Describe the appearance of the vascular tissue. Does it have a pattern?

   Vascular tissue appears as veins within plant roots, stems and leaves. In dicots, xylem and phloem form a bundle that forms a ring when viewed as a cross-section in the roots or stems.

5. Where within the stem is the vascular tissue located? How might this location be beneficial to the plant?

   Vascular tissue is located in the center of the roots, stems and leaves. Water and nutrients are protected from evaporation in the interior of the plant.

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

How are plants organized? Do they have tissues, organs or organ systems?

Concepts

• Phytonomy (plant anatomy)
• Meristem tissue
• Ground tissue
• Plant physiology
• Dermal tissue
• Vascular tissue

Background

Plants are complex organisms with multiple levels of cellular organization. Plants have two organ systems: the shoot system and the root system (see Figure 1). The shoot system is above ground and includes organs, such as leaves, buds, stems, flowers and fruits. The root system includes those parts of the plant below ground and includes organs, such as roots, tubers and rhizomes.

Both the shoot and root organ systems are composed of four different types of tissues; meristem, dermal, ground and vascular tissues. Meristem tissues are very specialized areas within the root tips and shoots of plants. Within the meristem tissues, plant cells undergo mitosis to form new cells which then differentiate to form one of several types of cells that comprise the remaining plant tissues. Apical meristems are located at the root tips and shoot tips. Apical meristems are responsible for root growth and plant height. Cambiums are meristems located within plant stems and leaves that provide lateral growth (girth) to the plant.

Cells that differentiate into dermal tissue will cover the outer surface of the plant. The primary cell type in dermal tissue is epidermal cells. The function of the epidermal cells is to secrete a waxy cuticle that helps prevent water loss and acts as a barrier to infections. Another cell type in dermal tissue is the guard cell which controls water loss through the opening and closing of pores on the leaves called stomata (singular = stoma or stomate). Thousands of stomata occur on the underside of a typical dicot or on the upper surface of a plant whose leaves float on water. By adjusting the size of the opening the guard cells control the rate of CO₂ and O₂ uptake and the loss of water by the leaf. Therefore, the guard cells control the rate of photosynthesis in the leaf by controlling the diffusion of CO₂ into the cells where it is needed for photosynthesis. Guard cells also contain a few chloroplasts and perform photosynthesis. The guard cells swell when they are full of water, opening the stoma into air spaces that surround the middle layer of leaf cells which are ground tissue.

Ground tissue is the primary component of the plant body. The three most common cell types in ground tissue are; parenchyma, collenchyma and sclerenchyma cells. The function of the parenchyma cells is storage and photosynthesis. Palisade parenchyma cells are elongated cells located in leaves just below the epidermal tissue (see Figure 2). Mesophyll cells are located in the middle of the leaf (meso = middle, phyll = leaf). Mesophyll cells are a type of parenchyma cell with an irregular shape that provide surface area for a thin coating of water necessary for transpiration. As a result, mesophyll cells are often described as “spongy.” Collenchyma and sclerenchyma cells both function in plant support and fluid transport. Collenchyma cells are alive when mature and have a thick outer cell wall. While sclerenchyma cells are dead when mature and have a thick inner cell wall.
The last type of plant tissue is the vascular tissue, which is often called the vascular bundle (see Figure 3). Vascular tissue is subdivided into two types: xylem and the phloem. The cambium is an area sandwiched between the two types of vascular tissue. Xylem is composed of specialized sclerenchyma cells with some parenchyma cells. Some of the sclerenchyma cells are the typical supportive fibers like those found in the ground tissues. The remaining specialized sclerenchyma cells conduct water and minerals from the soil up through the stem and into the leaves. These conductive sclerenchyma cells are called either tracheids or vessels depending upon their morphology. Tracheids are long and tapered with thick walls ending in angled end walls that connect cell to cell. Tracheids do not have cytoplasm, and conduct fluids through perforations in the end walls or through pits in the side walls (see Figure 4). Vessels occur only in flowering plants (called angiosperms). These are thick-walled, dead, hollow cells, with pitted side walls. Unlike tracheids, however, vessels lack end walls. They are also typically much larger in diameter than tracheids and are therefore responsible for most of the water movement in the plant. Phloem functions in the transport of sugars, amino acids, and other small molecules from the leaf to the rest of the plant. Phloem is composed of specialized collenchyma cells. Some of the collenchyma cells serve as support fibers. There are two specialized collenchyma cell types in phloem: sieve-tube cells and companion cells. Sieve-tube cells are long, with thin walls, perforated end walls (called sieve plates) and have no membrane bound organelles (no nucleus, mitochondrion or chloroplast). Since sieve-tube cells do not have a nucleus or other organelles, these cells depend on an adjacent companion cell for their cellular needs. The companion cell has a nucleus and other organelles which provide cellular materials for itself and the sieve-tube cell. Nutrients and wastes are exchanged through strands of cytoplasm called plasmodesmata that extend from one cell to the other through the sieve plates.

Biological stains are used to enhance the contrast of cellular structures to be viewed using microscopes. When applied to freshly prepared specimens many general stains adhere to structures within the cell that were exposed by the preparation techniques. Most stains bind to the organic compounds within the cell and each stained molecule acquires one color, such as iodine, which stains starch a blue-black color. A few stains, called polychromatic dyes, produce different colors depending upon the type of organic compound they bind to within the sample. Sclerenchyma cells appear green to blue-green when stained with Toluidine blue O (TBO). Parenchyma and collenchyma cells appear reddish-purple when stained with TBO. TBO stains sieve tubes and companion cells purple.

Experiment Overview

After doing this laboratory, you should be able to:

• Identify plant cells and tissues and describe their functions.
• Make thin sections of stem, identify xylem and phloem cells; and relate the function of these vascular tissues to the structures of their cells.

In Activity 1, an epidermal peel of a leaf will be created and examined to determine the morphology of a stoma.

In Activity 2, a cross-section of a stem will be prepared and analyzed in order to examine the interior features of a plant stem.

Materials

Safety Precautions

Ethyl alcohol is toxic by ingestion due to the presence of a denaturing agent. Glycerin may cause an allergic irritation reaction to skin and eyes. Contact with strong oxidant may cause an explosion. Toluidine Blue O Solution is moderately toxic by ingestion. Hot paraffin wax may cause minor skin burns. Razor blades are very sharp—use extreme caution when handling the razor blade. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all normal safety precautions.

Procedure

Activity 1. Stoma Morphology

1. Remove a leaf from a plant.
2. Holding a piece of clear tape by one edge, press the tape onto the underside of the leaf.
3. Quickly remove the tape from the leaf.
4. Mount the tape on a microscope slide, gently pressing the tape onto the slide.
5. Place the preparation onto the microscope’s stage and focus on the torn edge of the leaf using low power.
6. Rotate to medium power and focus on the leaf’s edge using the fine focus knob. Reduce the amount of light using the diaphragm if necessary.
7. Rotate to high power and focus on the guard cells on the underside of the leaf. The two guard cells form a “donut” around the opening (stoma). Many guard cells contain a few green chloroplasts that are visible when the guard cell is swollen with water. Sketch the field of view on the Plant Morphology Worksheet, including both guard cells and epidermal cells and any intracellular organelles that may be visible.
8. Observe several stomata before answering the questions.

Activity 2. Stem Morphology

1. Label the three Petri dishes “ethyl alcohol,” “stain” and “water.”
2. Use a graduated pipet to place 5 mL of 50% ethyl alcohol into the ethyl alcohol dish.
3. Use a clean, graduated pipet to place 2 mL of Toluidine blue O stain into the stain dish.
4. Use a clean, graduated pipet to place 10 mL of distilled water into the water dish.
5. Obtain a nut and a bolt. Turn the nut end until it is 4 mm from the end of the bolt, forming a small cup. This is the part of the microtome that will hold the specimen.
6. Using a new, single-edge razor blade, cut 5 mm piece of plant stem from the base of a plant. Cut both ends of the stem. Make sure that this portion of the stem is free of foreign substances.
7. Carefully dip one end of the stem into melted paraffin wax and quickly place the stem into the nut and bolt microtome, wax end down. The wax will hold the stem ion the correct orientation while the remaining paraffin is poured into the microtome. Note: Be careful that the paraffin is not too hot or the wax will cook the stem.
8. Carefully pour melted paraffin into the nut until the wax fills the opening, completely covers the stem inside the microtome.
   {10798_Procedure_Figure_5}
9. Allow the paraffin to harden. (5–10 minutes).
10. Hold the head of the bolt horizontal on the table with one hand (see Figure 5). Holding the razor blade in your other hand, remove any excess wax on top of the nut by slicing down to the nut. This technique keeps your fingers out of the way of the very sharp razor blade.
11. Twist the bolt ¼ of a turn, so only a very small portion of the sample rises above the surface of the nut. Note: Very thin sections are necessary for light microscopy. A thin section that contains 75% of the sample is better than a thick section of the entire stem.
12. Use a fresh razor blade and a slicing motion (see Figure 5) to cut a thin cross-section from the sample. Use as much of the edge of the razor blade as possible by starting on one end and sliding down to the other end with each slice. Note: The same razor blade can only be used for a maximum of four cuts before it must be discarded. Dispose of used razor blades in a sharps container.
13. Place the thin cross-section of stem in the dish containing 50% ethyl alcohol for five minutes to remove the sap from the stem. While the cross-section of stem is in the ethyl alcohol, free the plant tissue from the surrounding paraffin wax.
14. Repeat steps 11–13 until eight to ten thin cross-sections of stem have been obtained.
15. Using clean forceps, move the stem cross-sections to the dish of stain for 1–2 minutes.
16. Using clean forceps, move the stem cross-sections to the dish of water briefly to rinse the excess stain off of the sections. Place the rinsed sections onto blotting paper in order to remove the excess stain and water.
17. Use a clean, graduated pipet to place one drop of glycerin on a microscope slide.
18. Use forceps to place a stem cross-section into the drop of glycerin.
19. Place a cover slip at a 45° angle into the edge of the glycerin. Carefully lower the cover slip onto the stem cross-section. If the preparation contains a bubble, carefully maneuver the bubble out from under the cover slip using a pencil eraser, being careful not to crack the cover slip or to damage the stem.
20. Observe the stem using a light microscope. If the section is too thick light will not penetrate through the tissue. Prepare a new “wet mount” of a new stem cross-section.
21. Once a good section is obtained, sketch the cross section of the stem on the Plant Morphology Worksheet. Identify and label each type of cell within the plant stem.
22. Answer the questions on the Plant Morphology Worksheet.
23. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10798_Student1.pdf