PlantingScience Celery Challenge Toolkit Image: findingthenow (Flickr) BACKGROUND: The PlantingScience Celery Challenge Toolkit provides background, materials lists, detailed procedures, and safety considerations for additional experimental methods related to osmosis, transpiration, and visualizing plant cells. These tools can provide students the opportunity to ask a wider range of research questions during the Celery Challenge inquiries than would otherwise be possible. Alternatively, teachers may select one or more of these methods as the basis for classroom demonstrations of plant physiology or morphology. CONTENTS: Page Cutting Transverse Sections of Plant Tissues for Microscopy ....................................................................... 2 Making Epidermal Peels for Microscopy ...................................................................................................... 4 Visualizing & Counting Stomata Using the Impression Method ................................................................... 6 Visualizing Plant Cells Using a Microscope ................................................................................................... 9 Determining the Electrical Conductance of a Solution ............................................................................... 12 Measuring Transpiration Using a Simple Potometer .................................................................................. 14 Quantifying Water Mass in Celery .............................................................................................................. 17 Plant Cell Staining Techniques .................................................................................................................... 19 . PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.org | Celery Challenge—Toolkit Page 1 of 22 Last Updated 7/2013 CUTTING TRANSVERSE SECTIONS OF PLANT TISSUES FOR MICROSCOPY Purpose: To create thin cross-sections of celery tissue for microscopic visualization. How the Method Works: Transverse sections are samples of plant tissue cut perpendicular to the long axis of the organ. For example, leaf transverse sections allow the visualization of a thin slice of internal tissues from the leaf’s upper surface to its lower surface. Here, the focus is on celery petioles and the visualization of internal structures such as vascular bundles and pith. The first method, hand sectioning, is fast and simple, but requires more skill. The second method describes how to build a “poor man’s microtome” – an instrument that can be used to cut very thin sections of plant material. Sections can then be examined with a microscope. If desired, use Plant Cell Staining Techniques (p. 19) to identify cell types and components. Technical Complexity: Simple for hand sectioning, moderate for the microtome. Time Required: 5 minutes for hand sectioning; 10 min. for building and using the microtome. Materials: Hand Sectioning: Poor Man’s Microtome: Celery petiole for observation Celery petiole for observation Single-edged razor blade Nut and bolt of compatible sizes Small dish of tap water Wax, in a small microwaveable container Toothpick Microwave Microscope slide Utility knife (Optional) Potato or carrot Microscope slide Make a Hand Section Hand Sectioning: Hand sectioning is best used on relatively firm tissues and may be difficult if celery is limp. This method takes practice but can produce excellent results. 1. Prepare a small dish of water, then select the desired plant specimen and a new razor blade. 2. Hold the part to be sectioned firmly between your thumb and index finger (see image). Sandwich small or fragile parts between pieces of potato or carrot. 3. Taking care to avoid cutting yourself, make a preliminary cut in the plane of tissue you eventually want to examine. PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.orgMake an | Epidermal Celery Challenge Peel—Toolkit Page 2 of 22 Last Updated 7/2013 • Slice towards yourself with the razor, using as much of the blade surface as possible. • This cut will expose fresh tissue; you can discard the older piece it removes. 4. Using the same cutting technique, quickly make a series of sections, each as thin as possible. 5. Place each section in your dish of water as it is made. • Avoid tearing the section as you move it. An ideal section includes the entire cross- section of tissue. • Keep the pieces, however! These are often the thinnest. 6. Lay out a glass slide and place a drop of water onto it using a disposable pipette. 7. Transfer the thinnest section to the slide, using the flat side of a toothpick if needed. 8. (Optional) If you want to stain the cells, use a procedure from Plant Cell Staining Techniques. Poor Man’s Microtome: Although microtomes can be purchased, they are usually quite expensive. This method can be used much more cheaply and will give better results than can be achieved by hand sectioning alone. Step 1 Step 2 Step 3 Step 5 Step 7 Step 8 Steps in Using a “Poor Man’s Microtome,” as Described in the Instructions Below 1. Cut a small, cubical piece of plant material, such as from a stem. 2. Place the plant piece into a nut that is barely fastened to a bolt. 3. Melt a small amount of wax in a microwave for about 2 min, then pour the melted wax onto the plant material, filling the space around it in the nut. 4. After the nut is filled completely, let it sit for about 2 min while the wax cools and hardens. 5. With the bolt on its side, cut straight down the nut with a utility knife to make a thin section. • In all steps using the utility knife, take proper care to avoid cutting yourself. • This first segment of plant tissue plus wax can be discarded. 6. Screw the nut slowly onto the bolt to raise a thin section of fresh wax and tissue, then cut again. • Repeat until you have enough thin sections to work with. 7. Discard the wax from the section(s), and place the best section on a slide. 8. Place one drop of water onto the plant section or (optionally) carry out a staining procedure. PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.org | Celery Challenge—Toolkit Page 3 of 22 Last Updated 7/2013 MAKING EPIDERMAL PEELS FOR MICROSCOPY Purpose: To remove samples of the outer cellular layer of plant tissue for microscopic visualization. How the Method Works: Epidermal cells make up the “skin” of a plant. The cells form a distinct tissue layer that restricts water movement out of the plant; this layer also contains specialized cells called stomata that regulate gas exchange and transpiration. Quantifying and measuring epidermal and stomatal cells is only possible with a microscope, and this can provide information about the plant’s ability to move water. Two Make a Hand Sectionmethods for collecting epidermal samples for microscopy are described here. Epidermal peels are fast and make excellent samples for quantification, but theyMake require some a Hand Section skill. Epidermal scrapes are much easier, but they collect only a few cells at a timeMake a Hand Section . If desired, use Plant Cell Staining Techniques (p. 19) to identify cell components. Technical Complexity: Simple. Time Required: 5 minutes. Make an Epidermal Peel Materials: • Celery petiole or leaf Make an Epidermal Peel • Single-edged razor blade or scalpelMake an Epidermal Peel • Toothpick • Microscope slide Epidermal Peels: Single layers of epidermal cells can be obtained using this procedure. It will likely be easier to remove the epidermis from the smooth, concave side of the celery than from the ridged, convex side. 1. Break the petiole in half so that only the epidermis connects the two pieces. o Cutting partway through the opposite side from the one you are sampling will help, as shown in the figure above. Take care to avoid cutting yourself. 2. Gently pull one half of the petiole down the side of the other so that the epidermis is pulled away from the tissue of the latter. 3. Lay the exposed epidermis on a drop of water and cut away the remaining tissue. 4. Use the flat side of a toothpick to transfer the exposed epidermis to a microscope slide. 5. (Optional) If you want to stain the cells, use a procedure from Plant Cell Staining Techniques. 6. (Optional) Assess the density and condition of stomata and epidermal cells with the last section of Visualizing & Counting Stomata Using the Impression Method, and quantify the sizes of cells and stomatal pores using Steps 5-7 of Visualizing Plant Cells Using a Microscope. PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.org | Celery Challenge—Toolkit Page 4 of 22 Last Updated 7/2013 Epidermal Scrapes: Individual cells, or small groups of cells, can be obtained by scraping the celery surface. If you have difficulty getting epidermal peels from the ridged side of a celery petiole, this alternative may be helpful. Unfortunately, you will not be able to measure cell density with this approach. 1. Taking care to avoid cutting yourself, use a scalpel, razor, or wooden toothpick to scrape the epidermal tissue you wish to examine. 2. Rinse the scraper in a drop of water on a microscope slide. 3. (Optional) If you want to stain the cells, use a procedure from Plant Cell Staining Techniques. 4. (Optional) You can quantify the sizes of cells and stomatal pores using Steps 5-7 of Visualizing Plant Cells Using a Microscope. PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.org | Celery Challenge—Toolkit Page 5 of 22 Last Updated 7/2013 VISUALIZING & COUNTING STOMATA USING THE IMPRESSION METHOD Purpose: To determine stomatal density and examine