Procedures - Cells & Osmosis-1

Procedures – Cell Structure, Cell Functions & Osmosis ES 1111 Environmental Botany Laboratory 1 Part I. Cellular Organelles Elodea is a widely distributed photosynthetic pondweed (also frequently found in fish aquariums) that consists of green, submerged stems surrounded by many narrow, flat leaves attached in a tight spiral around the stem. Each leaf is two cells thick, except along the margins, where it is one cell thick. All of the cells are more or less rectangular in outline, but the cells in the upper layer are larger than those in the lower layer, and it is the larger cells we want to examine closely. Sprigs of Elodea are provided. Procedure: 1. Obtain an Elodea leaf, glass slide, and coverslip * . *Note: Specimens can be viewed without a coverslip using the dissecting microscope, but a coverslip must always be used with a compound microscope 2. Living material is often prepared for observation using a wet mount. Prepare a wet mount of Elodea following the steps below: a. Place a drop of water in the center of a clean microscope slide. b. Remove a single leaf of Elodea and place it on the slide in the drop of water. For best results, remove your leaf from just a few millimeters below the growing tip, and also be sure that the upper surface of the leaf you place in the drop of water is facing up (note how the leaf was oriented on the stem). c. Using forceps (or your finger), place a coverslip at a 45 ο angle above the slide with one edge of the coverslip in contact with the edge of the water droplet. Lower the rest of the coverslip gently until it makes a sandwich of the leaf with the slide, being careful not to trap air bubbles in the droplet. If the leaf is not completely surrounded by water when you have done this, add a little more water at the edge of the coverslip-it will run under on its own. 3. Using the scanning (4X) power objective of your microscope, bring the cells of the upper layer into sharp focus with the coarse focus knob*. *Note: Use the coarse focus only with the 4X objective, use the fine focus with the longer objectives or you may break the slide. Now switch to the 10X and finally 40X objectives (you should feel them click into place). Remember the light microscope is parfocal, meaning when you switch from a lower magnification to a higher magnification, the image should require only slight adjustments with the fine focus for a sharp image. If you find yourself having a hard time refocusing between objectives, go back to the 4X objective and start over. 4. The majority of plant cell organelles will be too small to see with a light microscope; however, you should be able to identify the structures listed below which are visible with a compound microscope. Note: To enhance resolution and contrast, be sure to examine the cells with the iris diaphragm closed so that it admits just enough light to be able to distinguish objects. To close the iris diaphragm, move the iris diaphragm lever to the right. a. Locate the cell well , the thin semi-rigid outer framework surrounding the cell. This structure gives the plant cell a definite shape and support. It is not found in animal cells. Sandwiched between adjacent cell walls, but not visible is the middle lamella which holds adjacent cell walls together. Be able to point cells walls out to your instructor. b. Cytoplasm is the material within a living cell, exclusive of the nucleus. The cytoplasm is quite thin and confined to the vicinity of the cell wall, although in some cells thin strands of

Procedures – Cell Structure, Cell Functions & Osmosis ES 1111 Environmental Botany Laboratory 2 cytoplasm, called cytoplasmic bridges , may extend across the vacuole. The cytoplasm is bounded by an invisible membrane, the plasma or cell membrane. c. The central vacuole is a large membrane-bound sac within the cytoplasm that is filled with water and dissolved substances. It often takes up the majority of the plant cell with its membrane pushed up against the cell’s membrane. This structure serves to store metabolic wastes and various other substances. It gives the cell support by means of turgor pressure. Some vacuoles occasionally will appear pink due to the presence of water- soluble anthocyanin pigments . These pigments are responsible for some, but not all, of the pink to red to blue and purple colors in flowers, ripe fruits and some leaves. Be able to point the central vacuole out to your instructor. d. Chloroplasts are the green, spherical organelles often seen moving within the cytoplasm. They are one of three types of plastids found in plant cells, each of which contains a different substance. Chloroplasts contain the green pigment chlorophyll that is the primary pigment involved in photosynthesis. Note: A pigment is any molecule that can absorb wavelengths of light—different pigments absorb different wavelengths of light. Be able to point chloroplasts out to your instructor. e. If at least some of the chloroplasts do not appear to be moving, ask to observe movement on someone else's slide. The movement is called cyclosis or cytoplasmic streaming . The chloroplasts are not moving under their own power but are being carried along by the river-like flow of the nearly invisible cytoplasm which moves when cell microfilaments contract . Cytoplasmic streaming helps orient chloroplasts for optimal exposure to light, which strikes the leaf cells at different angles during the day. f. The nucleus is often hidden by chloroplasts in Elodea cells, If, however, it is visible, it generally appears as a faint, flattened, grayish lump about the size of a chloroplast, or a little larger; often pushed up against the cell wall by the large central vacuole. 5. Draw Elodea cells and label the CELL WALL, CYTOPLASM, VACUOLE, and CHLOROPLASTS (NUCLEUS if you can find it). In the circle below, draw exactly what you see (do not make your sketch larger or smaller than what you see in the microscope’s field of view) at a total magnification of 400X. To the left of each circle identify the specimen, specifically, and include the total magnification. * Remember: Total magnification equals the ocular magnification X the objective magnification. Specimen _____________________________Mag. ______X 6. Remove your slide from the microscope’s stage and save to use in Part II.

Procedures – Cell Structure, Cell Functions & Osmosis ES 1111 Environmental Botany Laboratory 4 (known as parenchyma ) of the potato tuber. Nearly, all the cells of a common white potato are parenchyma cells that contain starch grains inside of amyloplasts. The starch grains are often clam-shaped in outline and may, when observed under high power, have faint concentric lines like a clam. Each line represents the limit of one day’s deposit of starch. Amyloplasts are quite small at first and increase in size as the starch deposited within the amyloplasts accumulates. Procedure-chromoplasts: 1. Obtain a sectioned, paper-thin slice of tissue from a red pepper to observe chromoplasts . 2. Make a wet mount of your section using a drop of water and following the same procedure used to make your Elodea leaf wet mount. If your coverslip is balancing precariously on the section rather than "floating" uniformly on the surface, your section is too thick. Obtain a thinner section or you will not be able to see individual chromoplasts. 3. Once you have your section focused clearly with the, scanning, low, and then high-power objectives, rotate the fine adjustment knob carefully to observe the internal structures of the cells. The cells should be filled with several tiny, oval, reddish, gold-pigmented structures. These are the chromoplasts. You may have to adjust your light level & contrast with the iris diaphragm to see them. Be able to point chromoplasts out to your instructor. 4. Make a sketch of a cell containing chromoplasts in the circle below at a total magnification of 400X. (Use red or orange colored pencils provided in lab to indicate the chromoplasts). Specimen ________________________Mag ______X 5. Properly discard of your chromoplast wet mount in red sharps container. Procedure-leucoplasts (specifically, amyloplasts) 1. Obtain a very thin wet slice of potato to observe leucoplasts . Make a wet mount. 2. Once you have your section focused clearly with the high-power objective, rotate the fine adjustment knob carefully, with low light level, to observe the internal structures of the fairly large, thin walled parenchyma (storage) cells. The cells should be filled with several unpigmented egg-shaped structures which are starch grains within amyloplasts. 3. Make a sketch of the cell with amyloplasts containing starch grains in the circle below at a total magnification of 400X.

Procedures – Cell Structure, Cell Functions & Osmosis ES 1111 Environmental Botany Laboratory 5 Specimen ________________________Mag ______X 4. Now add a drop of iodine* to the edge of the coverslip of your slide. Notice what happens to the starch grains in the amyloplasts as they absorb the iodine. Note: This is a very useful reaction often used in botany and biology to uniquely identify the presence of starch. *Be careful-Iodine will stain hands and clothes. 5. On your sketch above, use purple colored pencils to indicate the change to starch grains after they were stained with iodine.