1. Two or three fresh sprigs of Elodea
4. Single-edged razor blades
7. Beaker of fresh tap water
10. Two eyedroppers
2. One fresh onion
5. IKI solution (in dropper bottles)
8. One ripe tomato or red pepper
11. Plant cell models and charts
3. One small, fresh white potato
6. Two or three fresh Tradescantia flowers (or similar flowers with stamen hairs)
9. Bowl of pond water containing various algae and other aquatic organisms
Some Suggested Learning Goals
1. Be able to distinguish the various components of living cells visible with a light microscope.
2. Understand the difference between cyclosis and independent movement of microscopic objects.
Cells are the basic units of which all living organisms are composed. The living material of a cell is called protoplasm. Protoplasm is composed mostly of fluid cytoplasm, whose primary constituent is water. Cytoplasm also contains a variety of small bodies called organelles, membranes, particles, and dissolved substances. The most important organelle is a more or less spherical to ellipsoidal nucleus that contains DNA; the nucleus controls the activities of the cell.
Other organelles may include relatively conspicuous green chloroplasts that are often present in cells exposed to light, or chromoplasts, which are typically red to orange in color. Other important organelles that are not normally visible with light microscopes include tiny rod- to paddle shaped mitochondria that function in energy release; dictyosomes, which function as packaging centers for substances needed by cells; and endoplasmic reticulum, which forms a series of membranous channels connected to the nucleus. Endoplasmic reticulum occurs in a "rough" form, which has tiny, granular-appearing ribosomes associated with it, and a "smooth" form without ribosomes. Ribosomes play a role in the manufacture of proteins.
The cytoplasm of a plant cell almost always also includes one or more vacuoles. Vacuoles are flexible bags of watery fluid that are bounded by vacuolar membranes and that may occupy more than 95% of a plant cell's volume. Vacuolar membranes are similar to the plasma membrane that forms the outer boundary of the protoplasm. The plasma membrane is adjacent to the rigid or semirigid cell wall, which varies in thickness, depending on the type of cell. Cell walls are visible with a light microscope but vacuolar and plasma membranes are not.
Although cells are produced in a wide variety of sizes and shapes, all have these and other features in common. What features found in living cells are not found in dead cells? After obtaining the various items listed in the "Materials" section, attempt to answer these questions through direct observations, as indicated in the following paragraphs:
Elodea (Anacharis) is a widely distributed pondweed 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. Before obtaining an Elodea leaf, be sure that your glass slide and coverslip are completely clean (use detergent to clean them, or alcohol if fingerprints are present). Then, with an eyedropper add one drop of water from the plant bowl to your slide. The leaves at the tip of an Elodea stem may be immature; the older leaves farther down on the stem may have other organisms such as diatoms on their surfaces. Because of this, be sure you 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). Apply the coverslip to the slide by first dipping one edge in the water drop, and then lower the rest of the coverslip gently until it makes a sandwich of the leaf with the slide. 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.
Using the low-power objective of your microscope, bring the cells of the upper layer into focus. Now switch to high power and refocus. Are all of the cells roughly the same size and shape? Can you detect any movement of the contents of the cells? If at least some of the green 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 riverlike flow of the nearly invisible cytoplasm.
Locate a cell with numerous chloroplasts, and focus up and down very carefully and slowly with the fine adjustment. Note that at one point all the chloroplasts appear to be only along the margins of the cell. This is because the cells are box-shaped and have depth, even though the leaf may appear to be flat to the unaided eye. Each cell has a large central vacuole bounded by a vacuolar membrane. In addition to water, vacuoles sometimes contain pigments or crystals of waste substances. The chloroplasts are located only in the cytoplasm, and are plastered up against the six inner walls of the cell, leaving the large, clear vacuole to occupy most of the volume of the cell. When the cell first comes into focus, the chloroplasts appear to be spread across the wall of the cell, which they are. However, further focusing reveals that the cytoplasm is quite thin and confined to the vicinity of the wall, although in some cells thin strands of cytoplasm, called cytoplasmic bridges, may extend across the vacuole.
Locate the thin, semi-rigid cell wall and the vacuole. The nucleus is often hidden by chloroplasts in Elodea cells, If, however, it is visible, it generally appears as a faint, grayish lump about the size of a chloroplast, or a little larger; it is often up against the cell wall. Students who are both patient and determined to see a nucleus are often successful if they first look for a cell that has fewer chloroplasts. To enhance resolution and contrast, be sure to examine the cells with the microscope diaphragm closed so that it admits just enough light to be able distinguish objects. A nucleolus, which appears as a slightly denser spherical body within the nucleus, is often difficult to detect without special staining. The cytoplasm is bounded by another invisible membrane, the plasma membrane. Also not visible are the middle lamella, which is sandwiched between adjacent cell walls, and many smaller organelles present in the cytoplasm. As you move the slide around, you may occasionally observe cells whose vacuoles are pink. The color is due to the presence of water-soluble anthocyanin pigments. These pigments are also responsible for some, but not all, of the colors in flowers and ripe fruits.
Your instructor may demonstrate how to peel a single
layer of cells from an onion. Mount a segment of the onion peel in a drop
of water on a clean microscope slide. Are there chloroplasts present? How many nuclei are present in each cell?
The most abundant of plant cells are called parenchyma cells. Parenchyma cells occur in various sizes and are thinwalled. The cells usually have several sides (most frequently 14) at maturity. Nearly all the cells of a common white potato are parenchyma cells that contain starch grains. The starch grains are often clam-shaped in outline, and may, when observed under high power, have faint eccentric lines. Each line represents the limit of one day's deposit of starch. Each starch grain develops within a colorless leucoplast. Leucoplasts are quite small at first and increase in size as the starch deposited within accumulates.
With a sharp razor blade, make several paper-thin sections of potato, and keep the sections wet. Choose the thinnest section, and mount it in a drop of water on a slide. Add a coverslip and, if necessary, another drop of water at the edge of the coverslip so that the whole section is surrounded by water. Locate an area along one edge of the section where the tissue is thin enough for you to distinguish cells. Do not confuse the thin, usually dark and relatively straight cell walls with the numerous starch grains within them. To make the starch grains stand out, add a drop of IKI solution, which stains starch a dark blue-black color, to the edge of your coverslip.
Amyloplasts in the Potato Tuber
A tuber is an underground stem used for the storage of nutrients during plant dormancy.
Dormancy, a period of greatly reduced metabolic activity, allows many plants to survive winter. The nutrient reserve provides the energy for new growth in the spring. Starch is the common nutrient stored by plants. Recall that the plant organelles which store things are called plastids. Starch is stored in amyloplasts, a specific type of leucoplast (any unpigmented plastid). In this exercise you will observe amyloplasts in the storage cells of the potato tuber.
Use a sharp razor blade to slice a very thin section from the potato tuber. Do not use the "skin" portion.
A. Make a wet mount of your section using a drop of water. If your coverslip is balancing precariously on the section rather than "floating" uniformly on the surface, your section is too thick.
B. 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 and loosely packed storage cells. The cells should be filled with several unpigmented egg-shaped structures. These are commonly called starch grains, but we botanists know they are correctly referred to as amyloplasts.
C. Add a drop of iodine to the edge of the coverslip. What happens to the starch grains (amyloplasts) as they absorb the iodine? This reaction is a "famous" reaction which uniquely identifies starch. It is a very useful test in botany and biology.
D. Tradescantia Stamen Hair Cells
Tradescantia, commonly known as spiderwort, produces flowers with pollen-bearing stamens that have many fine hairs on their filaments (stalks). Each hair consists of a single row of connected beadlike cells that become sausage shaped as they expand.
Have a drop of water ready on a slide, and ask your instructor to give you a stamen hair from a Tradescantia (spiderwort) or related plant. Cover with a coverslip. If your cells don't have a faint lavender color, they may have been too old or crushed, or they may have been separated from the flower for too long before being immersed in water; if so, you should then obtain another hair. If you focus down carefully under high power, you will note that the surface of each cell is covered with fine striae, or lines, which are more or less parallel with each other. Inside the cell, observe the cyclosis occurring, and note that the cytoplasm crisscrosses the large central vacuole via narrow cytoplasmic bridges. Also observe the cytoplasmic granules in the cytoplasm. Locate the nucleus, the nucleolus, and the cell wall. Are any chloroplasts present?
Ripe tomatoes, red peppers, and several other red to orange fruits owe their color to organelles known as chromoplasts within their cells.
Cut a paper-thin slice of tissue from the ripe tomato or other red material provided, and mount in water. Locate the small orange or reddish chromoplasts in the cytoplasm. Are the chromoplasts similar in size and shape to Elodea chloroplasts? If not, how do they differ?
You have observed two different types of plastids so far in today's lab, the chlorophyll-containing chloroplasts which function in photosynthesis, and the unpigmented amyloplast, which stores starch. Now you will have an opportunity to observe a third plastid, the chromoplast which contains carotenes, the gold and orange pigments of plants.
Obtain a small piece of carrot, red pepper or tomato. Petals of bright orange flowers, such as marigold flowers, are also excellent sources of chromoplasts.
A. Use a sharp razor blade to slice a very thin section of your chosen material.
B. Make a wet mount of your section using a drop of water. If your coverslip is balancing precariously on the section rather than "floating" uniformly on the surface, your section is too thick.
C. 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 cells. The cells should be filled with several tiny oval, gold—pigmented structures. These are the chromoplasts. You may have to adjust your light level to see them.
How do the chromoplasts compare to the chloroplasts and amyloplasts you observed previously? Are the
chromoplasts of carrots different from those of red peppers or tomatoes?
Depending on the location, time of the year, and other environmental factors, pond water may contain a rich variety of microscopic living organisms, as well as larger plants and animals.
Stir the provided pond water, and using an eyedropper, place a drop of the agitated water on a slide, cover with a coverslip, and locate as many different kinds of cells or organisms as you can. (Don't confuse shapeless blobs or granules of debris with live cells or organisms.) Your instructor will help you with the names of the algae and other organisms observed. See charts for diagrams of some of the algae and cyanobacteria commonly found in pond water.
1. Draw one healthy Elodea cell and label the CELL WALL, CYTOPLASM, VACUOLE, CHLOROPLASTS, and NUCLEUS. If you have observed any other parts of a cell (e.g., NUCLEOLUS), label them also. Remember that each drawing must have a diameter of at least 3 inches.
2. Draw one or two potato parenchyma cells. Label the CELL WALL and STARCH GRAINS.
3. Draw a spiderwort stamen hair cell as viewed under high power. Label the CELL WALL, CYTOPLASM, CYTOPLASMIC BRIDGE(S), NUCLEUS, NUCLEOLUS, CYTOPLASMIC GRANULE(S), STRIAE, and VACUOLE.
4. Draw three different aquatic organisms observed in the agitated pond water.
Review questions 2
1. How many layers of cells are there in an Elodea leaf?
2. How should a coverslip be applied to a drop of liquid on a microscope slide?
3. When chloroplasts appear to be moving within a living cell, what is the cause of their movement called?
4. In most living cells, such as those of Elodea, where is the cytoplasm located? How extensive are plant cell vacuoles?
5. What are cytoplasmic bridges?
6. What parts of cells are normally visible with the aid of a compound light microscope?
7. If present in a cell, where are anthocyanin pigments located?
8. How are starch grains distinguished from parenchyma cells in a potato?
9. What are striae, and where are they located in a spiderwort stamen hair cell?
10. How does a chromoplast differ from a chloroplast?
1. In what part of a cell are chloroplasts located?2. What is cyclosis?
3. What is a vacuole?
What is the thin boundary of the vacuole called?_____ Is it visible?
4. Where would you look for the nucleus in an Elodea cell?
5. Anthocyanin pigments and chromoplasts may both be red in color. If you were to observe a cell that had both, how could you distinguish between them?
6. How can you tell a potato starch grain from a cell?
7. How would you distinguish a starch grain from a chloroplast?
8. What is a cytoplasmic bridge?
9. Specifically where do starch grains develop in a cell?
Where would you expect to find a
8. From what does a new cell wall develop?
9. Which cell organelle produces the materials for a new middle lamella?_________________________________
10. What is the function of a centromere?