MICROSCOPE AND PLANT CELLS
Objectives:
1. Know parts of compound light microscope and their functions.
2. Understand relationship between magnification and size of field of view.
3. Understand depth of field.
4. Know how to make a wet mount.
5. Know parts of plant cell and which ones are visible under our scopes.
6. Know effects of staining cells with iodine.
7. Estimate length of an onion cell.
We will use the light microscope to observe the general size and shape of selected plant cells, and to see just a few of the many organelles within the cell.
Activity 1: Your instructor will review the basic parts of the microscope and their functions. Do not use the scope without instruction! Your success in future lab exercises will depend on how well you listen to the instructions and learn to use this important laboratory tool.
Why we use the microscope:
The inability of the human eye to see microscopic objects is a consequence of the arrangement of receptor cells at the rear of the eye. Two objects must be at least 100 µm apart for their reflected light to fall on different receptor cells. If they are closer together than this their light falls on the same receptor cell and therefore they are perceived as one object. Resolution is the minimum distance that two points can be separated and still be distinguished as two separate points. At less than 100 µm apart the human eye can not resolve two points or objects. Resolution may be increased by increasing magnification, i.e. making objects appear larger. For most everyday objects this is quite simple, just move closer to the object or bring it closer to you. However, our eyes can not focus closer than about 25 cm. But putting a convex lens, such as a magnifying glass or microscope lens, between the eye and object provides a clear image at a much closer range. Because the object is closer, it projects a larger image on the back of the eye, and it appears larger. Since this larger image is spread over more receptor cells the resolving ability of the eye is improved, making it easier to see smaller objects.
Microscopes which use two or more magnifying lenses are called compound because the lenses work in tandem to magnify the image which ultimately appears on the back of the eye. The objective lens closest to the specimen magnifies and projects the image into the body tube of the microscope where it is further enlarged and projected to the eye via the ocular lens. Thus total magnification is a product of the magnifying power of the objective times that of the ocular.
When the parallel rays of a light source strike a convex lens the rays will concentrate or focus at a specific point, the focal point. If you have ever started a fire with a magnifying glass, you are acutely aware of the concentration or focusing effect of a lens. The distance between the center of the lens and the focal point is called the focal length. Focal length is related to lens power (magnifying ability). A powerful lens will have a short focal length, thus requiring that it be placed very close to the object being examined. This means that as magnification increases, the working distance between the objective lens and object decreases. Students often either forget or ignore this fact and broken slides and/or scratched objectives may result. Never use the coarse focusing knob on anything but low power.
Focal plane (depth of field) is another important concept you should become familiar with. Hold a page of text up at normal reading distance and focus on a few words. Note that background and peripheral objects, although noticeable, are out of focus. Now focus on a background object and note that the writing on the paper is no longer in focus. All lenses, including those in your eyes, have limited distances or ranges in which two objects on different planes (focal planes) may be simultaneously in focus. For purposes of microscopy it is important to note that as magnification increases, focal plane (depth of field) decreases. In fact at high magnifications, the focal plane is usually less than the thickness of the object being examined. Therefore to see all of an object it is necessary to focus through it, i.e. constantly focus up and down with the fine focus knob of the microscope.
USE OF THE MICROSCOPE
Microscopes are expensive, precision instruments with parts that easily get out of alignment. Even the slightest misalignment will greatly affect image quality and detract from your lab experience. Handle them with care!
1. Always carry the microscope upright with two hands.
2. Place microscopes gently on the lab bench, never near the edge, and never slide them across the table. Position for easy access by both lab partners and the instructor.
3. Carefully position electric cords to avoid tripping or having the microscope pulled off the table.
4. Clean all lenses with lens paper. Never use paper towels or kimwipes.
5. Turn on the illuminator, rotate the turret or nosepiece so no objective is over the stage opening, place the slide within the clips, and position specimen over the center of the opening using the stage adjustment knobs.
6. Rotate the low power objective into place (it should click) over the slide.
7. Watching from the side use the coarse focus knob to position the objective as close as possible to the specimen.
8. While looking through the ocular focus downward with the coarse focus knob until the specimen is in focus. Never focus upward with the coarse focus.
9. Use the fine focus knob to obtain a sharper focus. Always make sure the specimen is in focus under low power before moving to a higher power.
10. Regulate the light intensity with the iris diaphragm. Some microscopes have adjustable light sources as well. You may also need to move the substage condenser up or done to adjust the focal point of light passing through the condenser lens.
11. Prior to increasing magnification make sure the area to be magnified is in the center of the field of view. While watching from the side, rotate the next highest power objective into place. Make sure that it does not contact the slide.
12. Adjust the fine focus to sharpen the image. Never use coarse focus once you leave low power.
13. Before removing the slide always return to low power and move the objective away from the slide with the coarse focus knob.
14. When finished clean the lenses with lens paper, coil the electric cord, replace the cover, and return the microscope to its appropriate place in the storage cabinet.
Make sure you can identify and use the following parts:
substage condenser
diaphragm
mechanical stage
objective lenses
revolving nosepiece
ocular lens
coarse focus
fine focus
Activity 2: Field of View - Letter "e" slide
1. Obtain a slide with a small newsprint "letter e" glued onto it instead of a biological specimen. By convention, slides are placed on the stage with the label on the left. Observe the slide, beginning with low power. Draw what it looks like in the appropriate circle below. The circle represents your field of view. Pay attention to the size and orientation of the image.
2. Rotate the nosepiece until the medium power objective clicks into place. Refocus slightly if necessary with the fine focus knob. Notice there is very little space between the slide and the objective lens. If you use the coarse focus, you may smash the slide into the lens. Make sure your specimen is centered in your field of view. Notice that the end of the pointer, if your scope has one, is not exactly in the center. Draw your specimen in the space provided.
low
medium high
3. Rotate to high power. Refocus slightly if necessary. Adjust the light if necessary using the diaphragm control lever. Do you need to go brighter or dimmer?
Draw your specimen.
4. Compare your three drawings and answer the following questions:
a) How does the area of the field of view change as you increase the magnification?
b) How does the light change as you increase magnification?
c) How is the image oriented compared to the actual specimen? How does this affect your efforts to center the specimen or find a particular portion of it?
5. Obtain a small centimeter ruler. Without any slide on the stage, place the ruler flat on slide stage (under clips), and observe under low power. Approximately what is the diameter of the field of view, in mm.?__________ As you have seen, at higher magnification, the field of view is smaller. Magnification and diameter are inversely proportional. We can calculate the diameter of the field of view on high power using the following formula:
mag. low power = diam. high power
mag. high power diam. low power
What is the approximate diameter of our high power field of view? ____________
Activity 3: Depth of Field - Crossed threads slide
The purpose of this slide is to demonstrate the depth of field of the different objective lenses. Although all specimens on slides are "thin", they do have some depth or thickness, which affects the quality of the image. For most purposes, a thinner specimen makes a better slide. Thinner slides are also usually harder to make, and are more expensive as a result.
1. Observe the crossed threads on low power. Can you get all three threads in focus at the point where they intersect?
2. Switch to medium power. Do you need to refocus? How many threads are in focus at one time?
3. Switch to high power. How much of the specimen is in focus now?
4. How is depth of field affected as the magnification increases?
Activity 4: Making a wet mount to observe living plant cells.
1. Place a drop of water on your slide, approximately in the center. Obtain a single leaf from the tip of an Elodea plant, and carefully lay it flat in the water. Pick up a cover slip, holding by two opposing edges. Place an edge of the cover slip vertically on the slide, close to the water drop and specimen. Gently slide the cover slip towards the water until it touches the drop, and a film of water spreads along the bottom edge of the cover slip. Now gently lower the cover slip onto your specimen. The idea is to create a film of water over both surfaces of the specimen, without any air bubbles to refract the light. Air bubbles are usually outlined with a heavy black line.
2. After you have made a successful wet mount of the leaf, observe it under low, med., and high power. Remember to use good microscope techniques. Look at both the middle portion, and the edges. Draw your specimen in the spaces below.
low medium high
Look for the following : ( Look up the definitions in your text or notes) Put a check next to those parts that are visible with our scopes.
cell wall
plasma membrane
cytoplasm
vacuole
vacuolar membrane
cell sap
nucleus
chloroplasts
cytoplasmic streaming
Activity 5: Living non-green plant cells from onion bulb epidermis
1. Make a wet mount from a small piece of the epidermis of an onion bulb leaf. Make sure you strip the epidermis from the inside (concave) surface of the leaf. Place the inner side down on water drop. It may try to roll up, but it must be flat for observation. Throw the used piece away so no one else tries to use it.
2. Observe on low power and medium power.
a) Do these cells have the same basic shape and arrangement as the Elodea leaf cells?
b) Are there any chloroplasts? Why would you not expect to find chloroplasts?
c) Can you see the nuclei or cytoplasm? Can you see cytoplasmic streaming?
3. We will use iodine to stain these cells, in order to make certain parts more visible. Since iodine is poisonous, it will also kill the cells. Obtain a dropper bottle of iodine (IKI), and place one or two drops on your slide so they touch one edge of the cover slip. Place a tissue on the opposite side, and draw the iodine under the cover slip by capillary action.
a) What part of the cell has been stained most intensely?
b) Do you see any movement in the stained cells? Can there be cytoplasmic steaming in these cells? If you see tiny particles jiggling, it could be Brownian motion, which results from the random collisions of water molecules and suspended particles in an aqueous medium.
4. On high power, count the number of cells lined up end to end across the field of view. Using the information from Activity 2e, estimate the length of the onion epidermal cells on your slide. For comparison, a human blood cell is approximately 8µm, or .008 mm in diameter.
Activity 6: Other cell types
Look at the variety of other cells provided by the instructor. Some common examples are:
parenchyma cells- these are usually unspecialized, thin-walled cells with a multi-sided appearance (like a soccer ball, only with fewer sides). Often they are used for food storage; some may have chloroplasts. Make a wet mount of a very thin slice of potato; look along the edge of the slice to see the thin, straight cell walls and the oval starch grains (within plastids) inside the cells. You can stain the cells with iodine to make the starch grains turn blue-black.
tracheids and/or vessel elements - show the elongated structure and thick walls of water conducting cells
guard cells and other epidermal cells- guard cells enclose a pore for gas exchange, they can open and close the pore as their turgor pressure changes, they look like a pair of lips
pear tissue – the stone cells or sclereids have thick cell walls that are stained pink. Can you see the channels in the walls where the plasmodesmata occur?
petals- the edges of colored petals provide an opportunity to see the outline of the vacuole
Draw any of the specimens you wish to remember in the spaces above. Note magnification.
Questions:
1. How do you determine the total magnification of an image?
2. What parts of the Elodea cell are visible under our light microscopes?
3. Why did we use iodine on the onion epidermal cells?
4. What are the common characteristics of parenchyma cells?
5. In what ways are other cells specialized?