PART 1 THE MICROSCOPE

Transcription

1 Lab 2: INTRODUCTION TO MICROSCOPY (portions of this exercise have been adapted from Introductory Experiments in Cell Biology by K. Fleiszar and B. Wallace, Hunter Textbooks Inc., and Biology in the Laboratory by D. Helms, C. Helms, R. Kosinski and J. Cummings, Freeman and Company) INTRODUCTION The scientist has a variety of laboratory equipment which allows him to see what could not normally be observed with the naked eye. One such instrument is the microscope, which extends the visual range of the scientist. Part 1 of this lab will introduce you to two commonly used types of microscopes. In Part 2 of this lab, you will use both types of microscope to observe living specimens which you have prepared for viewing. Part 3 of this lab will ask you to examine the two general types of cells associated with living organisms, prokaryotic and eukaryotic cells, and make some general observations about them. PART 1 THE MICROSCOPE There are a variety of different kinds of microscopes each with different merits and uses. Two types of microscopes will be discussed in these exercises; the stereoscopic or dissecting microscope and the compound light microscope, for these are the two microscopes you will use during your undergraduate academic career. The Stereoscopic or Dissecting Microscope The dissecting scope (Fig. 1) available for your use magnifies objects only times. Material to be examined under a dissecting microscope is usually relatively thick. This scope is commonly used for performing dissections of small organisms. Note that it has two eyepieces, or oculars, for viewing. Thus, this microscope is said to be binocular. Each ocular contains a lens which usually magnifies objects to 10 times their size (10X). The distance between the oculars is adjustable. They can be positioned to suit your eyes by pulling then apart or pushing them closer together. Many, but not all, dissecting microscopes have a magnification changer that allows you to zoom in or out on the specimen within the set magnification range by adjusting another lens located within the body tube. The mechanical system of the microscope consists of the stage (which holds the specimen), the focus knob (for raising and lowering the optical system to bring the specimen into focus), and the base and arm (for easy carrying). Plug in the microscope and turn on the light. After correctly positioning the oculars, practice using the scope by viewing Drosophila specimens provided by your instructor (obtain at Station 1). Manipulate the focus knob to bring the organisms into focus. Record the structural details of the fly body that are discernable with the dissecting scope, being as descriptive as possible (use the space below). If you look carefully you will see two types of flies that differ in a physical feature. Describe the feature. When you have finished your observations, TURN OFF THE LIGHT SOURCE and set the microscope aside for future use.

2 variable magnification knob Ocular lenses arm body tube focus knob light source stage base on/off; light source selection Fig. 1. Dissecting microscope. (Courtesy Leica Corporation) The light source for your microscope may be a separate light or built into the microscope. Light can be transmitted through a specimen on the microscope stage from below. Light can be reflected from the surface of the specimen by positioning the light source above the specimen so that it shines downward onto the microscope stage. Which is your microscope capable of? If your microscope is capable of providing both types of illumination, which works better (e.g. allows you to see the most detail of your specimen)? The Compound Light Microscope The compound light microscope (Fig. 2) must be used if you wish to observe smaller and thinner specimens. This type of microscope has much greater powers of magnification than the

3 dissecting scope. The name of this microscope is derived from the fact that it utilizes two (i.e. compound) optical components and uses light as its source of illumination. Plug in the scope and turn on the light. The optical components are the ocular (eyepiece) and objective lenses. There are usually two four objective lenses projecting from the revolving nosepiece. Each objective has a different power of magnification (indicated on the side of the objective; see Fig. 3). The LOW POWER OBJECTIVE is the shorter of the two and magnifies objects 4 to 10 times (4X or 10X). The other objective(s) is a higher power objective and will magnify 40, 43 or 45X. An oilimmersion objective may also be present (100 X). This objective is only used with oil. Since the ocular lens is located at the very top of the microscope and the objective lenses are located just above the stage, the object being observed is magnified first by the objective lens and then this image is magnified ten times more by the ocular lens. Thus, if a 10 X low power objective is being used, the total magnification of an object is 10 x 10 or 100X. Magnification powers of 2,000 X are possible with more sophisticated compound light microscopes. COMPOUND LIGHT MICROSCOPE oculars revolving nose piece arm objectives condenser lens course & fine focus knobs condenser adjustment knob stage adjuster knob iris diaphragm/lever light source base Fig. 2. Compound light microscope. Calculate the total magnification if you are using the highest power objective of your microscope:

4 In addition to the ocular lens and objective lenses, the optical system includes the light source (necessary to illuminate the specimen), condenser (which contains a system of lenses that focuses the light on the specimen), an iris diaphragm (which is used to adjust the amount of light striking the specimen), and the body tube (which can be rotated on some microscopes). Some microscopes do not have a condenser. Others have a movable or a fixed condenser. If your microscope is equipped with a movable condenser, locate the knob that raises and lowers the condenser and add it to the diagram (identify it as such). The mechanical system consists of the stage, a course adjustment knob (used for initial focusing of specimens under low power), a fine adjustment knob (used for precision focusing at higher power), stage clips (for holding a specimen in place), base and arm (to allow easy carrying). You will be expected to locate the optical and mechanical parts of the compound microscope and discuss the function of each part. IT IS IMPERATIVE THAT YOU LEARN AND ALWAYS PRACTICE THE FOLLOWING PROCEDURE FOR USING THE COMPOUND LIGHT MICROSCOPE: 1. Use cotton swabs and lens cleaner to clean the ocular and objective lenses before and after use (if necessary). Do not use paper towels, Kleenex tissue, cloth, etc. Using something other than cotton swabs could smudge or damage the lenses. 2. Always carry the microscope in an upright position. Use one hand to grasp the arm of the microscope; use the other to support the base. The eyepiece (ocular lens) slides into the body tube in many brands and could fall out if the microscope is tilted. 3. When you are finished with an observation, turn off the illuminator and rotate the low power objective into viewing position. 4. To observe a specimen: a. Turn the illumination source on. b. Move the stage downward to its lowest position with the coarse adjustment knob. Place the microscope slide with specimen on the stage. Make sure the lowest power objective is in place (rotate the nosepiece until the low power objective clicks into place). c. Adjust the light so that you have enough illumination but not too much. Excessive light can cause eye strain. As you increase magnification you will need to increase the intensity of the light. d. If you wear glasses, remove them! Raise the stage to its highest position and, while looking through the ocular, scan slide until you see a region of color. If you have difficulty locating the specimen, use a systematic pattern to search the slide. Then, while looking through the ocular, slowly lower the stage until the object is in focus. Try to keep both eyes open. This will be less tiring for your eyes. e. Use the fine adjustment knob to bring the specimen into sharp focus. f. If necessary, readjust the amount of light with the light intensity control or iris diaphragm. g. Then, and only then, can you observe the specimen with a higher objective. h. Make sure to start on the lowest power objective lens and to focus the image at each magnification before advancing to the next one.

5 Practice using the compound light microscope by examining a prepared slide of Spirogyra or Oedogonium (available at Station 2). Move the stage/slide towards you. Which way does the specimen move in the field of view? Move the stage/slide to the right. Which way does the specimen move in the field of view? What is the relationship between the direction the stage/slide is moved and the direction of movement in the field of view? Count the number of cells you can see in one strand at 4X, 10X, and 40X:,,. What is the relationship between magnification and field of view? Do some parts of the specimen appear to be in focus when other parts are not? Why might that be? The magnification of an objective is indicated on the side of the objective. Other numbers will be visible also (Fig. 3). A second lens within the objective is responsible for limiting its resolving power (the ability to distinguish between two points). The unaided human eye can distinguish (resolve) two objects when they are at least 0.1 mm apart, whereas with the light microscope, the human eye can distinguish two objects as separate when they are up to 1,000 times closer than that. Resolution is indirectly proportional to numerical aperture (NA). The higher the NA value, the smaller R will be. And the smaller R, the better will be the resolution of the objective (and the more expensive!). As magnification increases, so must resolving power. Magnification without increased resolution is not advantageous for study specimens. Why?

6 Fig. 3. An Objective. PART 2 PREPARING A WET MOUNT The material studied with a compound light microscope is usually mounted on a slide. Permanently prepared slides (such as Spirogyra and Oedogonium) are commercially available, but to study living specimens, a scientist must prepare a temporary slide or wet mount. If the material has natural color, a thin section need only be placed on a microscope slide, a drop of water added to the top of the specimen, and a coverslip slowly lowered onto the specimen (Fig. 4). Care should be taken to avoid trapping air bubbles underneath the coverslip. If you examine such a slide for very long some of the water between the slide and coverslip will evaporate. You can avoid letting the specimen dry out by adding a drop of two of water to the edge of the coverslip when needed. Some specimens are colorless and difficult to observe with the microscope. These specimens are usually stained by adding a dye that reacts with one or more molecules contained in the cells of the specimen. Stain adds color to the material being viewed and greatly increases the scientist s ability to observe detail. Fig. 4. Application of coverslip. Make a wet mount of one Elodea leaf using the technique described above. Materials necessary will be available at Station 3a.

7 Examine your slide preparation USING THE DISSECTING SCOPE. Will it be necessary to stain these cells? Why or why not? (Note: The green color of the leaf comes from the presence of small, green spherical structures known as chloroplasts in the cells.) Can you see the cells of the leaf at the lowest magnification level? Zoom in on the leaf. Can you see the cells of the leaf at the highest magnification level? Can you see the individual chloroplasts? Provide an accurate diagram of what you see with this scope at the highest magnification level. Observe the same slide using both the low and high power objectives of the COMPOUND LIGHT MICROSCOPE. When going from low to high power be sure to follow the procedure for use of the compound light microscope. Can you see the cells of the leaf under the lowest power? Can you see individual chloroplasts under low power? If so, approximately how many chloroplasts can you see? Draw an accurate diagram of what you see under low power. Can you see the cells of the leaf under high power? Can you see individual chloroplasts under high power? Can you see individual chloroplasts under high power?

8 If so, approximately how many chloroplasts can you see? Draw an accurate diagram of what you see under high power. In some cells, the chloroplasts may appear to be moving within the cytoplasm. This phenomenon is called cytoplasmic streaming and is due to the movement of the cytoplasm within the cell. Wash your slide and coverslip with tap water. These will be reused in the next step. Prepare a wet mount of your own cheek cells. Obtain these cells by gently scraping the inside of your cheek with a toothpick. If you were to attempt to view these cells with the microscope, you would find them very difficult to see, since they are virtually colorless. To stain your cheek cells, place a drop of methylene blue stain (at Station 3b) at one edge of the coverslip. Place a Kimiwipe or paper towel at the opposite edge of the coverslip so that it absorbs water from between the coverslip and slide (Fig. 5). This will pull the stain under the coverslip. Observe the slide using the low power objective of your microscope. After locating cells, choose one and view it with the high power objective. You should be able to identify the dark-staining nucleus and the light-staining cytoplasm. Draw an accurate diagram of your cheek cells under high power.

9 Which is bigger an Elodea leaf cell or your cheek cell? Fig.5 Methylene blue application Once you have finished place the slide, cover slip and toothpick in the bleach solution under the fume hood. DO NOT wash the slide again. Part 3 - EXAMINING PROKARYOTIC AND EUKARYOTIC CELLS WITH THE COMPOUND LIGHT MICROSCOPE One of the fundamental features of life is that organisms are composed of many cells. As you may know, there are two basic types of cells: prokaryotic and eukaryotic. The distinguishing feature of these two cell types is an intracellular structure called the nucleus. The nucleus is a membranebound structure that encloses a cell s genetic material (DNA). Prokaryotic cells lack a nucleus, and their DNA is only loosely confined to an area within the cell. Eukaryotic cells possess a nucleus. Bacteria are single-cellular prokaryotic organisms. Present-day bacteria are extremely small (approximately 1 2 µm in diameter), and many are devoid of natural color. Morphologically, they are either round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilla). They are often found in clusters or in chains. To view bacteria with the compound light microscope, the cells must be stained, and one must use an oil immersion lens (100X). Even then, not much more than their basic shapes will be visible. Obtain prepared slides of Escherich coli, Staphylococcus aureus and Spriilum volutans from station 4. Observe a prepared slide of Escherich coli. Describe the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective

10 Observe a prepared slide of Staphylococcus aureus. Describe the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective Observe a prepared slide of Spriilum volutans. Describe the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective While eukaryotic cells are typically larger, they too can be difficult to examine with the light microscope. While some eukaryotic cells possess natural color, such as cells of the elodea leaf, many do not (e. g. cheek cells and root cells). In addition specimens viewed with a compound light microscope must be thin, consisting of one or two cell layer thickness. Thus, cells of multicellular organisms are often viewed by taking very thin sections. In both instances, stains must be used in order to observe cellular detail. Stains may be general, staining many parts of the cell, or specific, reacting only with particular biochemical macromolecules. Two cytochemical stains for DNA are Feulgen s stain and acetocarmine. By staining a tissue with one of these stains it is possible to see a cell s DNA. In cells that are not in the process of dividing, the DNA is enclosed within a membrane and is visible as a nucleus. Individual chromosomes will be visible in cells undergoing division. Obtain a prepared slide of whitefish blastula. Describe the appearance of cells (note cell shape, internal structures visible, colors or structures, etc). Record your observations below. Can you see chromosomes in any cells with this level of magnification? Can you count the number of chromosomes contained in whitefish blastula cell with this level of magnification? If so, how many chromosomes are present?

11 TO PREPARE FOR YOUR QUIZ ON THIS LAB YOU SHOULD MAKE SURE THAT YOU CAN ANSWER THESE QUESTIONS. 1. Briefly describe how to clean a glass component (e.g. eyepiece, lens) of a microscope. What materials should be used? 2. Using Fig. 1 (page 2) or a dissecting microscope at your bench, point out/identify the following parts: ocular, base, body tube, arm, stage, focus knob, light sources and switches, variable magnification know. 3. What is the function of each part of the microscope identified in Fig. 1 (page 2)? 4. You are viewing a specimen with the low power objective of the compound light microscope. As you move the stage/slide to the right, in what direction does the viewed specimen move? 5. Describe the difference between magnification and resolution. Which part of the microscope is important is determining its resolving power? If magnification increases, what must happen to resolution in order to obtain a sharp image? 6. Using Fig. 2 (page 3) or a compound light microscope at you bench, point out/identify the following parts: ocular, objective, base, body tube, nosepiece, arm, condenser lens, condenser adjustment knob, iris (and lever), stage, coarse adjustment knob, fine adjustment knob, light and light switch. 7. What is the function of each part of the microscope identified in Fig. 2? 8. What is the largest cell that you observed during this exercise? The smallest? How do/did you make this decision? 9. You are given the option of using three different compound light microscopes. Each of the scopes has a low (10X) and high power objective. The high power objectives for each scope are shown below. Which is the best microscope and why? 43/ / / / / / What is the total magnification available if the first high power objective in question 9 (above) was added to the microscope that you used to complete this exercise?