Homework answers / question archive / LAB TOPIC 4 Part 1 Microscopy: Theory and Practice Objectives After completing this exercise, you should be able to 1

LAB TOPIC 4 Part 1 Microscopy: Theory and Practice Objectives After completing this exercise, you should be able to 1

Biology

Share With

LAB TOPIC 4

Part 1 Microscopy: Theory and Practice

Objectives After completing this exercise, you should be able to 1. Identify the parts of the compound microscope and state the function of each 2. Describe and demonstrate the correct way to : a. Carry a microscope b. Clean the lenses of a microscope, and prepared slides c. Find and focus on the image of a specimen using low and high power objectives on the compound microscope d. Estimate the size of a microscopic specimen 3. Briefly explain the concepts of inversion, and depth of field. 4. State the contributions of the following people to the development of cell theory: Galileo, Kepler, Leeuwenhoek, Hooke, Schleiden and Schwann.

Introduction During most of man’s history, it was not possible to “see” any of the small units that make up living things. The first compound microscope (a magnifying tube with two lenses) was invented by a father and son Dutch team, Hans and Zacharias Janssen between about 1590 and 1600. By 1609 the Italian Astronomer and physicist, Galileo (1564-1642) had heard of the invention and had constructed his own magnifying tube having a power of 32X. Galileo sent one of his magnifying tubes to the German astronomer, Johann Kepler (1571-1630) who used it as a telescope but who also was stimulated to describe a compound microscope (one with more than one lens). Microscopy became all the rage in scientific circles and Marcello Malphigi, an Italian physiologist, used it to study frog lungs (opening the way for an understanding of respiration) and bat wings, resulting in his describing tiny blood vessels later called "capillaries", from the root meaning "hair like". Anton van Leeuwenhoek (1632-1723), a Dutch microscopist, was a contemporary of Malphigi who is perhaps the most interesting observer of microbiological life in the early days of microscopy. He discovered protozoa, bacteria and was the first to describe sperm. His findings greatly impressed and influenced the Royal Society of England. Robert Hooke (1635-1703), secretary of the Royal Society and himself an 88 accomplished microscopist, built microscopes according to Leeuwenhoek's design and was impressed with the Dutchman's discoveries. Hooke is the individual who coined the term "cell" to describe the small chambers he observed in the cork of tree bark utilizing his own compound microscope. Many other investigators working with microscopes studied all kinds of living things. They too identified the cells that Hooke had seen. In 1838, Matthias Schleiden (1804-1881), a German botanist developed a theory that all plants were composed of living units called cells. A year later, a German physiologist, Theodor Schwann (1810-1882), developed what he called the "cell theory" stating that animals as well as plants had cells as the basis for their living structure. Microscopes are precision instruments and essential tools in the study of cells. Please handle microscopes carefully: carry them with two hands, one hand under the base and the other grasping the arm. Microscopes have one or more lens systems, light sources, and mechanisms for adjusting the distance between the lens systems and an object. This exercise provides an opportunity for you to develop expertise in your knowledge and use of microscopes. Outline Section 1: The Compound Microscope Activity A: Identifying the Parts of the Compound Microscope Activity B: Viewing a slide: Letter e Activity C: The Size of the Field: Estimating the Size of Microscopic Organisms Activity D: Depth of Field Section 1. The Compound Light Microscope The name compound light microscope indicates that it uses two sets of lenses and light to view an object. The two sets of lenses are the ocular lenses located near the eyes and the objective lenses located near the object. Illumination is from below, and the light passes through clear portions but does not pass through opaque portions of a specimen. This microscope is used to examine small or thinly sliced sections of objects. Activity A: Identifying the Parts of the Compound Microscope Obtain a compound light microscope from the storage area, and place it securely on the table. Plug the microscope’s wire into the nearest electrical outlet. Using the following descriptions and instructions, identify the following parts on your microscope, and label them in Figure 4.1. 89 Figure 4.1 Compound light microscope. Compound light microscope with binocular head and mechanical stage. Label the drawing of this microscope with the help of the text material. 1. Ocular lenses (eyepieces): You should adjust the eyepieces by carefully moving them apart or together, to match the separation of your eyes. Each eyepiece is marked with a number followed by an X.. That number is the magnifying power of the eyepiece lenses. What is the magnifying power of the ocular lenses on your microscope?___________ 2. Body tube: Holds the revolving nosepiece at one end and eyepiece at the other end; conducts light rays. 3. Arm: Supports upper parts and provides carrying handle. 4. Nosepiece or turret: Revolving device that holds objectives. 5. Objective lenses (objectives): a. Scanning objective: This is the shortest of the objective lenses and is used to scan the whole slide. The magnification is stamped on the housing of the lens. What is the magnifying power of the scanning objective lens on your microscope? b. Low-power objective: This lens is longer than the scanning objective lens and is used to view objects in greater detail. What is the magnifying power of the low-power objective lens on your microscope? 90 c. High-power objective: This lens is used to view an object in even greater detail. What is the magnifying power of the high-power objective lens on your microscope? d. Oil immersion objective: Holds a 100 X lens and is used in conjunction with immersion oil to view objects with the greatest magnification. Your instructor will discuss its use when the lens is needed. As the magnifying power increases, what happens to the length of the objective? To find the total magnifying power from the specimen to your eyes, you must multiply the magnification of the ocular lens by the magnification of the objective lens. Thus when using the 4 X lens, the total magnifying power is 4X x 10X (for typical oculars) = 40X. What is the total magnifying power if you are using the 10X lens?_____________ 6. Coarse-adjustment knob: Knob used to bring an object into approximate focus. With no slide in place, turn the knob. Notice that it lifts and lowers the objective lens A LOT. Use this knob only with the low-power objective. 7. Fine-adjustment knob: Knob used to bring object into final focus. 8. Stage: Holds and supports microscope slides. 9. Mechanical stage: A movable stage that aids in the accurate positioning of the slide. 10.Mechanical stage control knobs (not shown): Two knobs usually located below the stage. One knob controls forward/reverse movement, and the other controls right/left movement.Turn the knobs to decide which one moves the slide in which direction. 11.Condenser: Lens system found below the stage that “gathers” light from the light source and directs that light through the hole in the stage. There is a knob under one side of the stage that controls the height of the condenser: the condenser works best if it is in position at the top (nearest the stage) at all times. 12.Light source: An attached lamp that directs a beam of light up through the object. Turn it on using the switch on the front of the bse of the microscope. 13.Variable light control: wheel-type control at one side of the base. Rotating the wheel from lower to higher numbers increases the brightness of the light. 14.Diaphragm or iris diaphragm control lever (not shown: near 11): Moving the lever regulates the amount of illumination passing upwards through the specimen. 91 With your eye to the side, look at the hole in the stage. Rotate the variable light control wheel through the numbers. What do you notice about the light coming through the hole in the stage with regard to the numbers?____________________ Set the wheel at number 5 or 6. Again with your eye at the side, move the iris diaphragm lever back and forth. What do you notice about the light coming through the hole in the stage? __________________________________________________________________ Notice that you have two different ways to control the amount of light that travels through the specimen. Activity B. Viewing a slide. 1. Turn the nosepiece so that the lowest power objective on your microscope is in straight alignment over the stage. 2. Always begin focusing with the lowest power objective on your microscope (4X [scanning]). 3. With the coarse- adjustment knob, lower the stage until it stops. 4. Go to the front table and take a slide labeled “letter e” from the tray. 5. Hold the slide up to the overhead lights and notice the position of the letter e. Draw that position in the left circle below. 6. Pull the vertical cylinder to open the spring arm on the stage, and insert the slide. Use the two control knobs located below the stage to center the e over the hole in the stage. 7. Again, be sure that the lowest-power objective is in place. Then, as you look through the eyepieces, decrease the distance between the stage and the tip of the objective lens until the object-in this case, the letter e-comes into view, or focus. 8. Once the object is seen, you may need to adjust the amount of light. To increase or decrease the contrast, rotate the iris diaphragm lever slightly, or use the numbered wheel on the base and go to a higher or lower number. 9. Now carefully use the fine-adjustment knob to sharpen the focus if necessary. 10.Practice having both eyes open when looking through the eyepiece, as this greatly reduces eyestrain. 11.Move the slide around until the entire letter e is within the field of view. In the circle at the right, make a rough drawing of what you see. 92 Orientation with naked eye How it looks through the microscope Observation: Inversion 12.What differences do you notice between the e on the left (naked eye) and that on the right (microscope)? ________________________________________________________________ 13.Move the slide to the right. Which way does the image appear to move? _______________________________________________________________ Summarize what the microscope does to specimens. Higher Power Objectives Compound light microscopes are parfocal; that is, once the object is in focus at a low power, it should also be (almost) in focus with the next highest power. 1. Make sure that the letter e is centered in the field of the lowest objective. 2. Move to the next higher objective (low power [10X]) by turning the nosepiece until you hear it click into place. Do not change the focus (move the stage); parfocal microscope objectives will not hit normal slides when changing the focus if the lowest objective is initially in focus. 3. Now pick a portion of the e you would like to study more carefully. Move the slide around until that portion is in the center of the field of view. With your eye at the side of the microscope, slowly turn the high power [40X] objective into place. Again, do not move the stage. 4. Look through the ocular at the letter e. If any adjustment is needed, use only the fine-adjustment knob. (Note: Always use only the fine-adjustment knob with 93 high power.) On your drawing of the letter e (above), draw a circle around the portion of the letter that you are now seeing with high-power magnification. 5. When you have finished your observations of this slide (or any slide), rotate the nosepiece until the lowest-power objective clicks into place, and then remove the slide. If it is a purchased slide, return it to the slide tray in which it belongs. If you have made the slide, put it into the “used slide” containers near the sink. Activity C: The Size of the Field: Estimating the Size of Microscopic Organisms When using each objective, you must know the diameter of the field to estimate the size of observed objects. Estimate the diameter of field for each magnification of your microscope as described in the section that follows. Record your results below. 1.Turn the nosepiece until the scanning (4 X) objective is in position. Place a clear plastic ruler under the objective so that the millimeter lines go horizontally across the field of view. The edge of the ruler should extend across the diameter of the field. Focus on the metric scale with the 4X objective, and move the ruler so one of the millimeter marks is at the left edge of the field. Estimate and record the diameter of the field at a total magnification of 40X (remember, 10 X ocular by 4 X objective lens) by counting the spaces and portions thereof visible in the field. ______________mm diameter at 40 X. 2. Use these equations to calculate the diameter of the field at 100X and 400X. A. Field diameter at 100 X ( in mm) 40X x diameter (mm) at 40 X 100X 1 B. Field diameter at 400 X ( in mm) 40X x diameter (mm) at 40 X 400X 1 94 Results C. DIAMETER OF FIELD a. Indicate the estimated diameter of field at each total magnification for your microscope. Magnification Diameter of Field 40 X mm 100 X mm 400 X mm b. Using the diameters determined above, decide on the estimated lengths of objects that extend across: 1. Two-thirds of the field at 40 X mm µm 2. 25% of the field at 100X mm µm 3. One-half of the field at 40OX mm µm 4. 80% of the field at 40X mm µm (1 mm = 1,000 µm) Activity D: Depth of Field When a specimen is prepared for microscopic examination it is very thinly sliced to allow light to pass through it. Even though the specimen is thinly sliced, it still has some thickness (think of a piece of cellophane -- very thin but it still has some thickness). The amount of material that is in focus when a specimen is viewed under the compound microscope is called the depth of field. The higher the magnification the thinner the depth of focus. This concept is important when viewing objects under high power because the depth of field is thinner than the thickness of the specimen. Therefore the lens must be raised and lowered using the fine adjustment knob to see all levels in the specimen. 95 Figure 4.2 In Figure 4.2 above, under the 10X lens the letters x, y and z would be in focus (and magnified 100X); under the 40X lens (400X magnification) only letter y would be in focus -- in order to see x and z clearly the objective lens must be raised and lowered (respectively) using the fine adjustment knob. Activity From the demo table obtain a slide labeled silk threads. On the slide are three differently colored, cross mounted silk threads. The threads are made of many smaller and finer fibers. With the 10X lens in place, find an area where you can see all three colored threads at once. Focus very slowly up and down with the fine adjustment -- as each thread comes into focus, you will see the fibers of which it is made more clearly than those of the other threads. See if you can determine which thread is on the bottom, which is in the middle and which is on top. Repeat the process using the high power (40X) lens. You may check your answer with the key available from your instructor.