Lab 4 - The Microscope Part A: The parts of the compound microscope. Obtain a microscope as indicated by your instructor. Always carry the microscope with one hand under the base and the other hand holding the arm of the microscope. Below is a list of the major parts of the microscope. Place the labels on the photograph of the microscope on your lab sheet. When transporting a microscope you should always pick it up by grasping the large arm with one hand and the support base (lowest part) with the other hand. The arm supports an inclined head or binocular body that in turn supports the magnifying elements of the microscope. At the upper end of the head are the two ocular lenses or eyepieces one or both of which may have an ocular focus knob. It is called a binocular microscope because you can, and should, use two eyes for effective use of this instrument. Attached to the arm on the underside of the head are the four other magnifying lenses that are mounted into the revolving nosepiece. These four lenses are called objective lenses and they include a scanning objective (4X), a low power objective (10X), a high power objective (40X) and an oil immersion lens (100X) although some microscopes have a second high power objective (60X) instead. Underneath the tube and revolving nosepiece is the stage. This is a square flat plate with a central opening that is the surface upon which the specimen (i.e. a slide) is placed. Directly under the opening in the center of the stage is a large condenser. The condenser serves to focus and concentrate light from the illumination lamp (mounted in the support base) to increase the intensity of the light that contacts a specimen on the stage. A small screw on the right hand side of the condenser holds the condenser in place (it should never be loosened as its release will cause the condenser to fall and smash on the bench or 30
floor). The correct amount of light is critically important for the effective operation of the microscope. As such, controls are available for carefully adjusting the light levels. The iris diaphragm is immediately beneath and attached to the condenser. This device will be adjusted every time that you change an objective lens. The diaphragm control lever is a black metal bar that is located below the condenser and it controls the iris diaphragm. Moving the bar to the left will open the diaphragm whereas moving it to the right will close the diaphragm. Orienting and centering a specimen for examination using a microscope requires practice. All specimens that you will examine using this microscope will be mounted on glass slides. The stage is equipped with a slide holder that is mounted on the stage itself. The slide-holder is comprised of two upright bars near the back of the stage on the right hand side. Squeezing together these upright bars enables placement of your slide in the slideholder. This allows you to insert a slide and gently release the bars such that a spring will hold the slide in place. The position of the specimen can now be adjusted in two dimensions using the stage adjustment knobs. These are the two knobs that are mounted on a vertical bar that hangs from the right hand side of the stage. The upper knob moves the stage (and the specimen) horizontally from front to back whereas the lower knob moves the stage horizontally from left to right. This will allow you to center a specimen for examination. The final dimension for orienting the specimen is in the vertical plane. This serves to bring a specimen into focus and is performed by raising or lowering the stage using the stage adjustment knobs. The coarse focus adjustment knob is a large knurled knob that is present on both sides of the microscope near the bottom of the arm. The fine focus adjustment knob is a smaller knob mounted in the center of the coarse adjustment knob. Finally, these microscopes are equipped with a built in light source. Once plugged into and outlet, and on/off switch at the front or back of the support base controls power to the illumination lamp. The intensity of light produced by the lamp is controlled using the illumination control knob that is mounted on the side of the support base. Each objective lens has at least four different numbers printed on its shaft. Using the 40X objective as an example, the first number is the magnification (40), the second is the numerical aperture (0.65), the third is the length of the tube in millimeters (160), and the last is the proper coverslip thickness in millimeters (0.17). 31
B. How to use the Microscope Now that you know the parts of the microscope you will learn how to use it properly. Obtain a slide of the letter e and follow the procedure below step by step. These steps are to be followed each and every time that you use a microscope. Note: Microscopes are precision instruments and the lenses must be kept clean and properly aligned. It is important to avoid getting fingerprints or any fluids on lens surfaces. Foreign materials can damage lens mounts and can reduce resolution and distort the observed image. Check that your microscope is clean and in good order and tell your instructor if there is a problem. 1. Place the microscope a few inches from the edge of the bench with the microscope arm facing away from you. It should sit squarely in front of your left shoulder if you are right-handed or squarely in front of your right shoulder if you are left handed. 2. Make sure that the scanning objective lens (4X) is in the optical axis (i.e. pointing straight down at the opening in the stage). The lens should click into place when properly aligned. 3. Make sure that the sub-stage iris diaphragm is fully open by sliding the diaphragm control lever fully to the extreme left. The stage should be positioned at least ¼ turn of the coarse adjustment knob below its highest point. 4. Set the illumination control knob to the lowest setting before you plug in the microscope. 5. Plug in the microscope power cord and turn on the power using the toggle switch at the front or back of the support base. Adjust the light intensity to a comfortable setting for your eyes. 6. Place the specimen slide (the slide with the letter e ) on the stage as follows. Squeeze the two upright metal bars of the slide holder and insert your slide such that the spring will hold the slide in place. Use the stage adjustment knobs to center the specimen on the microscope stage and center the e directly under the objective lens. 7. Look through the microscope. Raise the stage slowly wing the coarse adjustment knob, until the specimen comes into view. Learn and remember which direction the stage moves when you turn the knob toward or away from you. 32
Note: If you fail to locate the specimen before the stage reaches its highest point, lower the stage slowly for one full turn of the coarse adjustment knob. If you still do not see the specimen, check to see if the specimen is in the center of the field and try again. 8. Customize your microscope for proper and effective use (How to focus on a specimen): a. You must adjust the distance between the eye tubes that hold the ocular lenses in order to customize your microscope for proper use. Every person had a different distance between the pupils of their eyes and this must be adjusted so that you can comfortably view a specimen using both eyes. b. Many microscopes have ocular lenses that are focusable in order to compensate for the difference between your eyes. This enables you to customize your microscope for proper use because it allows you to compensate for the refractive differences between your eyes. For example, you might wear glasses with lenses of different prescriptions. You do not need to use glasses to use these microscopes since you can focus each eye tube separately. Everyone should perform the following steps to ensure that the microscope is in focus for you. Your eyesight will be different than the person that used the microscope before you. Look for a focus ring on one of your ocular lenses (if there is one on both lenses you can start the next procedure with either eye). Close the eye on the side that has the focus ring (i.e. if the focus ring is on the left, close your left eye). Looking through the other ocular lens, focus the microscope using the fine focus adjustment knob to bring the letter e into sharp focus. Now close that eye and open the other. Do not touch the fine focus adjustment knob. Using the focus ring on your ocular lens bring the letter e into sharp focus for that eye. The letter e should now be in sharp focus for both of your eyes. 9. Now that you have learned to focus your microscope you will practice increasing the magnification. a. First, you must position the slide so that the area of the slide that you wish to examine in more detail is in the exact center of the field of view. 33
b. Without changing the focus, rotate the revolving nosepiece until the 10x low power objective lens clicks into place. c. Remove one of the ocular lenses from an eye tube. Close the iris diaphragm control lever until you can detect a hexagonal shape in the field of view. Re open the diaphragm control lever until the hexagon just disappears from view. Opening further with result in loss of contrast in your specimen. The diaphragm must be adjusted every time you change magnification. d. Focus on the specimen using the fine focus adjustment knob. The specimen should be nearly in focus before you adjust, so this should only require small movements in one or the other directions. 10. To increase the magnification from 10X to 40X follow the same procedure as above. Be sure to adjust the diaphragm as outlined above to ensure that the proper amount of light is directed through your lens. Note: As you change the power of the objective lens the field of vision changes. This is why you must ensure that the specimen is in the center of your field of view before moving to a higher power lens. 11. Once you have finished viewing the specimen you should turn the light intensity down and turn off the power of the microscope. The revolving nosepiece should be used to place the 4X-scanning objective into the optical axis before removing the slide. C. Using the Microscope 1. Orientation of the letter e : Place the slide with the letter e on the stage such that the e is read properly as you face it without using the microscope. Use the procedure outlined above to bring the e into sharp focus using the 10X objective lens. How does the e appear to be oriented when viewed through the microscope? In which direction does the e move when you move the slide: to the right? To the left? Away from you? Towards you? Answer questions 1-5 on your lab report sheet. 34
2. Understanding depth using silk strands. Next, you will learn how to observe the third dimension (depth) of an object and how to tell the top of a specimen from the bottom. Look at the stage of your microscope and observe whether the stage is raised or lowered as you rotate the coarse focus adjustment knob. Note: A clockwise rotation using the knob on the left side will move the stage in the same direction as a counter-clockwise rotation on the right side. Follow the steps outlined above to examine the slide of the three colored threads. Center and focus the point where the 3 colors of thread cross by using the 4X, the 10X and the 40X lens. Be sure to adjust the diaphragm after switching to the 10X and 40X lenses. Use the 40X objective to determine which thread is on the top (as opposed to appearing to be on the top), which is in the middle and which on the bottom: Raise the stage slightly above the focal point of the 40X objective lens by turning the right hand fine focus adjustment knob ¼ turn clockwise (the threads should be out of focus). Turn the fine focus adjustment knob ¼ turn counter-clockwise to lower the stage slowly. The first thread that will come into clear focus will be the one that is on the bottom and the last thread to come into focus will be the one on the top. If the specimen does not come into view after lowering the stage, raise the stage and switch back to the 10X lens. Re-center and re-focus on the specimen before returning to the 40X objective. A good microscopist will focus up and down continually while observing a specimen, and you should acquire this habit as soon as possible. Note: Never raise the stage to its maximum height when using the 40X objective; this could smash the cover glass and slide and will damage the lens. 3. Preparing a wet mount. Now you are ready to look at biological material. Remove one leaf from the stem of Elodea (a common fresh-water plant). The young leaves at the growing tip are best for this exercise. Place the leaf in one or two drops of water on a slide. Now take a cover slip lightly by the edges. Drag the cover slip along the slide surface until it 35
contacts the water and the water spreads along its edge. Lean the cover glass slowly over the water drop and let it gently lower over the leaf allowing all of the air to escape from under the cover slip. This will take some practice but when mastered will allow for the preparation of wet mounts that are free of air bubbles. a. Examine your mounted leaf under 4X and note the outline of the leaf. Switch to the low power lens (10X) and observe the shape and appearance of the cells of the leaf. (Be sure to center, focus and adjust the diaphragm every time you change magnification). Use your 40X objective to count the number of cell layers in the leaf by using the fine focus knob to slowly focus up and down through the stack of layers. Start with the stage just above the plane of focus and slowly lower it. As you proceed from the bottom layer to the layer above it, you can see changes in the size, shape and position of the cell walls. The number of cell layers equals the number of different patterns of cell walls that you see as you change the focus. Note: Layers below the top will be blurred because light from deeper layers must pass through other cell structures to reach your eye. b. Once you have counted the number of layers in the blade of the leaf, change to the 10X objective and move the slide so that the edge of the leaf crosses the middle of your field of view. Switch back to the 40X objective. Look at a cell that is at the boundary between the leaf and the water. The pattern of cells should not change as you focus up and down because there is only on layer of cells present here. 4. Observations of cell structures and organelles in the plant Elodea. Observe a single cell located just in front of a spine cell at the extreme outer edge of the leaf. One side of this cell must form the boundary between leaf and water. Focus carefully and look for the following structures. Answer question 5 on your lab report sheet. All living organisms are composed of basic units called cells that carry out the processes fundamental to life. In multicellular organisms, cells are specialized and grouped together to form tissues. The structure of each type of cell is intimately linked to the function of the tissue as a whole. In a cell on the extreme outer edge of an Elodea leaf, you should be able to see most of the following features that are typical of a plant cell. 36
Cell wall: a thick barrier surrounding the cell. This will be easiest to see on the side of the cell that is touching the water around the leaf. Vacuole: a large transparent fluid-filled area that fills the central part of most mature plant cells. A vacuole appears to be an empty space within a cell. It occupies most of the volume of the cell. (If your cell does not have a large transparent vacuole, ask for help, you may be looking at the wrong part of the leaf). Cytoplasm: the contents of the cell in the extremely narrow space between the vacuole and plasma membrane (excluding the chloroplasts and other inclusions). The cytoplasm usually appears slightly granular due to the presence of structures that are too small to be readily visible in the light microscope (i.e. mitochondria or ribosomes). Chloroplasts: bright green organelles that are found in plant cells (but not all plant cells; e.g. onion cells). Nuclear membrane: the membrane that surrounds the nucleus. This may be difficult to see in the Elodea leaf. Nucleus: a relatively large transparent structure surrounded by a nuclear membrane. The nucleus is often roughly spherical and contains the cell s genetic material (DNA). It may be difficult to see in some cells if it happens to be near the top or bottom surface of the cell (remember cells are 3D and therefore have depth). Observe the nucleus in the onion cells on demonstration (if available). Plasma membrane: the membrane separating the cell contents from the environment. In plants, it lies tightly adhered against the cell wall and it cannot be observed unless the cell is placed in a concentrated salt solution. Once you have completed the drawings and measurements outlined in your lab sheet and the next section you can transfer a 20% sodium chloride (NaCl) solution to your slide to observe the membrane. Apply two drops of this solution to one edge of the cover slip. Touch a small piece of Kimwipe to the opposite edge of the cover slip to draw the fluid under the cover slip. Repeat this procedure once more to be sure the original water has been replaced by the salt solution. Examine the slide using the 40X objective to observe the effects. 37
Lab Report for Microscope Lab 1. The parts of the compound microscope. Label the parts of the microscope pictured below. 41
2. When examining the letter e using the microscope, how far has the image been rotated? Draw a picture of the image as seen through the microscope. 3. When you move the stage to the right which direction does the image move? What if you move the stage towards you? 4. When you examined the slide of the three colored threads: Which color was on top? Which was in the middle? Which was on the bottom? 5. Draw a cell at the edge of your Elodea leaf and label five of the features that are typical of plant cells. Your drawing should be between 5 and 15 cm long. Include with this drawing a title and labels for all visible structures and organelles. 42