04.Prokaryotes and Microscopy (1).pdf

Lab 4 Microscopes and Prokaryotes Part 1: Microscopy Review Objective After completing this lab, you should be able to: 1. Name the parts and functions of the compound microscope. 2. Know the proper procedure for looking at specimens. 3. Know how to safely us...

Lab 4 Microscopes and Prokaryotes Part 1: Microscopy Review Objective After completing this lab, you should be able to: 1. Name the parts and functions of the compound microscope. 2. Know the proper procedure for looking at specimens. 3. Know how to safely use and put away the microscope. Introduction 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 cannot 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 cannot focus closer than about 10 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 the microscope 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. Only clean lenses with lens paper. Never use paper towels or kimwipes. 5. Make sure that the stage is lowered, the lowest power objective lens is in place, and the light intensity knob is turned low. Turn on the illuminator, gradually turn up the light intensity knob, and place the slide into the slide holder. Use the stage control knobs to position the specimen over the light source. 6. Watching from the side, use the coarse focus knob to raise the stage so that objective is as close as possible to the specimen. 7. While looking through the ocular, lower the stage and focus downward with the coarse focus knob until the specimen is in focus. Never focus upward with the coarse focus. 8. 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. 9. Regulate the light intensity with the iris diaphragm. You may also need to move the substage condenser up or down to adjust the focal point of light passing through the condenser lens. 10. 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. 11. Adjust the fine focus to sharpen the image. Never use coarse focus once you leave low power. 12. Before removing the slide always return to low power and move the objective away from the slide with the coarse focus knob. 13. 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: diaphragm stage objective lenses (4x, 10x, 40x) (We do not use the 100x lens) revolving nosepiece ocular lens (10x) coarse focus fine focus Estimating the size of objects on the microscope using a ruler Note: this method works best when the item of interest is along the vertical dimension of the stage. Compare your results using this method by using an object of known size (one square of graph paper). 1. Get the object in focus and in the center of the field of view. 2. Look through the right ocular with your left eye (or vice versa), and your other eye shut. 3. Hold a ruler upright against the edge of the stage (same distance from your eye as the slide). 4. Slowly open your other eye, and try to look at both the image and the ruler at the same time. “Measure” the length of the item using the ruler to the nearest cm or 0.5 cm. 5. Calculate the actual size by dividing the measured length by the total magnification. Actual Length = Measured Length / Total Magnification Microscope Practice 1. Graph Paper: Estimate the length of one square of graph paper using the method above. 2. Amoeba: Practice getting an amoeba in focus at low, medium, and high power. Exercise 5: Bacteria Cells Related reading: Campbell Biology in Focus (2e or 3e): Chapter 24 Related reading: Photographic Atlas of Biology (6e): Chapter 3, pages 27-32 Objectives After completing this lab, you should be able to: 1. Recognize the structure and shape of bacterial cells and colonies. Procedure 1. Examine each of these three types of bacteria to see representative cell shapes. 2. Draw a picture of the cells. 1. Bacillus, a rod-shaped bacterium. 2. Rhodospirillium, a spiral-shaped bacterium. 3. Streptococcus, a chain-shaped bacterium. Some species of streptococcus are pathogenic (strep throat, meningitis, etc.), while other species are a normal component of the “flora” of the human mouth and skin. Note: this one is very small and hard to find.