THE MICROSCOPE One important technique for a Microbiologist to master is use of a binocular light microscope. Because Cellular Biology is a prerequisite for Microbiology, you should have some experience with the care and use of a light microscope. The small size of bacteria, however, makes them especially challenging subjects to view on a microscope. Your first challenge will be to locate the bacteria on a slide. Once located, it can be easy to lose them, especially when changing objective lenses. I. Use and Care of the Microscope: A. Care of the microscope 1. Carry the microscope with BOTH hands, one on the arm and the other under the base. Do not tilt the microscope when carrying it. 2. Clean the lenses with lens paper only (not a paper towel or Kim wipes). Lens paper is very finely textured and will not scratch the lenses. You should clean your lenses at the end of every laboratory session. Dirty lenses do not allow the light to pass through your microscope properly and will compromise the image. Clean top eyepiece (ocular) lens occasionally as eyelashes leave an oily film. 3. Always remove the slide and clean lenses when finished. 4. Clean stage and condenser if oily. Keep clean. 5. Do not force parts. Call your instructor if you should require assistance. 6. Always raise the objective (or lower the stage) while focusing so that the specimen and lens move apart from each other. 7. Store your microscope with the low power objective down and no oil on lenses or stage. B. Using the accompanying diagrams (Fig. 1), review of the parts of the microscope 1. Ocular lens(es). Each ocular contains an ocular lens, which magnifies ten times (10X). The distance between your oculars can be adjusted to your pupil distance. 2. Objective lenses. Three lenses are located on the nosepiece. Each lens magnifies by a different power and each lens has a different name. The low power lens magnifies 10 times (10X), the high dry lens magnifies 40 times (40X) and the oil immersion lens magnifies 100 times (100X). The total magnification of the microscope is the product of the magnification of the ocular lens times the magnification of the objective lens being used. 3. Rotating nosepiece. Turn it carefully and note that each objective lens clicks into place directly above the opening that lets light through the stage. 4. Mechanical stage. Note the spring-loaded lever which acts to hold a slide in place. by turning the Mechanical stage adjustment knobs, your slide can be moved from side to side and front to back. 5. Coarse focus adjustment knob. This knob raises and lowers the nosepiece. Never use this knob with the 40X or 100X objective in position. This will prevent you from forcing an objective lens through a slide. 6. Fine focus adjustment knob. This knob is used while looking through the eyepiece to refine focus on an object. An experienced microscope user always has their hand on this knob and will constantly focus up and down in order to see a specimen in three dimensions. 7. Condenser lens. The condenser lens focuses light from the light source onto the specimen on the slide. The position of the lens is adjusted with the condenser adjustment knob. 1
Figure 1. A compound microscope. II. Principles of Microscopy: A. MAGNIFICATION is the result of one or more lenses. The objective lens (nearest the specimen) magnifies and produces a real image. The eyepiece or ocular lens magnifies the real image producing a virtual image that is seen by the eye. The total magnification is determined by multiplying the magnification of the objective by the magnification of the ocular lens, i.e. if you use 4X objective, the magnification will be 40X. 2
B. RESOLUTION is the ability of a microscope to distinguish between two closely adjacent points and is determined by the wavelength of light from the illuminator. The area that you see through the ocular eyepiece is called the fields of view. When the specimen becomes blurry as the magnification increases, the size of the field of view decreases. However the resolution of the image remains unchanged do fine focus knob must be adjusted to bring the image into focus again. C. WORKING DISTANCE is the space between the objective and the specimen when the specimen is in focus. The higher the magnification, the smaller or the shorter this distance. CAUTION: Due to the closeness of the 40X and 100X lenses to the slide, you CANNOT focus using the coarse adjustment knob. In addition, care must be taken even with the 10X lens. If a slide is too thick, the 100X objective lens will strike the coverglass. This could damage BOTH the lens and the slide. D. PARFOCAL AND DEPTH OF FOCUS The objective lenses are PARFOCAL. This means that when an object is in focus in the VERTICAL center of one objective lens, it will be approximately in focus with all other objective lenses. The lenses of a microscope provide a limited depth of focus. This means that only part of the object is in sharp focus; areas above and below that part will be slightly out of focus. III. Procedure A. Viewing prepared slides 1. Obtain a prepared slide. Place the prepared slide on the stage, specimen side up. 2. Center the specimen over the stage light hole as carefully as possible. 3. Open the diaphragm and lower the low power objective until it cannot be lowered further. 4. Slowly raise the objective (or lower the stage depending upon the type of microscope you are using) using the coarse adjustment until the specimen comes into view. 5. Bring the specimen into sharp focus with the fine adjustment. Reducing the diaphragm opening may be necessary. Generally, the diaphragm will be almost closed when using the low power objective. 6. Shift to the high power objective by rotating the nosepiece until the objective clicks into place. Focus using the fine adjustment only. It may be necessary to open the diaphragm about halfway. 7. After bringing the specimen into focus using the high power objective, you are ready to use the oil immersion objective. Focusing of the oil immersion objective requires more care than that of the other objectives, but the procedure is essentially the same. 8. Rotate the nosepiece slightly so that you can place a small drop of immersion oil on the portion of the slide that will be directly under the objective. Then rotate the nosepiece until the objective clicks into place. The tip of the objective should be immersed in the drop of oil. Using only the fine adjustment, slowly focus until the image appears. More than likely, the diaphragm will need to be opened further to obtain optimum illumination. 9. Draw a few representative cells of each type of bacteria in your lab journal as they appear under oil immersion. Make the drawings reasonably large. Be sure to label your drawings. HINT: One trick to assist you in focusing on bacteria on prepared slides at low magnifications is to begin by focusing on the edge of the cover slip. Then, move toward the center of the cover slip. You should now only need to fine focus your slide. When viewing stained slides WITHOUT cover slips, make a mark with a grease pencil near the smear (where the bacteria will be) and focus on this. Again, once you have done so, you can move to the area of the smear and it should be in approximate focus. B. Making a wet mount slide of pond water Under the 10X objective and with reduced light, make a survey of the drop of pond water. Identify as many of the organisms as you can. 3
Fig. 2. Protozoans commonly found in pond water Amoeba Paramecium Stentor Fig. 3. Unicellular algae commonly found in pond water Euglena Chlorella Diatom Diatom Fig. 4. Filamentous and colonial algae Spirogyra Oscillatoria Pediastrum Fig. 5. Invertebrates commonly found in pond water Rotifers Hydra Daphnia 4
Study Guide 1. What is the total magnification with the following objective lenses in place? 10X 40X 100X 2. In your own words, what are the steps you would take in order to find a bacterium and then observe it under oil? 3. Why are the lenses of a microscope ALWAYS cleaned with lens paper and NOT with Kim Wipes? 4. How many different structures do you see in pond water? Sketch at least 3 different organisms observed in your sample of pond water. 5. When scanning for an object, which objective should you use? 6. List some similarities among the cheek cells, onion cells, and organisms found in the pond water. 5