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Laboratory 1: Use and Care of the Microscope USE OF THE MICROSCOPE The compound microscope is a precision, highly specialized instrument that is designed to allow the viewing of objects that are too small to be seen by the unaided eye. The resolving power of your microscopes is approximately 0.2m (micrometers or microns), which means it can discriminate objects down to a size of 0.2 (1 micrometer is 1/1000 of a millimeter). Unfortunately, many users fail to properly adjust the optical system resulting in unsatisfactory results or resolving power. We will spend one lab period learning how to properly use a microscope. For some of you this may be a review, for others, it may be the first time you were properly instructed on the proper use of a microscope. MAJOR PARTS AND FUNCTIONS Optimum clarity of images produced by the microscope cannot be obtained if the elements of the optical system are dirty. Before using the microscope, always clean the lenses with lens paper provided for that purpose. Never substitute paper towels of facial tissues as they contain grit and will scratch the lenses. Dust may be blown from the lenses with a bulb. Do not touch the surfaces of the glass lenses with your fingers. The compound microscope is basically a tube with magnifying lenses at each end; the objective lens at the lower end, and the ocular lens (or eye pieces) at the upper end. The tube is attached to a supporting frame that is equipped with coarse and fine focusing screws. There is also usually a stage to support the specimen being examined. In the case of binocular microscopes, there is an internal image splitting prism and mirror arrangement to deliver the image to both oculars. Most microscopes have more than one objective lens attached to a revolving nosepiece, which allow the selection of objectives of different magnifications. These objectives are constructed in such a way that they are parfocal; that is, they may be rotated into position for viewing without danger of touching the specimen slide and should require only minor adjustment to bring them into sharp focus. The light necessary for viewing the specimen may be either natural or artificial, and may be directed into the optical system by either a mirror or be beamed directly through the specimen from below. When the mirror is used, the flat side is usually utilized with low magnification and the concave side with higher magnifications. Modern microscopes also have a set of lenses between the light source and the objective called the condenser. The condenser is equipped with an adjustment screw so that the light can be focused to provide the most effective illumination of the study specimen (always use the flat side of the mirror if your microscope has a condenser). Immediately below the condenser is an iris diaphragm (a lever on the condenser) which may be opened or closed to control the total light passing through the optical system. On microscopes with built-in artificial light sources, there is usually a rheostatic control which regulates the intensity of the light beam illuminating the specimen. Parts of the Microscope Label the microscope with the following terms. arm - this attaches the eyepiece and body tube to high-power objective - a large the base. base - this supports the microscope. body tube - the tube that supports the eyepiece. coarse focus adjustment - a knob that makes large adjustments to the focus. diaphragm - an adjustable opening under the stage, allowing different amounts of light onto the stage. eyepiece - where you place your eye. fine focus adjustment - a knob that makes small adjustments to the focus (it is often smaller than the coarse focus knob). lens with high magnifying power. inclination joint - an adjustable joint that lets the arm tilt at various angles. low-power objective - a small lens with low magnifying power. mirror (or light source) - this directs light upwards onto the slide. revolving nosepiece - the rotating device that holds the objectives (lenses). stage - the platform on which a slide is placed. stage clips - metal clips that hold a slide securely onto the stage. ADJUSTMENT OF THE MICROSCOPE The following procedures should be performed to adjust your microscope at the start of each laboratory session: 1. Carefully carry a microscope back to your lab bench. Plug it in. You are responsible for the care and maintenance of this microscope, so please be careful. 2. Clean all glass surfaced carefully with lens paper. 3. Place the 4X objective in position, turn the lamp on at approximately one-half power. Obtain a prepared “letter e” slide and place it on the stage with the specimen centered in the light path. Make sure the slide clips are snuggly against the slide NOT ON TOP OF IT. While looking at the stage, bring the stage to a position near to the objective lens. 4. Look through the oculars. Move the oculars farther apart or closer together until the oculars match your inter-pupillary distance. DON’T LEAN YOUR EYES ON THE EYEPIECES AND MAKE SURE YOU ARE LOOKING THROUGH BOTH EYES! What is your inter-pupillary distance? _____________ 5. Lower the stage with the coarse adjustment slowly until the specimen comes into sharp focus. If both oculars are not in sharp focus, follow these procedures. Close your eye that is over the diopter adjusting ring (usually the left). Use your coarse and fine focus to bring the image into sharp focus with your other eye (usually the right). Now close the other eye, and use the diopter adjusting ring to bring the image into sharp focus. You will probably have to do this every time you use these microscopes, particularly if other people are using them in the meanwhile. 6. Gently close the iris diaphragm. 7. Raise or lower the substage condenser until the edges of the diaphragm are in sharp focus when viewed through the oculars. 8. Slowly open the diaphragm until the edges disappear from the field of view. 9. Your microscope is now adjusted and ready to use. 10. Look critically at the prepared slide. Does the specimen appear the same when looked at with the naked eye as it does when you look at it through the microscope? How is it different? ILLUMINATION Increase or decrease the light intensity of the lamp by turning the variable rheostat knob on the base of the microscope. Generally, you should avoid moving the substage condenser to adjust the light intensity. However, for some purposes at lower powers better contrast can be achieved by lowering the substage condenser. OBJECTIVE LENSES 1. Magnification of your specimen is a determined by the magnification of your objective lenses (i.e.4X, 10X, 40X, and 100X) times the magnification of your ocular lens (ours are 10X). Therefore, fill in the following information: Objective Lens 4X 10X 40X 100X Ocular Lens 10X 10X 10X 10X Total Magnification 2. Always start your study with the lowest power objective. Do not try to focus on a fresh slide with an objective higher that the 10X. Start with the prepared slide at 4X. Make sure it is focused and centered in the field of view (the illuminated circle you see when you look in your microscope). What percentage of the total field of view does the specimen take up? _______ Now, switch to the 10X objective and use the fine focus to bring it into focus. What percentage of the total field of view NOW does the specimen take up? ________. Do it again for the 40X and again notice the percentage of the total field of view that the specimen takes up. OIL IMMERSION OBJECTIVE- Think about but do NOT do. 1. The 100X lens is meant to be used with immersion oil. We won’t be doing this today as the procedure is messy and it is very easy to break specimen slides. 2. Don’t attempt to look at specimens under 100X as the image will NOT resolve without the use of immersion oil. 3. However, if you wanted to look at very small specimens such as bacteria, you would have to use 100X and immersion oil. To do this, place 1 drop of immersion oil on the specimen in the field of view. Swing the 100X lens into the oil and carefully use your fine focus to locate the specimen. NEVER GET IMMERSION OIL ON ANY OTHER LENS EXCEPT 100X. Always wipe ALL the oil off the 100X lens immediately after use, for if the oil dries on the lens, it can ruin it. GENERAL CARE 1. The microscope is a precision instrument. Carry the microscope carefully, avoiding any bumping or jostling that may knock it out of alignment. 2. Keep the microscope dusted and the optics clean. 3. At the completion of the laboratory or study session, turn the lamp off, place the 4X objective in position, lower the stage with the coarse adjustment knob, move the mechanical stage clamp to the extreme left position, and replace the microscope dust cover. 4. Inform an instructor if your microscope is not working properly. Do not attempt repairs yourself. Live Specimens 1. 2. 3. 4. 5. To make a wet mount, first obtain a clean microscope slide. Using your disposable Pasteur pipette, obtain a drop of aquarium water from the bottom of the container. If you get the drop directly off the bottom you have a better chance of finding live specimens. Place a drop of water/specimen on your glass slide. Gently place a cover slip over the water/specimen. Start at 4X and try and locate a living specimen. Carefully scan the entire slide. When you find a specimen, draw a picture and label as many parts as possible. For each organism you find, write what environment (water source you found it in), some descriptive characteristics, how does it move and anything else you find interesting. Repeat for the other water/specimens. If you find more than 4, you can draw them and describe them on separate paper. Draw a specimen here: Write descriptions/ water source and locomotion next to the drawing. Draw a specimen here: Write descriptions/ water source and locomotion next to the drawing. Draw a specimen here: Write descriptions/ water source and locomotion next to the drawing. Draw a specimen here: Write descriptions/ water source and locomotion next to the drawing.