Basic Microscopy for Plant Biology
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1 Page 1 of 8 Basic Microscopy for Plant Biology OBJECTIVES After completing this exercise, you should be able to do the following: a. Name the parts of the compound microscope and the functions of each. b. Describe how total magnification is calculated. c. Know the correct procedure for using and caring for the compound microscope. d. Understand the concept of depth of focus. PARTS OF THE MICROSCOPE Read through this information carefully. Be sure you understand each item before proceeding to the next step. 1. Review how to carry a microscope. Obtain a microscope. 2. The major parts of the compound microscope are shown in Figure 1. As you read this text, refer to this diagram and label the parts that appear in bold print. (Your microscope may be slightly different from that illustrated.) 3. The base rests on the table and, in most microscopes, contains a built-in light source and a light switch. The arm rises from the base and supports the stage, lens system, and control mechanisms. The stage is the flat surface on which microscope slides are placed for viewing. A mechanical stage holds the slide in place with a clip and can be adjusted side to side and forward and back with control knobs. 4. Most microscopes have a condenser located below the stage. It concentrates the light on the object and may be raised or lowered by the condenser control knob. Usually the condenser should be raised to its highest position. Check to see that it is. 5. An iris diaphragm is built into the base of the condenser. The iris diaphragm control lever varies the amount of light entering the condenser and lens system. Move the lever back and forth to see how this works. 6. The body tube is supported by the arm and has a pair of ocular lenses at the upper end. Notice that the ocular lenses move apart and together to adjust for the distance between your eyes. 7. Now find the revolving nosepiece with attached objective lenses at the lower end. The nosepiece is rotated to bring different objectives into viewing position. The objectives will click into place when they are in viewing position. Try this now.
2 Page 2 of 8 8. The magnification of each objective is indicated on the objective itself. An objective with a magnification of 4x is called a scanning objective. A low-power objective has a magnification of 10x. A high-power objective has a magnification of 40x. Finally, the objective with a magnification of 100x is an oil-immersion objective. 9. Look at your microscope and list the objectives it has here: 10. There are two focusing knobs. The coarse-focusing knob has the largest diameter and is used to bring objects into rough focus when using the 4x and 10x objectives. The fine-focusing knob has a smaller diameter and is used to bring objects into fine focus. It is the ONLY focusing knob used with the high-power and oil-immersion objectives. 11. Check to see that all parts of Figure 1 are labeled. MAGNIFICATION 1. The magnification of a microscope is determined by the power of the ocular and objective lenses being used together. The ocular of your scope has a magnification of 10x. You have a list of the objectives above (in item 8). Total magnification is determined by multiplying the power of the ocular by the power of the objective. 2. Enter the objective and ocular magnifications from your microscope in the table below. Calculate the total magnification when each of the four objectives is being used. OBJECTIVE scanning OBJECTIVE MAGNIFICATION OCULAR MAGNIFICATION TOTAL MAGNIFICATION low-power high-power oil-immersion USING A MICROSCOPE The following exercise is designed to help you develop skill using the compound microscope. Follow the steps and check them off as you go. Write answers to all of the questions. 1. Carry the microscope with two hands: one on the arm and one under the base.
3 Page 3 of 8 2. Place the microscope on the lab bench with the arm away from you. 3. Use a piece of lens paper to clean the ocular and objectives of your microscope. Only use specially prepared lens paper on the lenses. You could damage the optics using anything other than lens paper. 4. Plug the microscope cord into the outlet at your lab station. 5. Swing the scanning lens into place. You will hear the nosepiece click when the objective is in position. 6. Looking from the side, NOT INTO THE SCOPE, rotate the coarse adjustment knob one-half turn. Rotate it the other way. What happens to the stage? 7. In the same way, rotate the fine adjustment knob one-half turn. Rotate it the other way. How far did the stage move this time compared with the coarse adjustment? 8. Turn the coarse adjustment so that the stage is as near to the scanning objective as possible. Using a piece of paper, mark off the distance between the tip of the objective and the stage. This is a rough estimate of how far the objective will be from the slide. 9. Now swing the low power objective into place, making sure you hear the click. Mark off the distance to the stage using the same piece of paper. Is it larger or smaller than that for the scanning objective? 10. Repeat the measuring procedure after swinging the high power objective into place. Watch to see that it doesn t hit the stage. What happens to the distance between objective and slide as magnification increases? 11. Why should you NEVER USE THE COARSE ADJUSTMENT WHEN USING THE HIGH POWER OBJECTIVE?
4 Page 4 of 8 THE FOLLOWING PROCEDURE IS THE EXACT SEQUENCE OF STEPS THAT YOU ALWAYS FOLLOW WHEN USING A MICROSCOPE! PAY ATTENTION!!! 1. Put the scanning objective into place. 2. Turn on the light of your microscope. 3. Raise the condenser to its highest position. 4. Looking through the eyepiece, sweep the iris diaphragm control all the way from right to left. What happens? 5. Move the control about midway between the extreme left and right positions. You should now have even illumination over the field of vision. The amount of light will have to be adjusted for different specimens and different levels of magnification. 6. Take a slide of the letter e. Place the slide on the mechanical stage so that the clip holds it securely in place. 7. Using the knobs on the mechanical stage, center the letter over the stage opening. 8. Show how the letter e is oriented on the slide as it appears WITHOUT LOOKING THROUGH THE MICROSCOPE by drawing it in Figure 2. Figure 2. Slide of the letter e as seen without using a microscope. 9. Turn the coarse adjustment knob to position the stage as close to the scanning objective as possible. 10. Look through the ocular and adjust the light so that there is even illumination across the whole field of view. 11. Slowly turn the coarse adjustment toward you until you have the letter in view. If after a few turns it doesn t appear, try centering the slide and adjusting the light again. Repeat the focusing procedure. Now use the fine adjustment for getting sharp focus.
5 Page 5 of Compared to the picture that you drew in Figure 2, is the letter e right side up or upside down? 13. Does it face the same way or is it backwards? 14. Using the knobs on the mechanical stage, move the slide to the right slightly. Which way does the image move? 15. Move the slide up. Which way does the image move? 16. In your own words, summarize what happens to the orientation and movement of objects when viewed through the compound microscope. 17. Draw the letter e as you see it in Figure 3. Try to get it about the right size and in the right orientation. Figure 3. Image of the letter e at 40x total magnification
6 Page 6 of Before changing to a different objective, make sure that the object is in the center of your field of view and in sharp focus. Looking at the SIDE OF THE MICROSCOPE, swing the 10x objective into place. 19. Now use the fine adjustment knob to bring the image into sharp focus. You should not have to adjust it more than ½ turn at any time. It may be necessary to increase the light intensity when you use this objective. 20. How large is the letter e compared to what you saw at 40x total magnification (using the scanning objective)? 21. At this point have your instructor check your work and initial your paper. 22. Draw the letter e in Figure 4. Again show the approximate size and orientation. Figure 4. Image of the letter e at 100x total magnification 23. Looking at the SIDE OF THE MICROSCOPE, carefully swing the high power objective into place. You should not have to touch the adjustment knobs. 24. Remember the coarse adjustment knob is NEVER used with high power. Focus with the fine adjustment knob only. 25. What happened to the light when you changed to high power?
7 Page 7 of Draw what you see in Figure 5. Give a title to this figure. Figure Swing the scanning objective back into place then remove the slide. 27. Check here with your instructor and have her/him initial your paper. MAKING A WET MOUNT 1. Watch carefully as your instructor demonstrates the process. 2. Obtain a clean slide, coverslip and toothpick. 3. Place your sample on the slide. 4. Place the slide on a piece of paper towel. Cover the sample with one drop of mounting medium (ex: water, sucrose solution, etc). 5. Hold the coverslip by the edges between your thumb and index finger. Place one edge of the coverslip over the specimen supporting it with a toothpick as shown in Figure 1. When the coverslip is nearly parallel to the slide, withdraw the toothpick so that the coverslip is floating on the drop of liquid. Figure 1. Making a wet mount 6. Observe your specimen using proper microscope technique.
8 Page 8 of 8 BEFORE PUTTING THE MICROSCOPE AWAY 1. Remove the microscope slide. 2. Turn off the light. 3. Clean all lenses using lens paper. 4. Move the mechanical stage so that there are no parts sticking out. 5. Place the cover on the microscope. 6. Carefully wrap the cord electrical cord around the microscope. 7. Put the microscope in the proper cabinet.
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