3. The Compound Microscope Purpose of Laboratory
Identify the parts on a microscope
Learn proper care and use of a microscope
Observe prepared slides and bacterial smears
Use proper technique with oil immersion
A few warnings: If a lens is not dirty, don’t clean it. Every time, a lens is cleaned, it does get slightly scratched. The only lens that is routinely cleaned is the oil immersion lens. Lower the stage before removing a slide to avoid touching the objective lens. Start with the lowest magnification objective and move up. It will save time and aggravation. Do not waste time with the 40X objective. It does not work without a coverslip. Always bring the stage up before focusing and focus while lowering the stage. Leave the condenser all the way up, close to the slide. You are looking at bacterial smears, not tissue sections.
Set up Refer to diagram and fill in the blanks.
4. If the oculars appear dusty or smudged, notify your instructor who will provide you with a cotton swab or compressed air to clean these lenses. Since the oculars are not equipped with protective shields they are likely to become dusty. Do not attempt to clean the oculars with lens tissue.
1. Obtain a microscope from the cabinet and place it on the bench. Be very careful as you remove the microscope from the cabinet. Grasp the arm firmly with one hand and support the base with your other hand. The scanning objective (with red ring, 4X) should be above the stage, the stage should be in the lowest position, and the electrical cord secured.
5. Place the slide on the stage and secure it within the clamps. Adjust the slide so that the specimen is centered over the beam of light coming from the condenser.
2. Unwrap the electrical cord, plug it into one of the outlets at your bench, and turn on the illuminator. Adjust the brightness control to a little below full brightness (about 70% intensity).
6. With the scanning objective (red ring) in place (vertical over the condenser) and the stage elevated to the stop point, look through the oculars and bring the specimen into focus by moving the stage down with the coarse adjustment. (With bacterial preparations this procedure will
3. While looking through both oculars, adjust the interpupillary distance by moving the ocular objectives closer or farther apart until a single bright circle can be viewed.
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only allow you to see the stained smear, individual organisms will not be seen.).
specimen. Obtain a bottle of immersion oil and, with the applicator, place a small drop of oil on the slide, directly over the stage opening (With most bacterial preparations, the oil is applied directly on the smear, no cover slip is used.). Now carefully rotate the nosepiece and swing the oil immersion objective (with white ring) into the oil. Using ONLY the fine adjustment, bring the specimen into sharp focus.
7. Enter the specimen in the field of view by adjusting the mechanical stage without touching either of the focus adjustments. 8. If the field of view is too bright, the light intensity can be reduced, by adjusting the brightness control. With live mounts this is a necessity. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get.
15. Increase light intensity with the diaphragm. It will improve resolution.
Do not use the iris diaphragm of the condenser until oil immersion is used. The iris diaphragm allows increased resolution with high-powered objectives.
16. When you have finished your observations, turn the oil immersion lens out of position, lower the stage, remove the slide from the stage, and gently clean the oil from the oil immersion lens with a piece of dry lens paper.
9. Rotate the nosepiece so that the 10X low power objective (yellow ring) is brought into place.
17. Be sure to use the lens paper supplied. If you cannot find any lens paper, do ask the instructor. Do not use kimwipes or paper towels. It is the same as using sand paper.
10. View the specimen and use first the coarse adjustment to bring the specimen into focus. The fine adjustment is used to obtain maximum resolution. 11. This step is used only if there is a coverslip. Center the specimen and rotate the nosepiece so that the 40X high power objective (blue ring) is brought into place.
18. Clean any oil from the stage or other objectives. Don’t use a slide that is covered with oil with a dry objective.
12. Use only the fine adjustment to bring the specimen into sharp focus. The lenses are parfocal, that is, when you switch magnification, the specimen remains in focus or close to focused.
Don’t call instructor if you have a problem
Skip the scanning objective
13. If appropriate (greater magnification would be useful), you are now ready to use the oil immersion objective.
Focus using the condenser lens instead of the focusing adjustment
How to waste time and guarantee frustration when using a microscope:1
Start focusing at any stage position and hope that you will eventually find the specimen
Focus at high power using the coarse adjustment and blame the broken slide on either
FOLLOW THIS STEP VERY CAREFULLY 14. Without moving either of the focusing knobs, rotate the nosepiece so that the high power objective is just out of position, a little to one side of the
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The author gratefully acknowledges previous students for the content of this paragraph.
poor quality glass or the microscope or your lab partner, preferably all three.
3. The scanning objective is in the vertical position;
Use plenty of oil so that it drips onto the condenser and takes forever to clean up
4. The stage has been lowered to the stop point.
Clean up
5. The electric cord is wrapped and secured with the rubber band.
1. All oil has been removed. Several pieces of lens paper will be necessary.
6. The scope is back on the shelf, stage facing the back of the cabinet.
2. Turn off the light switch.
Observations 1-Observation of Prepared Slides Choose 4 to 5 slides from the microbiology bacteriology box and observe them with the scanning objective, under low power and high power. View only slides of eukaryotic organisms. No oil immersion is necessary.
Microbiology Slides: Eukaryotes Stagnant water Foraminifera
Protists with shells
Trypanosoma gambienses
Protozoa, agent of sleeping disease, hard to find, note RBC and granulocytes
Hemogregarina
Intracellular parasites of RBC in frogs, note that the cells are nucleated
Vorticella
Protist
Penicillium
“Bread mold”
Staurastrum
Chlorophyta (green algae)
Oedogonium
Chlorophyta (green algae)
Fucus
Pheophyceae (brown algae)
Fuligo
Slime mold
Albugo candida
Fungus, parasite of grass, oomycetes
Schizosaccharomyces
Same family as baker’s yeast, ascomycetes
Puccinia graminis
Fungus, parasite of grass, basidiomycetes
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2-Observation of Pond Water Ponds are nothing more than depressions in the ground where water collects. They are quiet, shallow bodies of water that allow enough sunlight to reach their bottom.
Ponds often support a large variety of animal and plant life. Wind and streams can carry in eggs, seeds, and organisms that develop into various life forms. Pond animals include birds, crayfish, fish, frogs, insects, and turtles. Microscopic organisms also thrive in most ponds. This sample comes from a shallow backyard pond and contains mostly microscopic organisms, algae, and insects larvae. The sample is collected by scraping the surface of aquatic plants and rocks.
Material and Methods Samples of pond water
Concave slides and Regular slides
Transfer pipettes
Vaseline for hanging drop
Cover slips
Toothpicks for hanging drop
Procedure 1. Make wet mounts of the living culture
once you have found something to examine.
2. Squeeze the bulb of the pipette firmly BEFORE inserting into culture. Pull from the bottom
5. You must be patient to find organisms, but it is worthwhile.
3. Put one drop of culture on slide, this is usually sufficient unless specified differently.
6. Although you don’t have to write a report on your observations, try to sketch a few organisms.
4. Observe the drop of pond water under Low Power to scan and find the organisms. High power (400X) is useful
No report due on pond water, but we will log on these sites to identify what we are observing in class.
References http://www.virtualcolony.com/micro/ http://ebiomedia.com/gall/drop/dropmain.html http://www.microscopy-uk.net/mag/indexmag.html?http://www.microscopyuk.net/mag/artjan99/haem.html www.microscopy-uk.org.uk/pond/
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