• Use the terms listed and the figure of the microscope in the to complete Pre-lab questions 1-12 or Part A of the Lab Report.
• Arm
• Condenser
• Coarse focus knob
• Fine focus knob
• Head
• Iris diaphragm
• Iris diaphragm lever
• Lamp
• Objective lenses
• Ocular lenses
• Rotating nosepiece
• Stage
General Instructions
• The microscope is a precision instrument that must be handled and maintained properly. A few initial instructions are in order.
1. Carry the microscope in the upright position and with both hands. This prevents moveable parts, like the ocular lenses, from falling and breaking.
2. The glass of the lenses is soft and scratches easily. Use only lens paper to clean the lenses. Avoid the use of handkerchiefs, facial tissue, paper towels, or Kimwipes. Avoid touching the ocular or objective lenses as the glass is easily corroded by acids present in fingertips
3. Always cover moist, living, or preserved materials with a cover slip. Always maintain a safe distance between the cover slip and the objective lens in order to avoid damage to the lenses.
4. Always begin an examination of a slide with the scanning-power (4x) objective. Always look at the objective lens when you swing it into position. These precautions will prevent cracked slides and damaged objective lenses.
5. Report any problems (parts missing, loose parts, blurred images when looking through the microscope, burned-out light bulbs) to the instructor immediately. Don't put a problem microscope back on the shelf.
6. At the completion of the lab exercise, please store the microscope properly:
a) Remove all materials from the microscope stage and wipe the stage surface free of dirt or water.
b) Make certain the scanning objective (4x) lens is locked in place over the hole in the stage and lower the stage as far down as it will go.
c) Turn the voltage control dial to 1 and then turn off the light.
d) Wrap the power cord around the microscope base and tuck the power cord plug into the appropriate place.
e) Return the microscope to the numbered slot in the cabinet that corresponds to the number on the back of the microscope.
Setting up the Compound Light Microscope
1. Position the microscope on the table with the ocular lenses pointing toward you. Adjust yourself so that you can look through the ocular lenses easily without having to tilt the microscope towards you.
2. Unwind and insert the plug into the nearest electrical outlet. Wrap and excess cord around the base of the microscope. For safety reasons, do not let the cord hang over the table.
3. Turn on the light and adjust the voltage control dial to 7 or 8. This will give you enough light to view through the scanning-power objective lens without irritating your eyes.
4. Clean the ocular lenses, objective lenses, condenser glass surface showing through the hole in the stage, and the lamp surface with lens paper.
5. Click the scanning-power objective lens in place. You will hear a "click" as the objective snaps in place.
6. Check the position of the condenser. Using the condenser knob, raise the condenser to the top surface of the stage and then back it down a short distance to ensure that it is not protruding above the stage. You don't want the condenser surface scratched by microscope slides.
Obtain a letter "e" slide from your instructor.
• The following exercise uses the printed letter "e" to demonstrate how to center, find and focus a specimen under the microscope. As you work, complete Part B in the Lab Report.
1. Place the letter "e" slide on the microscope stage with the labeled surface up. Open the pincer-like clip of the slide holder with your right hand and slide the "e" slide squarely into place. When you release the tension on the pincer-like clip, it should hold the slide firmly in place. Center the "e" over the stage opening using the mechanical stage controls.
2. Lock the scanning (4X) objective lens in place. While watching from the side (not though the ocular lenses), use the coarse adjustment knob to raise the stage as far up as it will go. This is very important. You must bring the specimen near enough to the objective lens so that the objective actually sees the specimen.
3. While looking through the ocular lenses with both eyes, turn the coarse adjustment knob slowly until the letter "e" comes into view. If you cannot find the "e" move the slide right-left or toward-away from you using the mechanical stage control dials until the letter is centered in the field of view.
4. Sharpen the image with the fine focus knob.
5. Another important point to remember when trying to find and focus specimens is that these microscopes are parfocal. Parfocal means that when an image is focused with one objective lens, it will be at, or near, focus with the other objective lenses. As long as you are in focus with one objective lens you can move to the next highest magnification without damaging the lens or slide as long as you don't move the stage.
a) Now switch to the low power (10x) objective lens. Use ONLY the fine focus knob to focus the letter "e".
b) Center the image in the field of view using the mechanical stage control dials.
c) Now switch to the high power (40x) objective lens. Use ONLY the fine focus knob to focus the letter "e".
d) Center the image in the field of view.
• Use this procedure for every slide you study. Taking the time to find the specimen with the scanning power, low power and then high-power objective lenses will save you time and you won't break the slides or scratch the objective lenses.
• When you are done viewing the letter "e" return to the scanning objective lens, bring the stage down, remove the letter "e" and return it to your instructor.
• Many of the mutualistic microorganisms that populate humans live in the digestive tract. Most of these microorganisms are members of the Domain Bacteria. There are many bacteria that live in the human mouth, no matter how clean you think your mouth is they are there.
• The variety of bacterial populations in the mouth is dependent on factors such as diet, toothpaste, mouthwashes, and cigarette smoke.
• Some environments will support large diverse bacterial communities while other will support very few.
• In the exercise, you will use the microscope to visualize the bacteria living in YOUR mouth.
Procedure
1. With a toothpick, scrape some material from between your teeth.
2. Smear the collected material over the center of a flat slide in an area about the size of a dime.
3. Allow the smear to dry.
4. Pass the dried slide, smear side up, slowly through the flame of a lamp twice to kill the bacteria and adhere the bacterial cells to the slide.
5. At the staining station, cover the smear with crystal violet stain and let stand 60 seconds.
6. Do NOT allow the stained smear to dry completely; add enough drops to prevent drying.
Use Caution: Crystal violet can stain your skin and your clothes!
7. Using a squeeze bottle of water over a sink to gently rinse the slide. Direct the water above the smear instead of directly on it. Be gentle, as vigorous rinsing may wash the bacteria away.
8. Do not add a cover slip. Observe the smear under the microscope. Bacteria are very small (0.2-10um), search for purple areas under low power.
9. When you can visualize a clump of bacteria, move it to the center of the field of view before turning to the high-power objective lens. The organisms form colonies. Try to locate individuals in the mass of bacteria.
10. Sketch the bacteria in your Lab Report.
11. Place your slide in the red bucket for disposal.
• Ponds and puddles contain vast arrays of diverse organisms. In this exercise, you will examine pond water under the microscope to visualize protozoa, which are single-celled eukaryotic organisms, and various small multicellular organisms. Like any ecosystem, a microecosystem has trophic levels and food chains too. You may be able to distinguish the primary photosynthetic producers seeking light at the surface of the pond water from the consumers, who will be eating dead organisms or other living organisms deeper in the pond water.
1. Make 3 slides. The first slide should use 2-3 drops from the surface of the pond water, the second slide should use a sample from the middle of the pond water and the third slide should be from the from the bottom of the pond water. Doing this will allow you to distinguish between the different ecosystems within the pond water. Work quickly as the slides will dry out. Be sure to use a cover slip on each slide.
2. Observe the pond water under the microscope. Note: many of the organisms are colorless and will best be viewed under dim light conditions, with the iris diaphragm partly closed.
3. Use Figure 2.3 to identify the organisms in the pond water samples.
4. When you are done place your slides in the red bucket for disposal.
• Answer questions in Part D of the Lab Report.
Non-majors College Biology Lab Manual © 2021 by Marie McGovern Ph.D. is licensed under CC BY-NC 4.0