Experiment 3: The Use of a Compound Light Microscope
Objectives:
The purpose of this lab is to learn how to properly use a compound light microscope, and to make measurements with the scope.
Introduction:
A compound light microscope is the basic tool that biologists use to look at the microbial world. Antonie van Leeuwenhoek is considered to be the father of the microscope. He was the first person to use such a device to look at objects too small to be seen with the unaided eye. Although his microscope was nothing more than a thick piece of glass that he placed between the specimen and his eye, it magnified objects about 266 times (about one-third of the power of today’s scopes).
The light microscope of today is much different than van Leeuwenhoek’s. It now consists of a series of glass lenses stacked together in a tube and magnifies the specimens a little more than 1000 times. The beauty of the light microscope is its ability to resolve the fine detail of small objects; this feature is known as the resolving power.
Specimens that are viewed under the microscope are mounted on a variety of glass slides. Depression slides are glass slides with a depression in them and are typically used for viewing live specimens that would otherwise be crushed under the cover glass (an example would be a flatworm-platyhelminthes); wet-mount slides which are temporary slides used to examine live organisms which cannot resist slide preparation (pond water); and permanent slides which undergo numerous treatment procedures and can be viewed long after initial preparation.
In your study of biology, it will be necessary for you to estimate the length and width of some of your specimens. To measure objects under the microscope, a unit called the micron (m) is used. One micron is equal to 0.001 millimeter. This exercise is designed to get you acquainted with the compound light microscope, and the ways it is used in the laboratory.
Figure 1-1. The compound light microscope. This microscope illustration was taken from http://nationaloptical.com/products/compound/models/160.html
Below is a list of parts common to many microscopes and a description of what the parts do. Be sure to know the names of each item, what it does, and where it is found on the microscope.
Nosepiece—allows you to switch from one objective to another.
Iris diaphragm—regulates the amount of light passing through the specimen being viewed.
Light source—directs light upward through the diaphragm and the stage opening.
Ocular—contains lenses that typically magnify the image from the objective an additional ten times.
Body tube—holds the ocular and objective lenses at the proper working distance from each other.
Arm—supports the body tube and coarse adjustment.
Objective—each objective contains lenses that magnify to a certain known degree.
Mechanical stage—this consists of a clamp for holding a slide and two knobs that enable you to move the slide back and forth or right and left.
Stage—supports a slide over the opening that admits light from below.
Coarse adjustment—moves the nosepiece and objective up and down to approximately the correct focus.
Fine adjustment—permits the exact focusing by moving the nosepiece and objectives up or down very slightly.
Base—Firm support that bears the weight of the microscope.
Stage clips—hold slide in place when it is on the stage.
Materials:
1. Microscope
2. Lens paper
3. Glass slide
4. Coverslip
5. Water
6. Soft cloth or paper towel
7. Scissors
8. Fine newsprint
9. Medicine dropper
10. Dissecting needle
11. Transparent metric ruler
12. Toluidine O stain.
13. Male pollen cones
Methods:
Part A. Identifying and using the parts of a microscope.
1. Take a microscope from the storage area. Carry the microscope with one hand under the base and the other hand grasping the arm.
2. Place the microscope on the laboratory table. The microscope should be about 10 cm from the edge of the table.
3. Look at the drawing in Figure 1-1, and identify the parts of your microscope and their functions.
4. Carefully clean the eyepiece and objective lenses with lens paper. Using Figure 1-1, locate the nosepiece and gently turn it so that the low-power objective is in line with the body tube. The nosepiece will click into place when the objective is in the proper position.
5. Keeping both eyes open, look through the eyepiece. Turn on the lamp so that you can see a circle of light. This circle of light is the field of view. To make the circle of light as bright as possible, you may have to adjust the diaphragm.
Part B. Preparing and observing a wet mount.
1. Rinse the glass slide and coverslip with water. Wipe both sides of the slide and coverslip with a clean cloth or paper towel. The cleaned slide and coverslip should be handled by their edges.
2. Cut out a small piece of newspaper that contains the letter “e.” Place the letter “e” in the center of the slide. Using the medicine dropper, place a drop of tap water on top of the letter “e.”
3. Place the coverslip at about a 45° angle over the drop of water. Using the dissecting needle, gently lower the coverslip onto the slide as shown in Figure 1-2. If air bubbles appear, gently tap the coverslip with the eraser of a pencil.
Figure 1-2. Placing the coverslip on the slide.
4. Place the wet mount of the letter “e” on the stage of the microscope with the letter facing you, as you would read it. Adjust the slide so that the letter is above the opening of the stage. Use the stage clips to hold the slide in place.
5. Look at the slide at eye level. Carefully observe the space between the slide and the low-power objective. The low-power objective should be in line with the body tube. Slowly turn the coarse-adjustment knob, lowering the low-power objective so that it almost touches the slide.
*CAUTION: Never lower an objective while looking through the eyepiece; you may hit it and damage the slide.
6. Look through the eyepiece and slowly turn the coarse-adjustment knob toward you until the letter “e” comes into focus. In the circle, sketch what you see in Figure 1-3a.
Magnification: ______
Diameter of Field of View: ______
Figure 1-3a. The letter as seen through the microscope under low power.
7. To observe a specimen under high-power magnification, turn the nosepiece until the high-power objective clicks into place. Use only the fine-adjustment knob to bring the specimen into focus. Sketch what you see in the circle in Figure 1-3b.
Magnification: ______
Diameter of Field of View: ______
Figure 1-3b. The letter as seen through the microscope under high power.
Part C. Measuring an object under the microscope.
1. Place the millimeter scale of the transparent plastic ruler over the center of the stage opening of the microscope.
2. Use the low-power objective of the microscope to locate the millimeter lines of the ruler. Place these lines in the middle of the field of view and use the coarse-adjustment knob to bring them into focus. Use the fine-adjustment knob to make the image clearer. The distance between two lines on the ruler represents 1 mm.
3. While looking through the eyepiece, move the ruler so that one of the millimeter lines is seen at one end of the field of view as shown in Figure 1-4.
Magnification: ______
Diameter of Field of View: ______
Figure 1-4. The millimeter line as seen through the microscope.
4. To determine the diameter of the field of view under low power, count the number of millimeter lines that are visible. You may have to estimate the diameter to the nearest tenth of a millimeter.
5. Because under high power the width of the millimeter takes up practically the entire field of view, it is difficult to estimate the diameter of the field of view under high power. The diameter under high-power can be calculated on paper, however.
a. Divide the magnification of the high-power objective (eg. 40x) by the magnification of the low-power objectives (eg. 10x).
b. Now divide the diameter of the low-power field of view in microns by the answer obtained in a. If the diameter of the low-power field is 2 mm, the calculation would be: a. 40/10=4 and b. 2000m/4=500m.
6. Make a wetmount preparation of pollen. Observe the pollen under low and high-power.
7. Calculate the length of one pollen grain by estimating how many could be placed end to end across the field of view. Divide the diameter of the field of view by this number.
8. Before returning the microscope to the storage area, remove the slide from the stage. Click the low-power objective into place and lower the body tube as far as it will go.
9. Place the stage clips in their proper position and carefully carry the microscope to the storage area.
Questions:
1. Looking through the microscope, in what direction does the letter “e” appear to move when you move the slide in the following directions?
a. to the right
b. to the left
c. away from you
d. toward you
2. Determine the total magnification of your microscope under the following magnifications.
a. low power
b. high power
3. What happens to the focus of the letter “e” as you change from low power to high-power magnification?
4. How many times is the magnification increased when you change from low power to high-power magnification?
5. What happens to the field of view when you change from low-power magnification to high-power magnification?
6. Describe the change in the image of the letter “e” when you switch from low power to high power magnification.
7. How many microns are there in 1 millimeter?
8. What is the diameter of the field of view under low power?
9. Show your calculations for the diameter of the field of view under high power.
10. What is the length in microns of one pollen grain?
References:
Mills, Verne M. Laboratory Manual for Cell Biology. Pp. 1-7. 1993.
Hampton, Carol D., and Carolyn H. Hampton. Laboratory Manual: Prentice-Hall Biology. Pp. 11-17. Prentice-Hall. Englewood Cliffs, NJ. 1986.
Lacey, A.J. Light Microscopy in Biology: A Practical Approach. Oril Press. Oxford. 1991.