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Biology 100 – Concepts of Biology
Spring, 2006
Microscopy
One of the fundamental tools of biology is the microscope. Its purpose is simple: to enable one to view tiny objects. Many living organisms are too small to be seen with the unaided eye. In multi-cellular organisms, individual cells are also too small to see without a microscope. In many areas of the sciences microscopes are a fundamental tool with which to test hypotheses. There are many kinds of microscopes. In this exercise we will concentrate on the compound light microscope.
Drawings help you to remember.
It is often necessary to obtain a written record of what is observed with a microscope. The simplest way to do this is to make a drawing of what you see. In this exercise, you will practice sketching what you see through the microscope. Drawing forces you to examine objects more carefully than you would otherwise.
It is often necessary to estimate the size of the object you are viewing. In this exercise you will learn how to estimate the size of a microscopic object.
Caution! The eyepieces will easily slip out of these microscopes. Be careful when removing the dust covers to assure that the eyepieces do not fall out.
I. Parts of the Compound Microscope
Your instructor will explain the parts of the microscope and their functions. Below is a diagram of the Olympus CX-31 compound microscope, which is the microscope we will be using.
A. Eyepiece (ocular)
B. Objectives (4x, 10x, 40x)
C. Stage
D. Substage condenser
E. Field Lens
F. Fine focus knob
G. Coarse focus knob
Your instructor will point out additional parts of the microscope including: substage condenser iris diaphragm, mechanical stage knobs, main power switch, light intensity knob. Be sure you are conversant on the parts of the microscope. Make special note that each microscope has a pointer in the right eyepiece and a micrometer in the left eyepiece.
Magnification
The magnification of a compound microscope ranges from about 100x to about 1000x, depending on the lenses used. The total magnification of a microscope is easily calculated with the following formula.
Magnification of the eyepiece x magnification of the objective lens being used
For technical reasons, 1000x is about the maximum magnification one can obtain with a compound microscope and still have good resolution, i.e., sharpness of the image. Magnification of the objective lens is 10x.
II. Setting up Koehler Illumination on the compound microscope
Setting the adjustments of a compound microscope such that the optimal resolution is obtained is called setting up "Koehler Illumination." It takes only a minute or so to do this.
1. Turn on the microscope. Set the light intensity knob so it is comfortable for your eyes. Place a prepared slide on the stage.
2. Start with the 4X objective lens (blue ring) in place. Use the mechanical stage knobs (right side below stage) to orient the prepared slide so that it is centered below the objective lens.
3. Use the course and fine adjustments to focus on the slide. Now switch to the 10X objective lens.
4. While looking through the microscope use the knurled ring on top of the field lens to close the field diaphragm. You will see the edges of the diaphragm close in the field of view.
5. Focus the edge of the field diaphragm by raising or lowering the substage condenser (black knob on left side under stage). The substage condenser should now be near the upper end of its range of travel. Refocus the specimen.
6. Open the field diaphragm until the light fills the field of view.
7. Open the diaphragm of the substage condenser (front of scope beneath stage); now close it until the image of the specimen just begins to darken. If you close it further the contrast will increase but your resolution (ability to see detail) will decrease.
Guidelines for the Safe Use of the Compound Microscope
1. Always carry the microscope with two hands.
2. Never touch the lens surface.
3. If any of the lens need cleaning, alert your instructor and she/he will show you how.
5. Always observe any specimen first under low power (4x). When you are ready to
switch to high power (10x or 40s), do so carefully, watching the lenses as you rotate the revolving nosepiece. Remember, the high power objective is longer than the low power objective.
6. If you spill any liquid on the stage, wipe it up immediately.
7. When unplugging the microscope, pull on the plug, not the cord.
8. When finished,
a. remove the slide from the stage,
b. carefully wrap the cord around the base of the microscope,
c. cover the microscope with the dust cover.
III. Practice with the Compound Microscope
The letter 'e'.
1. Obtain a small piece of newspaper with text that contains a lower case letter 'e' Cut out the 'e' and place it on a clean microscope slide. Place a cover slip over the newspaper. You have just made a ‘dry mount’ (so called because there is no water).
2. Place the slide on the microscope stage so that the 'e' is in normal reading position. Observe the slide under low power. Draw the 'e' as it appears in the microscope.
3. What is the relationship of the orientation of the object to that of the image?
4. While looking through the eyepiece, slowly move the slide to the left. Which way does the
image appear to move?
Move the slide to the right. Does the image move the direction you expected it to move?
While looking through the eyepiece, slowly move the slide away from you on the stage. Which way does the image move?
What do you think will happen if you move the slide toward you on the stage? Try it.
Practice moving the slide and focusing until these actions become comfortable. When you are finished, discard the letter 'e' and the cover slip, but save the slide for later use.
HYPOTHESIS: What hypothesis have we tested by observing the letter 'e'? Prediction. Did we make a prediction? What was it? |
Colored threads.
1. Obtain a prepared slide containing three pieces of thread positioned so that they cross at a common point. Observe the slide under low power and locate the point at which the three threads cross. Adjust the diaphragm lever and focus. Can you focus on all three threads simultaneously?
2. Without moving the slide, carefully switch to high power. Compare the depth of focus with that under low power. Which is greater?
3. Switch back to low power. Adjust the focus until the slide and the low power objective are barely separated (about 2 mm apart). Look through the eyepiece; all threads should be out of focus. Adjust the fine focus until the first thread comes into focus. Note its color. Continue focusing until the second, and finally, the third threads come into sharp focus, noting their respective colors.
What color was the first thread to come into focus? Second? Third?
From your observations, which thread do you think is on top? On the bottom?
Your instructor will tell you the correct order of the threads. Is it what you predicted? Is the
image inverted vertically?
4. What hypothesis(es) have we tested with the colored thread exercise?
HYPOTHESIS: What hypothesis have we tested by examining the colored threads? Prediction. Did we make a prediction? What was it? |
IV. Measurement
Sizes of microscopic objects are usually given in microns (abbreviated µm;
1000 µm = 1mm). How many microns in a meter?
These microscopes have an ocular micrometer (a little ruler) in right eyepiece. These ocular micrometers are calibrated as indicated below.
Objective lens Smallest division on micrometer
4x 25 µm
10x 10 µm
40x 2.5 µm
Practice with the microscope
1. Thickness of hair
Pull a hair from your head and make a dry mount. Place the slide on the stage, find the hair with low power, and then switch to high power. Sketch what you see and estimate the diameter of your hair. Develop an hypothesis about your hair and test it by examination with the microscope.
2. Human blood cells
Obtain a prepared slide containing preserved human blood cells. Find a white blood cell. The white blood cells will be much rarer than the red blood cells, and will be larger and stained purple. Estimate the diameter of a human white blood cell.
3. Other prepared slides
There may be other prepared slides available for examination.
4. Diversity of life in pond water
The purpose of this exercise is to introduce you to the variety of microscopic organisms. Do not worry about trying to identify what you see; just look and enjoy.
Making a wet mount: Obtain a clean microscope slide and cover slip. Place a drop or two of pond water onto the slide and carefully lower the cover slip onto the slide, edge first.
Your instructor will demonstrate!
Examine your slide. When you find something interesting, show your neighbor and instructor. Develop a hypothesis about an organism that you find on the slide and test it by making additional observations.
What color are most of the organisms you see?
If organisms are clear (colorless), what can you do to increase your ability to see them with the microscope?
Sketch two organisms that you see in the space below.
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