As you move from lower magnification to higher magnification field diameter will

Laboratory Notes for BIO 1003

© 30 August 1999, John H. Wahlert & Mary Jean Holland

USE OF THE MICROSCOPE

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The microscope is at tool that allows us to see the structures and tissues that comprise organisms in very fine detail. Often it is possible to understand function of a structure on the basis of its microscopic morphology. For example, seeing the orientation of muscle cells in the body wall of an earthworm allows one to understand how it moves and burrows. You will also see strong evidence in support of the cell theory.

Instructions for use of the high power microscope:

• Carry the microscope with one hand grasping the neck and the other supporting the bottom.
• Always have the shortest, low power objective lens facing the stage when you put a new slide on the stage or remove a slide.

To focus the microscope:

1. Hold a prepared slide up to the light and see where the specimen is located under the cover slip.
2. Make sure the microscope light is turned on and the low power objective lens that faces the stage is clicked into position. Raise the condenser lens system that is under the stage to its topmost position. The condenser lenses focus the light through the hole in the stage.
3. Center the specimen over the light that is coming up through the hole in the stage.
4. Raise the stage to its topmost position. (Low power objective is in position!)
5. Look through the ocular lens of the microscope and adjust the light so that it is comfortable for your eyes. All microscopes are equipped with an iris diaphragm under the stage that reduces the aperture through which light passes; there may be a rheostat that dims and brightens the bulb. Now lower the stage slowly with the large, coarse focusing knob.
6. At some point the specimen should come into sharp focus.
7. You may now switch to a higher power objective. These microscopes are parfocal and thus remain in focus.
8. Readjust the iris diaphragm so that the light is comfortable for you and you can see detail.
9. At high power, focus only with the small, fine focus knob.

Getting the best possible image:

• Use lens tissue, which we supply, to clean the ocular and objective lenses; do not use any other kind of paper. You may also need to clean the slide.
• Always begin to focus the microscope with the low power, coarse focusing knob. These microscopes are parfocal. This means that once you have focussed carefully with the low power objective, the other objectives will be nearly in perfect focus. You must have the object centered before you change objectives to increase the magnification, because the field of view becomes smaller; if the object is off to the side, it may disappear when you go to higher magnification.
• For best viewing at high power, white light is essential. Use the rheostat to increase the voltage to the bulb. As it brightens, the light becomes less red and more white. Closing the aperture through which the light passes increases the resolution of detail that you can see; use the iris diaphragm, which is operated by a lever among the condenser lenses, to change the size of the aperture. The higher the power of the objective lens, the less will be the depth of field.

Magnification:

The total magnification that you see can be calculated. Find the magnification imprinted on the ring around the ocular lens--it is probably 10x. Then find the magnification imprinted on the objective lens that you are using--it is probably either 4x, 10x, or 40x. Multiply the magnification of the ocular lens times that of the objective lens; this is the total magnification that you see. The quality of the microscope is in its objective lenses. If they are awful, magnifying what they see by the ocular lens will make no improvement. It is important that you do not get the objective lenses wet. Dry them promptly if water or stain is transferred from the slide to the lens.

Preparation for clean up:

1. Be sure that the low power objective is in position before you remove a slide from the stage and before you put the microscope away.
2. When you unplug the microscope, grasp the plug, not the cord.
3. Microscopes are numbered and should be put away in the niche bearing the same number in the microscope cupboard.
4. Return prepared slides to boxes, and put them in the slot whose number matches that in the upper right corner of the slide.

Slides:

Letter e. Hold this slide up to the light and you will see a small piece of typewriter paper under the middle of the cover slip. It has an e typed on it. Start with the low power objective (the shortest) in place. Center this piece of paper in the light coming through the stage of your microscope and focus the e with the large, coarse focusing knob. You will see immediately that the e is upside down and backwards, just the reverse of the way you oriented it on the stage. The microscope lenses are responsible for this reversal. With the e centered, raise the magnification to medium power. The focus is nearly correct; recenter the e and sharpen the focus, again with the coarse focusing knob. Now raise the magnification to high power. Adjust the focus with the smaller, fine focusing knob only. Never use the coarse focusing knob, when you are using high power, because you can easily crunch the objective through the slide�a costly mistake. Always begin the process at low power and then raise the magnification of the objective lenses. You will have noticed that the higher the magnification of the objective lens, the smaller and dimmer the field of view. On high power, you will need to maximize the brightness of the light source and regulate the iris diaphragm.

Bolting silk. This is a small square of fine silk fabric. Examine it with low power, and then raise the magnification. On high power use the iris diaphragm to improve the resolution. You see much more detail when the light is passed through a very tiny aperture. Remember this fact when you are looking at nearly transparent organisms in future labs. With the fine focussing knob raise and lower the stage to get a sense of the thickness of the fabric.

Millimeter rule. Put the transparent millimeter rule on the stage of the microscope. Observe the length of the diameter in millimenters of the field of view at each available magnification. Fill in the following chart:

The Cell Theory

In 1665, Robert Hooke used the word cell, meaning little rooms, to describe the small cavities separated by walls in cork, which is the bark of a tree. Matthias Schleiden, a botanist, published (1838) his conclusion that all plants are made of cells; in the following year (1839), Theodor Schwann extended the observation to animal tissues and proposed a cellular basis of all life. The pathologist Rudolf Virchow added an important extension of the theory in 1858 that all living cells arise from pre-existing living cells; there is no spontaneous creation of cells from nonliving matter. As you look with the microscope at tissues representing the 5 or 6 kingdoms of organisms, you will confirm the validity of the cell theory. It seems odd that something so obvious to us with modern technology had to be discovered and proposed as a theory.

Animal cells--cheek. Take a clean microscope slide, and place a small drop of iodine stain on it. Use a sterile wooden stick, and rub the tip gently across the inside of you cheek. Cells that were about to slough off have been collected on the end of the stick. Swirl the end of the stick in the iodine on the slide to transfer and stain the cells. Place a cover slip on the preparation, and view with the microscope; remember to start with the low power objective in place. The cells are irregular; you can see flat surfaces where they met other cells in your cheek. The boundary of a cell, the plasma membrane, is so thin you can only see where the cell ends. The nucleus is stained an orangish brown and is near the center in each cell. Tiny dots on the surface are probably bacteria.

Plant cells--onion. Place a drop of iodine stain on a clean microscope slide. Take a layer of onion, and use a scalpel tip to peel the very thin layer of cells that line its inner curvature. The peel held between your finger tip and the scalpel should be like extremely thin cellophane. Place the peel in the iodine on the slide, and put a cover slip over it. View the preparation with the microscope. These cells are quite regular in shape, and there is a thick cell wall surrounding them. Since the cells secreted the wall material, the plasma membrane of the cell is inside the wall. Nuclei are stained as in the animal cells, but they are at the side of the cell. The center of each plant cell is occupied by a large transparent organelle called the central vacuole. You cannot see it, but you can see the result of its presence: the nucleus is off to the side.

Cell dimensions: Use the information in the chart above, in which you determined the diameter of the field of view at each magnification, to measure approximately the average diameter of a cheek cell and the length and width of an onion cell.

Protista. If cultures of the single-celled organisms are available, put a drop on a new slide, and gently place a cover slip on top. Examine with the microscope.

What happens to the diameter of field when the magnification is increased?

What happens to the size of the field as you move from low magnification to high magnification?

What is the relationship between magnification and the diameter of the field?

Why does the field diameter get smaller as the magnification increases?