How do you use a microscope?
Introduction
     In almost every type of biological research, the microscope plays a fundamental role.
Scientists in each field rely on it to study the fine structures of cells and tissues — things too small to see
with the unaided eye. Were it not for the microscope, our understanding of life would be far different from
what it is today. In this lab, you will learn how to :
          a. use a compound microscope;
          b. measure the size of a specimen you see under the microscope;
          c. know the relative sizes of the low and high power fields.
     In future labs, you will observe microscopic organisms using the techniques you learned in this lab.

Prelab Preparation
     It is essential that you learn the name and function of each part of a microscope and how to properly
care for your microscope. Study the diagram of the microscope and learn the names of all its parts

Standard Compound Microscope

     Your microscope is expensive and fragile. It is important for you to use it correctly to avoid
damaging it and to avoid breaking slides or destroying specimens. When you are using your microscope,
it should rest securely on your table or bench, away from the edge. When you carry your microscope,
always use two hands. Hold its base with one hand and its arm with your other hand as shown.

How to Hold a Microscope

     Always use an appropriate light source. If your microscope has a lamp, plug it in and turn
the lamp on. Make sure the diaphragm is sufficiently open so enough light can get through. (This
will be especially important if you look through the eyepiece and see nothing). Always keep both
eyes open as you look into the eyepiece. This is important because it reduces eyestrain.
Keep the lenses on your microscope clean. Never touch them with your fingers. If the eyepiece
or objective lenses get dirty, clean them with a piece of lens paper moistened with alcohol (or
xylene). Wipe the lens in a light circular motion and change the lens paper as it picks up the dirt.
Make certain that you leave no streaks on the lens. NOTE: Cleaning the lens with anything
other than lens paper, or wiping too hard, will scratch the lens.
     The purpose of the microscope is to magnify your specimen. This microscope uses two
lenses—the eyepiece and an objective—to magnify the image. Such microscopes are called
“compound” microscopes. The magnification of any microscope is the number of times the size
of an object appears increased. If the magnification of an object is 10X, it will appear 10 times
larger than it really is.
     The magnification of your microscope is equal to the product of the separate magnifications of
the eyepiece and the objective. (The magnification of each lens is written on the lens case.) If the
eyepiece is 10X and the low power objective is 10X, then the magnification under low power is
100X. In equation form, this is written:

     (Eyepiece magnification) x (Objective magnification) = Total microscope magnification

     1. If the magnification of the eyepiece is 10x and the magnification of the high power
          objective is 40x, what is the total magnification under high power?
     2. Why is this type of microscope called a “compound” microscope?

     NOTE: Your microscope has two additional lenses - a “scanning” power (4X objective), that
gives a very low magnification and an “oil immersion” power (100X objective) that gives a very high
magnification. The scanning power is useful for rapidly locating a specimen on the slide, but in
many cases it is not powerful enough for observing much detail. The oil immersion power is useful
for viewing very small microorganisms, particularly bacteria.
     For purposes of this lab LOW power will always refer to the 10X (yellow) objective lens and
HIGH power will always refer to the 65X (blue) objective lens.

Procedure
Part 1: Using your microscope
A. Obtain a microscope. Make certain that you are familiar with all of its parts and that it is
     functioning properly.
     3. What are the magnifications of your microscope:
          a. under low power?                     b. under high power?
     The first thing you will observe under the microscope is the letter “e”. It is very easy to focus on
     and it helps demonstrate what a microscope does to the image of an object.
B. Prepare a wet mount of an “e” by first cutting a letter out of a newspaper. As shown in the
     illustration, place the "e" on the slide. Add a drop of water onto the "e". Place one edge of
     a coverslip on the slide, in the water, next to the “e”. Gently lower the coverslip onto the “e”.
     Do not press directly down on the coverslip.

C. Adjust the diaphragm to obtain the appropriate light for viewing.
D. Become familiar with the high (blue) and low (yellow) power magnifications of your microscope. Put
     your wet mount on the microscope's stage and clip it in place. Turn the nosepiece so that the low power
     objective is in place. Focus under low power, using the coarse focus knob first and then the fine focus
     knob. You should be able to clearly see the letter “e” on the slide.
E. To use the high power objective, first focus on the letter “e” under low power and move it to the center of
     the field of vision. Without changing the focus, turn the high power objective into place. If your microscope
     is properly adjusted, the image should be almost in focus. Use only fine focus on high power. Move the
     slide around and focus to see small irregularities in the “e”. NOTE: Slides are easily broken by using the
     coarse focus with high power.
     4. Which magnification, low or high, is more appropriate for looking at the whole “e”?
     5. How does the “e” appear to be oriented compared to the way it looked before you
          put it under the microscope? (ie. right side up, upside down, reversed left to right, etc)
     6. Move your slide to the left and then right. Which way does the image of the “e”
          appear to move?
     7. Move your slide towards you and then away from you. Which way does the image
          of the “e” appear to move?
F. Center the “e” in the middle of the low power field. Now switch to high power and focus. (Remember to
     use only the fine focus under high power.)
     8. Why isn’t the coarse adjustment knob used when viewing under high power?
     9. Do you see more or less of the “e” on high power than on low power?
G. To learn how to see the depth of a specimen, obtain a piece of colored thread about 2 cm long.
     Place two pieces of thread in an "X" position on a clean slide. Without adding a cover slip, clip the slide in
     place on the stage. Focus up and down using both low and high powers.
     10. Why do only parts of the thread (ie. top or bottom) appear sharp and clear at any given time?
     Adjusting the diaphragm opening increases or decreases the amount of light present which changes
     the contrast and aids in making more accurate observations.
H. With the slide from step G in place, put your high power objective in place. Observe the thread carefully
     as you slowly open and close the diaphragm.
     11. What differences did you observe under the various diaphragm settings? (ie. consider color
          sharpness, clarity of details, etc)
I. When you are finished, put the low power objective in place. Clean your slide and cover slip with water
     and paper towels. Now begin with Part II of this lab.

Part II: Microscopic Measurement
     You will now measure the diameters of the low and high power fields. This will enable you to estimate the
actual size of the specimens you will observe later.
J. Put a clear plastic ruler on the microscope stage so that you can see the millimeter scale under low power.
     Place one millimeter marking of your ruler at the far left hand side of the low power field. You should see
     one other millimeter marking in your field of view. This means that your low power field is between 1 and 2
     mm in diameter. If you see two other millimeter markings then your low power is 2 mm or more in diameter.
     12.To determine the diameter of the low power field of your microscope, approximate, to the
          nearest 0.1 mm, what fraction of a second or third millimeter is in your field of view.

In this sample field of vision the ruler reads 1.3 mm.

     13. Divide the high power magnification of your microscope by the low power magnification
          to determine how many times larger your low power field is than your high power field.
     14. Now determine the diameter of your high power field by dividing the diameter of
          your microscope's low power field by the number you obtained above.
K. Many of the specimens you will observe under the microscope will be smaller than 1 mm in size.
     Because of this, microscopic measurements are often expressed in microns (u m or micrometer).
     One millimeter equals 1,000 microns.
     15. What are the diameters of each of your fields in microns:
          a. Low power?                     b. High power?
     For the remainder of this lab, express all of your measurements in microns. For example, if you
     measure a cell to be 0.2 mm wide, then report it as 200 microns. Since nearly all cells are less than
     1 mm in diameter, you can see that it is better to think of them in terms of microns than millimeters.

Part III: Microscopic observations
     In addition to whole cells, you will often wish to observe the parts of a cell. A good example
of a part of a plant cell is a grain of starch. Starch grains appear white in the normal plant cell and
are not easy to see. To make them more visible, you will stain them with iodine, which turns them
black. Stains are commonly used in microscopy to make certain types of cells or cell parts easier
to observe.
L. Prepare a wet mount of starch grains by first peeling a section of a potato. Then, use a
     single-edged razor blade or scalpel to gently scrape the peeled surface of the potato (not
     the skin). You should see a whitish liquid substance on the edge of the blade.
M. Place a drop of this fluid onto a clean slide and spread the drop to make a circle about the size
     of a dime.
N. Add one drop of iodine and place a coverslip on top of the iodine. Examine your slide under low
     power. You will see numerous black circles and ovals. These are starch grains from the potato.
O. Move the slide around to find a field that has about 100 starch grains. (or where the grains are
     the thinnest in number)
     16. Count and record the number of starch grains on one field of view under low
          power. (NOTE -> Do NOT move the field of vision once you start counting)
     17. Without moving the slide, switch to high power. Count and record the number
          of grains in one field of vision.
     18. a. Which power of the microscope, low or high, has a larger field of vision?
          b. Would you expect to see more grains of starch under low or high power?
          c. Divide the number for the high power lens by the number for the low power lens,
               and express the answer as a fraction.
          d. Divide the number of grains under low power by the number of grains under high
               power, and express the answer as a fraction.
          e. Explain why these two fractions should be equal?

     To observe the size and shape of plant cells you will examine a piece of cork. Cork is a mass of
     dead plant cells in which only the cell wall remains. These cell walls will appear as compartments
     under the microscope.
P. Slice a small piece of cork with a single-edged razor blade or scalpel. Make your slice as thin as
     you possibly can. Place your slice on a slide but do not add water or cover it with a coverslip.
Q. Observe the thinnest part of your cork under low power. (If your cork slice is extremely thin, you
     might be able to make useful observations under high power as well.)
     19. Draw a diagram of what you see. Label the cell wall (outer wall) and cell cavity (inner
          space). Make your drawing as accurate as you can.
     20. Measure the average size of a single cork cell. To do this you must:
          a. Determine how many cork cells are needed to reach from one side of the visual
               field to the other. (This number will be an estimation.)
          b. Divide this estimated number of cork cells (part "a" above) into the diameter of your low
               power field. (determined this diameter in #15. a.)
          c. This number is the average size of a single cork cell.

Postlab Analysis
21. Which of the following specimen sizes are appropriate for observing under your microscope:
     a. 35 um           b. 425 um           c. .07 um           d. 3829 um

22. Why are drawings the most common method for communicating observations made under
          a microscope?

23. Why didn’t you make use of the:
          a. the scanning lens (4X)?
          b. the oil immersion lens (100X)?

24. Assume that you are looking at a yeast cell, and you estimate that under high power it would
          take 40 yeast cells to reach across the field of vision. What is the size of this yeast cell?
          (SHOW MATH)