are useful for viewing objects that are too small to see clearly
without magnification. This exercise is designed to familiarize
students with the use of a compound light microscope and a binocular
Before using the microscope, read the document on microscope care using the link below.
Compound Light Microscope
compound light microscope uses two sets of lenses to magnify the
object. Illumination is provided by a light source on the base of the
microscope. The magnification typically ranges from approximately
40 X to 1,000 X. They can be used with objects that range in size from
about 100 nm to 2 mm.
Parts of the Light Microscope
The stage is a platform that holds the slide containing the specimen to be
viewed. A mechanical stage (see the photographs below) has a mechanism
for moving the slide.
A light microscope must have a light source. This is usually a light bulb located beneath the stage.
An adjustable diaphragm located beneath the stage is used to regulate the amount of light that passes through.
A condenser contains two sets of lenses that concentrate light. It is located
directly underneath the stage. Light from the light source passes
through the diaphragm and condenser before continuing up through the
specimen to be viewed.
The body tube contains an ocular lens (eyepiece) and a nosepiece with several objective lenses.
Each objective lens is used for a different magnification and is moved
into place by rotating the nosepiece. The image is brought into focus
by adjusting the coarse and fine focus knobs.
Click on the photographs to view enlargements.
After viewing, press the "Back" key to return here.
microscopes (below) have two eyepieces; monocular microscopes (above)
have one. The distance between the two ocular lenses of a binocular
microscope can be adjusted to fit the distance between your eyes.
optical devices such as binocular telescopes and field glasses also
have two ocular lenses that adjust in a manner similar to the
microscope. Binocular lenses can be adjusted individually, making it
unnecessary for many people to need their glasses when using them. If
you wear glasses and are unfamiliar with adjusting binocular lenses to
correct for your own eyes, see the section titled "Adjusting the Ocular Lenses" below.
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light microscopes contain two lens systems, an objective and an ocular.
The total magnification of an image is calculated by multiplying the
magnification of the ocular by the magnification of the objective. The
microscopes we will use each have a 10X ocular lens and four different
objective lenses listed in the table below.
|Objective||Magnification ||Total Magnification |
|Scanning||4 X||40 X|
|Low Power||10 X||100 X|
|High Power||40 X or 43 X||400 X or 430 X|
|Oil Immersion ||100 X||1000 X|
bends when it passes from glass to air or from air to glass because air
and glass have different refractive indices. The bending of light as it
passes through the glass slide to the air and then to the glass lens
decreases the resolving power. At high magnification (1000X) it can
prevent a clear image from being viewed. This decrease in resolution
can be prevented by putting immersion oil between the slide and the lens because immersion has the same refractive index as glass.
condenser also increases the resolving power of the microscope. When
using the oil-immersion lens, the condenser (located beneath the stage)
should be raised to a position very close to the stage for maximum
The link below may be helpful before using the microscope.
CAUTION - Never use cloth or paper products (paper towels, tissue paper, etc.) to clean
the lenses. They will scratch the coating and decrease the resolving
power of the lens. Use only lens paper.
microscope to the lowest magnification or raise the objectives from the
stage before inserting a slide. This will prevent the objective lens
from being accidentally scratched by the slide.
Place the slide to be viewed on the stage and center the specimen over the opening.
Begin with either the scanning lens or the low power objective lens.
Raise the stage (or lower the lens) all the way so that the slide is as close as possible to the objective lens.
the coarse adjustment know to slowly raise the lens from the stage
while viewing the image. Fine focusing is not needed when using the
lowest magnification (scanning or 4X objective). If you are using any
of the other objectives, it will be necessary to use the fine focus
after using the coarse focus.
Adjust the condenser so that a
sharp focus is produced. This step is important at the highest
magnification (oil immersion or 1000X).
Adjust the iris diaphragm. This will need readjustment after changing to a different magnification.
To Increase the Magnification
The microscopes are parfocal, meaning that after you adjust the focus, the image will remain approximately in focus if you change the magnification.
the object before switching to a higher power objective. This will help
you find the object after switching the objective.
Switch to the
next highest power. It will be necessary to center the image again. The
image should be approximately in focus but it will be necessary to use
the fine focus. The coarse focus should not be needed after switching
Adjust the diaphragm.
This procedure is repeated each time you switch to a higher magnification.
100 X objective (1,000X total magnification) requires that a drop of
immersion oil be placed between the slide and the lens.
focusing the specimen under high power (400X or 430X, see above),
rotate the high power objective out of the way and place a drop of
immersion oil on the slide. Rotate the oil immersion objective into
place so that it touches the oil.
Adjust the fine focus, condenser, and iris diaphragm as previously described.
After viewing with oil, the lens must be cleaned with fluid designated for this purpose. Remember, use lens paper only. Never use cloth, paper towels, or other paper products on coated optics.
Practice Using the Microscope
- Obtain a slide of colored threads and view them under the scanning and low power. Use the focusing procedure described above.
1) Can you tell which thread is above the other?
the threads under high power (400X or 430X). Use the fine focus to
focus to determine the order of the threads from top to bottom. As you
rotate the fine focus, different strands will go out of focus while
others will become more sharply focused. This procedure will therefore
enable you to determine the order of the threads.
2) Are all of the threads in focus at the same time?
3) What is the order (from top to bottom)?
of field" refers to the thickness of the plane of focus. With a large
depth of field, all of the threads can be in focused at the same time.
With a smaller or narrower depth of field, only one thread or a part of
one thread can be focused, everything else will be out of focus. In
order to view the other threads, you must focus downward to view the
ones underneath and upward to view the ones that are above.
What happens to the depth of field when you increase to a higher
magnification (increases, decreases, or remains the same)?
5) Explain how the slide with threads could be used to answer the question above.
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- Obtain a slide of the letter e and view it under the scanning objective. Move the slide to the left.
6) What way did the image move when the slide was moved to the left?
7) How does the orientation of the image compare to the image on the slide?
a prepared slide of paramecium and view it using the scanning lens
(40X). After the light and focus are adjusted, center a paramecium and
increase the magnification to 100X. Next, adjust the lighting, use the
fine focus, and then center the paramecium. Increase the magnification
to 400X and then adjust the light and adjust the fine focus.
procedure described above (adjust the light, adjust the focus, center
the specimen, increase the magnification) should be used whenever you
are trying to view a small specimen that is difficult to find.
8) Draw the cell. Label the cilia and the oral groove.
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Determining the Size of a Specimen
is useful to know the size of the field of view so that you can
estimate the size of objects. If the field of view is 2 mm and an
object is about one half that big, then the object length is ? X 2 mm =
We can measure the diameter of the
field of view under low power using a ruler. You cannot use a ruler
under high power because it is too big.
Suppose you measure the low-power field of view with a ruler and it is 2 mm.
high power is 10X more magnification than the low power, the field of
view will be 1/10 as big. The field of view under high power will be 2
mm X 1/10 = 0.2 mm.
Suppose that the low power diameter (LPD) is 2.5 mm (2500 um).
LPM (low power magnification) = 100X (10X objective and 10X eyepiece)
HPM = 400X (40X objective)
What is the HPD? In other words, if 100X is 2500um wide, how wide is 400X?
Answer: It is 1/4 as wide. = 2500 X 100/400
a small transparent ruler on the stage of your microscope and measure
the diameter of the field of view using the scanning (4X) objective.
9) Record the diameter in millimeters.
10) Convert this number to micrometers.
11) Record the total magnification when using the scanning objective.
12) Record the total magnification when using the high power objective.
13) Calculate the diameter of the field of view under high power by using the formula below.
Making Wet Mounts
Wet mounts are useful for viewing living biological material.
make a wet mount, place the specimen on a slide and then add a drop of
water or stain. Stain is often used to make the specimen more visible.
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a cover slip at an angle so that it touches the drop. Slowly lower the
raised end of the cover slip. The diagram below shows that as the cover
slip is lowered, the drop of liquid moves to the right.
- Scrape the inside of your cheek with a toothpick and rub it on a dry slide.
- Add one drop of methylene blue to stain the cells. This will make them easier to see.
- Place a cover slip on the slide as described above and observe the cells under low power then high power.
14) Draw a cell under high power.
Below: Cheek cells 100X and 400X.
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Size of Onion Cells
a wet mount of fresh onion and stain it with methylene blue. Try to get
the thinnest piece that you can; it should be thinner than paper. It
might help if you take a thicker piece and bend it until it snaps into
two pieces. After it snaps, there is usually a very thin connection
remaining between the two pieces. This thin connecting layer can be
peeled off and placed on a microscope slide for viewing. After you view
this slide and answer the questions below, set the slide aside for use
later in the laboratory period.
15) Draw several onion cells as they appear under high power.
Estimate the length of a typical onion cell in micrometers (um). This
can be done by using the diameter of the field under high power
calculated earlier. The following is an example; your numbers will
probably be different. Suppose that a typical cell is approximately 1/3
the diameter of the field of view and the field of view is 450 um, then
the cell is 450 um/3 = 150 um. Show the numbers that you plugged into
Below: Onion cells.
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microscopes and stereomicroscopes have one ocular lens that is
adjustable (see photograph below). This enables you to adjust the
viewing for your eyes so that you do not need to wear your glasses.
Remove your glasses before doing the procedure below.
these lenses, first, cover the adjustable lens (or your eye) so that
you cannot see the image through it. Next, focus the microscope the way
you normally would so that a sharp image is produced through the ocular
lens. Cover this lens (or eye) and view the image through the other
(adjustable) ocular. Turn the ocular lens but not the focus knob and
adjust so that the image is sharp.
Binocular Dissecting Microscope (Stereomicroscope)
dissecting microscopes are useful for viewing material that is too
large to be viewed by compound light microscopes. The magnification of
these microscopes typically ranges from 8X to 40X.
microscopes (Stereomicroscopes) have two ocular lenses and produce a
three-dimensional image. If you wear glasses and are unfamiliar with
adjusting the ocular lenses of optical equipment to suit your eyes, see
the section titled "Adjusting the Ocular Lenses" below.
are several kinds of binocular dissecting microscopes available in the
laboratory room. The microscope shown below contains a zoom adjustment. By rotating the adjustment, the magnification changes from 8X to 40X. The microscope requires a separate light source (shown).
microscopes shown below have zoom magnification adjustments. The scope
on the left ranges from 20X to 40X and the one on the right ranges from
10X to 45X.
These two microscopes have a light source built in. The light adjustment knob (see photograph) enables the specimen to be illuminated from above, from below or from both above and below at the same time. The zoom adjustment has the total magnification written on the knob.
Practice Using the Dissecting Microscope
a dissecting microscope. If your dissecting microscope does not have a
lamp, you will need to also obtain a separate microscope lamp.
a plant leaf using the dissecting microscope by placing the entire leaf
under the scope. It is not necessary to prepare a slide.
The leaf will probably look best if illumination is provided from below.
17) Draw the leaf and write the total magnification on your drawings.
- View a clam shell using the dissecting microscope. The shell is opaque, so it must be illuminated from above.
18) Draw the shell and write the total magnification on your drawings.