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Lab 5
Microscopy
Introduction
The microscope is an instrument that contains one or more lenses and is used to view
objects that are too small be seen with the
unaided eye. A magnifying glass is a simple
microscope because it contains only one lens.
A lens functions by refracting (bending) light
rays coming from an object and focusing them
to from an image of the object. A compound
microscope contains more than one lens.
The microscope is useful for making observations and collecting data in scientific
experiments. Microscopy involves three basic
concepts:
1. Magnification - The amount that the image
of the specimen is enlarged.
2. Resolving power - How well detail is
observed and preserved during the magnification process.
3. Contrast - The ability to observe detail
against the background. Often, stains and
dyes are used on biological specimens to
increase contrast. Sometimes special optics
are used such as phase contrast lenses.
Focusing the microscope: There are two
round knobs on the arm of the microscope.
The larger knob is the coarse adjustment
knob. This knob is used to make large, coarse
adjustments when focusing on a specimen.
Use the coarse adjustment knob when using
the low power objective lens only. The lower
smaller knob is the fine adjustment knob.
Manipulating this knob makes fine adjustments when focusing on a specimen.
Carrying the Microscope
The microscope is an expensive instrument.
When removing the microscope from the
storage cabinet, always grasp it with both
hands. Place one hand around the arm and the
other hand firmly under the base. Hold it
close to your body for stability. Once you
reach the lab bench, set the microscope down
gently on the table. Do not slide the microscope across the table. Instead, lift it off the
table and gently place it back down where you
want it.
Depth of Focus
Parts of the Compound Microscope
The bottom of the microscope is the called
the base. Mounted on the base is the fixed
light source. Light then passes through the
diaphragm (iris, aperture) which you can use
to adjust the amount of light coming from the
light source. After the diaphragm, the light
then passes through the condenser which
focuses the light on the specimen. The object
(specimen) to be examined is placed on the
stage. Light passing through the specimen
then enters the objective lens. The objective
lenses are attached to the revolving nosepiece
which facilitates changing from one objective
lens power to another by revolving upon its
axis. Our microscopes have three objective
lenses: the scanning or low power lens (10X),
high power (40X), and oil immersion (100X).
We will not use the oil immersion lens in this
class. After the objective lens, light then
passes up the body tube. At the upper end of
the body tube is the ocular lens which focuses
the light on the retina of your eye. The magnification power of the ocular lens is 10X.
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The vertical distance that stays in focus at a
single time is called the focal plane. On high
power, the focal plane is more shallow than on
low power. As you focus up and down, you
will get an idea of the specimen’s threedimensional form.
Magnification
The magnification of each objective lens is
marked on the side of the lens. These lenses
are color coded. The scanning lens (10X) has
a yellow ring on it, the high power (40X) lens
has a blue ring, and the oil immersion lens
(100X) has a black ring on it. The ocular lens
has a magnification of 10X. The total magnification is obtained by multiplying the power
of the ocular eye piece (10X) by the power of
the objective lens used.
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but you also risk cracking a slide or an
expensive objective lens. The distance
between the objective lens and the slide
decreases with increased magnification.
Change to the high power objective by
revolving the nosepiece until the object is in
line with the tube. CAUTION: Keep your
head to the side to be sure that the lens does
not smash the slide! The lens is properly
seated when you hear and/or feel the “click”
of the indent stop.
Using the Microscope
First make sure the scanning (low power)
lens is in position. (NOTE: this is the way the
microscope is stored.) Next place the prepared slide on the stage in such a position that
the object of interest is centered over the hole
in the stage. With the scanning lens (low
power), there is little danger of smashing a
slide; nevertheless, make sure the objective
lens does not touch the slide. Turn on the
light.
You may have to twist the fine adjustment
knob to sharpen the image by turning slightly
upward or downward.
Look into the ocular lens. The field should
be evenly illuminated and bright. You are
now ready to focus the microscope. While
looking through the ocular lens, focus slowly
by turning the course adjustment until the
object is brought into view. If you continue to
focus and the object does not come into view,
you have passed the proper focal plane and
must start over again. If after two or three
attempts you are still unable to focus on the
slide, call your instructor for help.
Now that you know what to do, practice will
enable you to use the microscope with ease.
Remember, start with low power and scan for
objects on the slide. Do not hunt with high
power.
To avoid eye strain, practice using the
microscope with both eyes open. This is most
easily accomplished by covering the eye not in
use with your hand, and then removing your
hand while leaving your eye in “neutral”.
With practice you will soon learn to leave
your eye in neutral when using the microscope.
The focus may be sharpened with the fine
adjustment if necessary. When changing from
a lower power to a higher power objective,
very little adjustment is necessary. This is due
to the fact that these microscopes are parfocal, meaning that the subject remains nearly in
focus from one objective lens to the next.
Therefore, only use the coarse adjustment with
the scanning or low power lens. Never use the
coarse adjustment when using the high power
or oil immersion lens. Not only is it unnecessary due to the parfocality of the instrument,
Make sure the microscope is clean at all
times. Keep your fingers off the ocular and
objective lenses. If there is dirt on any of the
lenses, it must be removed with lens tissue
paper only!
Exercise 5.1 Specimen Orientation
1. Obtain a permanent slide with the letter “e” on it. Carefully looking at the slide with your
unaided eye determine if “e” is right side up or upside down. Remembering the orientation of the
letter, center the “e” in the field if view and carefully bring it into focus under the scanning lens.
2. Draw the “e” as you observe it through the microscope below.
3. Rotate the nosepiece to the high power (40X) objective lens and focus with the fine focus only.
4. Again draw the “e” as you observe it now.
5. With either objective lens in place, move the prepared slide to the right while watching the image
through the microscope.
6. Repeat step 5 only move the slide away from you.
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Exercise 5.1 Report
Name ___________________
1. Calculate the total magnification for the combined ocular lens and objective lens in the table
below.
Objective
Lens
Unaided eye
Lens Power Ring Color Ocular
on lens
lens
power
1X
none
1X
Scanning
4X
red
10X
Low Power
10X
yellow
10X
High Power
40X
blue
10X
Oil Immersion
100X
black
10X
Total Magnification
4X × 10X = 40X
2. Draw the “e” as seen under low and high power. Calculate the total magnification of each field
and write it on the line provided.
3. When you moved the slide to the right which way did the image in the microscope move?
4. When you moved the slide away from you which way did the image in the microscope move?
5. What is the relationship between the movement of the image and the movement of the specimen?
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Exercise 5.2 Report
Name ____________________
1. What is the order of threads on the prepared slide?
Top _________________
Middle ________________ Bottom ___________________
2. How did you determine the order of threads?
3. What diaphragm adjustment can be made to more easily determine the order of the colored
threads?
Exercise 5.3 Report
1. Draw & measure the diameter of your field of view using a Stage Micrometer.
Low Power
High Power
2. What is the diameter of the field of view under low power?
3. What is the diameter of the field of view you can observe under high power?
Exercise 5.4 Report
1. Draw the image of a cheek cell under high power
in the field diagram to the right.
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go (or it will not fit into the cabinet).
Returning the Microscope.
When you are finished using the microscope, it must be returned to the storage
cabinet in the proper condition.
•Turn the light off.
• It should be clean. If the lenses are dirty,
obtain lens paper from the cabinet and
clean them.
• Place the lowest power objective (yellow
ring) in the light path.
•Wrap the electrical cord loosely around the
base.
•Cover the microscope with the dust cover.
•Place the microscope (carrying it with two
hands) back in the correctly numbered
compartment of the storage cabinet.
• Move the body tube down as far as it will
Exercise 5.2 Depth of Focus
1. Obtain a slide with three colored threads mounted one over another.
2. Using low power (10X), find the point where all three threads cross. Then switch to high power
(40X).
3. Slowly focus up and down with the fine adjustment knob to observe the depth of the threads.
4. Notice that when one thread is in focus the others seem blurred.
5. Determine the order of the colored threads and enter the results below.
Exercise 5.3 Field of View
1. Obtain a microscope slide with a ruled stage-micrometer.
2. Draw what you observe under both low and high power and
determine the diameter of each field.
Exercise 5.4 Viewing Cheek Cells
1. Obtain a new microscope slide.
2. Place a drop of Methylene Blue stain in the middle of the slide.
3. Use a sterile toothpick and gently scrape the inside of your cheek with the flattened end of the
toothpick.
4. Swirl the flat end of the toothpick in the drop of stain on the microscope slide.
5. Obtain a cover slip and place it over the drop on the slide.
6. Observe the cells under the microscope under low power then switch to high power.
7. Under high power find the nucleus, a round centrally located body within each cell.
8. Draw the high power image on your lab report.
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The Microscope
Label the parts of the microscope below.
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