Lab 2: Microscopy

Name:

Prelab Exercises
*Complete this page as a Pre-­‐Lab Activity*
Turn in this page when you arrive at your lab session.

1. Copy down the equation for total magnification found on page 4 of this lab. (0.4pt)

2. Using the equation above, fill in the correct values for total magnification in the following
table (ocular magnification is 10x): (0.6 pt)
Scanning/low power 4x objective total magnification = _____ x
Medium power 10x objective total magnification = _____ x
High power 40x objective total magnification =_____ x

3. A Biology student is observing arthropods with a compound light microscope using 40x total
magnification. The large circle represents the field of view for the microscope at 40x total
magnification. If the diameter of this field of view is 4 mm, what is the approximate length of
the little critter she’s examining? (Show your work for full credit 1pt)

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 Lab 2: Microscopy

Preparation
1. Read the entire exercise before lab and mark anything you will need to ask about. This will
prepare you to work more efficiently in lab because you might not then have to read
through all of the background information while you could be doing the activities. We will
be able to tell who is prepared and who did not read their lab material thoroughly.

2. Study the included microscope diagram so that you can locate microscope parts by name.

Reference
The attached pages on microscope parts/function, microscope care, and how to prepare a wet-­‐
mount slide, will be a valuable resource for this lab and future labs during the semester.

Objectives

1. Know the names, functions, and correct operation of microscope parts.

2. Use good technique in preparing and studying fresh specimens.
3. Use good technique in drawing and labeling diagrams.
4. Estimate the true size of objects seen in the microscope.
5. Know the names of the basic tests for carbohydrates and proteins.
6. Be able to accurately interpret the results of the tests for biological molecules
7. Use your knowledge of the basic tests for carbohydrates and proteins to determine
what is in an unknown solution.

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Part I: Microscopy
Resolution and Magnification
Microscopes are magnifying tools used to produce enlarged images of tiny objects to bring them
into the range of resolution of the human eye.
Magnification refers to how many times bigger the image is than the object itself. It is
measured in terms of the number of times the diameter is increased—10x, for example, means
that the diameter of the image is ten times the diameter of the actual object.
Resolution, or resolving power, refers to the clarity of the image and is measured as the
smallest space that can be seen between nearby objects. The human eye cannot distinguish
between objects that are closer together than about 0.1 mm; they blur together and look like a
single object. The resolving power of the eye is therefore said to be about 0.1 mm (or 100 µm).
This also means that the unaided eye cannot detect an object less than 0.1 mm wide, which is
about the width of a human hair, because it blurs into the background. If the object is
magnified so that the eye looks at a larger image, it can be seen—if it is magnified accurately. A
microscope (or any other magnifying system) is useful only if its resolving power is better than
that of the eye. A microscope with poor resolution but high magnification simply produces a
large fuzzy image. As photographers know, if you want to blow up a picture to a very large size
and still have sharp details, you need to start with a high-­‐resolution image.
Resolving power depends on (1) the quality of the lenses and (2) on the wavelength of
light used. Excellent light microscopes can resolve objects that are about 0.2 µm apart. This
means also that objects only 0.2 µm wide can be seen with such a microscope—provided they
are magnified enough to bring the magnified image within the resolving power of the eye. This
would require a magnification of at least 500 times, which is a little more than the microscopes
we use in lab can produce. But our microscopes do not have lenses capable of resolving such
small objects, so higher magnification would be wasted.
Electron microscopes use a beam of electrons instead of a beam of light. The
wavelength of a beam of electrons is many times smaller than the wavelength of any visible
light, so an electron microscope can resolve much smaller objects. But an image cannot be
formed by focusing a beam of electrons on the retina of the eye. Instead, the image is formed
on a television screen, and then viewed and/or photographed.
Contrast is another critical factor in microscopic visualization. Contrast refers to
differences in color or intensity between different structures. If all of the parts of an image are
the same color, or the same shade of gray, they are difficult to distinguish. At the microscopic
level, there is very little natural contrast, and so material that is prepared for the microscope
must often be stained with something that will make structural differences show up better.
Stains can do this only if they color some structures more readily than others. Different
materials, therefore, require different stains. Inevitably, though, using a stain means changing
the specimen and stains must be used cautiously and studied carefully to be sure they are not
introducing meaningless differences.
Some advanced microscopes manipulate the light coming through an object so that
small differences are enhanced, increasing contrast. On compound light microscopes, like the

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ones we will use in lab, adjusting the iris diaphragm allows you to control the amount of light
passing through the specimen on the slide, thus affecting contrast.

Using Our Microscopes

During the course of the semester, we will use two kinds of microscopes in lab:

Light microscope Dissecting (stereoscopic) microscope

-­‐for slides -­‐for large, opaque, 3D objects.

Both are compound microscopes because there are two stages at which the image is
magnified on its way to your eye. (A magnifying glass is a simple microscope—there is only one
stage of magnification.) When using a compound microscope, the image is first magnified by an
objective lens (that is, one close to the object). Then, the lens through which you look—the
ocular lens, mounted in the eyepiece, magnifies the image again. Some microscopes are
monocular (one-­‐eyepiece) and some are binocular (two-­‐eyepieces).

When looking through a microscope, it is important to know how much the image you
see has been magnified. Since we have compound microscopes, meaning two sets of lenses are
working to magnify the image, we must calculate total magnification using the equation below:

Total magnification = magnification of the x magnification of the

objective lens ocular lens

Magnification of the objective lenses is written on the lenses themselves. For our
microscopes with 4 objective lenses the magnifications are 4x, 10x, 40x and 100x. The
magnification of the ocular lens is 10x. We will not use the objective lens marked 100x
because it requires a special technique, called “oil immersion” to produce a focused image.
For this lab and others, you will be asked to draw images of cells you are viewing under
the light microscope. Part of a properly labeled drawing includes reporting the total
magnification of the image, not simply the magnification of the objective lens.

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Light Microscope:

Table 1: Microscope Parts (counterclockwise from top)

Microscope Part Function
Eyepieces contain the ocular lenses (i.e., the one closest to the eye). On all our
microscopes, the ocular lenses magnify the image 10x
Revolving Nosepiece rotates to move different objective lenses into place above the slide
Objective lenses The lenses closest to the object. The three lenses of our
microscopes (from shortest to longest) magnify 4x (low power, or
“scanning lens”), 10x (medium power), and 40x (high power). The
magnified image from the objective lens passes through the body
tube to the ocular lens, which magnifies it ten times. Therefore
total magnification = objective magnification times ocular
magnification.

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Microscope Part Function
Objective lenses Each lens “sees” only what was in the center of the field at lower
continued: power.

When the image has been focused at a lower power, the next
higher lens can be rotated into place without moving the focus
knobs. Minor adjustment of focus may be necessary at the new
magnification.
Specimen Holder Holds the slide in place over the opening in the stage and helps to
on the Stage stabilize it when it is moved to view a new area.
The Stage supports the object being studied (normally a glass slide).
Condenser The lens located under the stage and above the lamp; contains the
aperture iris diaphragm ring. The lens focuses the light from the
light source on the specimen so that it is evenly illuminated. For
most purposes, the condenser should be raised at or near its
highest level.
Aperture iris Located on the condenser, operates diaphragm that opens and
diaphragm ring closes to admit more or less light from the lamp through the object.
Generally, very transparent objects and/or high magnification
require less light than intensely colored objects and/or low
magnification.
Lamp/Light source provides light that passes through the object; absorption and
refraction by the object alter the beam of light and produce the
image that is subsequently magnified as it passes through the
lenses. (Light switch and intensity adjustment knob are located on
right side of the base)
Feed knobs Move the Specimen holder forward and backward; left and right

Base Supports the optical parts of the microscope. The microscope must
always be placed on a flat, stable surface. Light switch and intensity
adjustment knob are located on right side of the base.
Coarse adjustment (on both sides of arm) move the objective lenses significantly closer
knobs to or further away from the object. Coarse focus knobs are never
used at high power because they move the lens too fast, and at
high power the lens almost touches the slide.
Fine adjustment (on both sides of arm) used to make delicate adjustments to focus,
knobs especially at high power.
Arm (not labeled) Handles on top should be used to carry the microscope.
Focusing knobs are located on the arm near the base of the scope.

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General Principles for Care and Use of Microscopes

Care of microscope:

Always carry a microscope with both hands as shown:

Fig. 1

• Clean lenses and slides with special lens tissue only.

• Always use a coverslip with wet preparations.
• When finished, remove slide; rotate low-­‐power lens into position.

Use of microscope:
• Always secure slide with the Specimen holder.
• Begin examination of slide with scanning-­‐power (4x) lens (shortest one), and specimen
(if visible with unaided eye) in center of opening in stage. Turn coarse-­‐focus knob all the
way forward (i.e., focus down), then turn it gradually towards you until image comes
into focus. Move slide until desired area is in center of field. (Microscope rotates image,
making it necessary to move slide in the direction opposite to the one that seems
required. Practice!)
• When image is focused at scanning power, rotate the medium-­‐power (10x) lens into
place without touching focus knobs until lens is in place. Then re-­‐focus, using either
focusing knob.
• Adjust light if necessary by moving the light intensity adjustment knob and the
aperture iris diaphragm ring.
• If higher magnification is desired (usually necessary for study of cells), move desired
area to center of field and carefully rotate high-­‐power (40x) lens into place. (Some
slides are too thick for this lens; don’t force the lens into place.) Use ONLY the fine-­‐focus
knob to adjust focus.
• Readjust the light if necessary. (Do this frequently to see if it improves image.)

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Skill Building Exercises:

Materials: for each pair of students

one light microscope
one slide with a printed phrase or word

Becoming confident using a microscope takes time and practice. No doubt there will be times
when you cannot find your image. Below are four common things to try in order to bring
your image into focus.

1. Slowly move the slide around on the stage. If the object you want to view is not centered on
the stage you will not be able to see it in your field of view. It might be just out of the field of
view. Remember the microscope rotates the image, so when you turn the stage knobs to
move the slide, it will be moving in the direction opposite to the direction you want the
image to move.

2. Go back to the lowest magnification where you last saw your object by rotating a different
objective lens into position. Always begin by finding your object in focus on low power then
proceed stepwise through medium and high power, making sure the image is centered and
focused before moving to the next magnification.

3. Change the focus. There are focus knobs on both sides of the microscope. The coarse-­‐focus
knob is used only at scanning and medium power; the fine-­‐focus knob is most useful at high
power, though it can be used also at other magnifications.

4. Change the amount of light coming through the object by moving the aperture iris
diaphragm ring. Generally, you will need more light as you go to a higher magnification or if
you use a slide with a lot of color. If your preparation is pale, too much light will wash it out.
Get in the habit of trying different light levels to see their effect on a particular slide.

How to find something on the slide

1. Hold the slide so that the label is to your left. Draw the label and the word or phrase on your
slide as they appear to your naked eye (i.e. do not put it under the microscope just yet).

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2. Check to make sure the scanning power objective lens (4x) is clicked into position. You
should ALWAYS begin looking at a slide under scanning power even if you eventually plan to
view the object under high power.

3. Now, put the slide on the stage so that the label is to your left. Pinch the projecting handle
to open the “jaws” of the specimen holder, position the slide securely, and then carefully
release the handle. Use the feed knobs to move the slide so that the word is over the hole in
the stage.

4. Adjust the coarse and then fine adjustment knobs as needed until one or more of the letters
in your word are completely in focus on scanning power. Draw the letter(s) as it appears
through the microscope. Label your picture with a title and total magnification.

Title:

Total Magnification:

5. Explain what this quick demonstration has taught you about how the lenses of the
microscope affect the orientation of your image (hint: it’s not just simply upside-­‐down). If you
need help figuring out what the lenses are doing, try this: turn the feed knobs so the slide
moves away from you. Watch in the ocular lens at the same time. Is your image in your field of
view also moving away from you? Now try moving the slide to the left on your stage. Is the
image in the field of view also moving to the left?

6. How to keep the object in view when you change magnification: When you increase
magnification, you look at a smaller part of the object, more highly magnified. Therefore,
you need to be sure that the specimen you want to study is in the center of the field before
you change magnification. Get the desired object in the center of the field and focused

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 with the scanning power objective (4x). Then without changing the focus, carefully rotate
the 10x objective lens (medium power) into position and re-­‐check the focus. Adjust it
carefully, using the fine-­‐focus knobs so you do not overdo it.

7. Now move to the high-­‐power lens (40x). DO NOT TOUCH THE COARSE ADJUSTMENT
KNOB. The high power lens will fit, although it will look like it might hit your slide. Make
sure it is clicked into place. Now look through the microscope. If you do not see anything
there are 4 things to try BEFORE calling over your instructor:
1. Adjust the FINE focus knob (DO NOT TOUCH THE COARSE KNOB).
2. Use the feed knobs to move your slide around slightly since you could be looking at
the space between letters.
3. Return to medium power (10x objective) and get your letter(s) in focus and centered
and then try again to switch to high power.
4. Clean the lenses of the objectives with lens paper and lens cleaner.

Title:

Total Magnification:

8. We will not be using the 100x Oil-­‐immersion lens

Estimating the size of an object seen with the microscope:

There are devices that can very accurately measure the size of microscopic objects. For our
purposes, however, an estimate is sufficient. A reasonable estimate can usually be made by
using the field of view (the lighted area you see through the lens) as a crude ruler.

Exercise 1: Determining the Diameter of the Field of View:

1. Using a slide with graph paper or metric ruler/grid, you will measure the diameter of the
field of view with the 4X and 10X objectives only. You cannot accurately measure the field
with the highest power objective (40x), because the markings are too far apart.

a. Place the grid of the slide over the hole in the stage of the microscope right over the
area where the light shines.
b. While looking through the eyepiece, align the grid to measure the diameter of
the circular field of view.

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 c. The lines are 1mm apart. Enter the diameter of the field of view for the 4X and 10X
objectives only in the table below:
d. Calculate the diameter of the field of view in microns (µm) and enter it in the table
below:

Objective Lens Total Magnification Field of View Field of View

(objective x ocular) Diameter (mm) Diameter (µm)
Scanning 4x
Medium power 10x
High power 40x

2. Because the slide cannot be used for the highest power objective, simple math can be used
to calculate the diameter of the field of view. Look back at your drawings of the word slides.
What happened to the size of the field of view when you increased the magnification?

As magnification increases, the field of view decreases proportionately. Knowing this, you can
use the following formula to calculate the field of view size for the 40X objective:

Mag4x X FD4x = Mag40x X FD40x Where: Mag = magnification of that objective

FD = field of view diameter of that objective

3. Fill in the last row of the table.

Exercise 2: Measuring an actual object

1. Obtain a slide with a word or phrase. Estimate the width of a letter “e” using the following
strategy:

a. Choose the best magnification at which the object (“e”) to be measured fits entirely
within the field.

b. Move the slide so that one edge of the object (“e”) is at the edge of the field.

c. Estimate how many objects of this kind could fit end-­‐to-­‐end (or side by side) across the
center of the field of view.

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 d. Determine the approximate diameter (or length, or width) of the object using the
following equation:

Estimated diameter (mm) = Field of View Diameter ÷ # times your object

(from table) could fit across the f.o.v.

Show your work below. You should include a drawing of your “e” at the edge of the field of
view (to scale; label total magnification) and write out the formula used.

Find the answer in mm and micrometers (µm). Ask your instructor to check your work before
continuing.

Formula used:
Total magnification:
Diameter in mm:
Diameter in µm:

Little swimmy critters!

Pond Water
Using a drop or two from the jar of pond water, prepare a wet mount according to the
directions below. Make two separate drawings of two different organisms you find in your
sample of pond water. Record the magnification at which you view the organisms, and
estimate the length (i.e., the longer dimension) of the organisms in micrometers (µm) using the
estimation technique you learned.

Preparing a wet mount

Have slide, coverslip, and pencil (with eraser) or finger tip ready.
• Place specimen (see previous section) in middle of slide and
cover with a drop of water or stain. If the specimen is in
water, like your pond water, adding water is not necessary.
• Put one edge of coverslip against slide at one edge of water
drop, and lower coverslip carefully onto drop.
• Press lightly against coverslip with pencil eraser to squeeze
out air bubbles.

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Using the Iris Diaphragm to Improve Contrast
When observing organisms in the pond water, you can increase the contrast by
adjusting/partially closing the opening of the iris diaphragm. Using this tool to adjust the light
going through your specimen can solve many focusing problems.

Pond Water Drawings:

Total Magnification: Total Magnification:

Estimated length: mm µm Estimated length: mm µm

Using Stain to Visualize Cell Structures:

Parenchyma cells of potato

Parenchyma cells are large cells commonly found in many different parts of a plant. Some types
of parenchyma cells may contain chloroplasts and carry out photosynthesis. Other parenchyma
cells may be adapted for storage of substances, like starch, which you’ll remember as being a
type of storage polysaccharide. Let’s look at how starch is stored in the parenchyma cells of a
potato:
1. Using a wet, single-­‐edged razor blade, cut an extremely thin slice from the freshly cut
surface of a potato.
2. Specimen Preparation: A good specimen will occupy no more than about one-­‐fourth of
the area under the coverslip, and it must be somewhat transparent in order to be seen.
For plant material, tear off a small piece or use a razor blade to shave a thin slice from a
larger specimen. It sometimes works to cut a wedge-­‐shaped piece first and put it on the
slide, then cut and save only the thin edge of the wedge.
3. Prepare a wet mount, in water, of this ultrathin slice of potato. If you sliced it thinly
enough, the cover slip will lie flat and even.
4. Examine the parenchyma cells under low power, and then increase the magnification. Look
for typical plant cell components.
5. Draw what you observe in the space provided (next page).
6. Without removing the coverslip, stain the potato slice with iodine according to the
following procedure:

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Staining a specimen that has already been prepared in water:
It’s generally a good idea to examine a specimen in water before staining it, in order to
understand the effects of staining. You can use low or medium power without a coverslip, and
simply add a drop of stain and a coverslip after the initial examination. However, if you need to
use high power before staining, you must put a coverslip on the preparation. The water can be
exchanged for stain (or for any other solution) without removing the coverslip, and even
without removing the slide from the microscope stage:

• Put a drop of the new solution at one edge of the

coverslip.
• At the other edge, hold a strip of paper towel. As the
paper towel draws water from under the coverslip,
the solution from the other edge will flow in.
• Repeat with additional drops as necessary.

Potato in water only Potato stained with iodine

400X (high power) 400X (high power)

Clean up:
1. Wash and dry the microscope slides. Return the dried slides to the boxes on
the counter.
2. Throw away the cover slips (regular trash is fine)
3. BEFORE PUTTING AWAY YOUR MICROSCOPE, HAVE YOUR INSTRUCTOR
CHECK TO MAKE SURE YOU HAVE PREPARED IT PROPERLY:
• Make sure that the last slide has been removed from the stage.
• Rotate the nosepiece so that the scanning objective is down.
• Disconnect the power cord from the microscope and the outlet, and
return the power cord to the top shelf of the cabinet.
• Put the dust cover back over the scope before returning the scope to the
cabinet.

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