Science in Motion Wilkes University Revised Fall 2010

Introduction to Microscopy
Standards:
PA ST& E Standards:
3.1.10.A1. Explain the characteristics of life common to all organisms.
3.1.B.A1. Compare and contrast the cellular structures and degrees of complexity of prokaryotic and
eukaryotic organisms.
3.1.10.A5. Relate life processes to sub-cellular and cellular structures to their functions.
3.1.B.A5. Relate the structure of cell organelles to their function.
3.1.10.A8. Investigate the spatial relationships of organisms’ anatomical features using specimens,
models, or computer programs.
3.1.B.A8. Recognize that systems within cells and multi-cellular organisms interact to maintain
homeostasis.
3.1.10.A9. (Science as inquiry) Know that both direct and indirect observations are used by scientists to
study the natural world and universe.
PA E & E Standards:
4.2.10.C. Explain the relationship between water quality and the diversity of life in a freshwater
ecosystem.

Guiding Questions:

1. What are three ways compound and stereoscopes differ and one feature they
share?
2. How are magnifications calculated for compound and stereo-microscopes?
3. What are the main parts and their functions for the compound microscope?
4. What steps are used to view a prepared slide, such as the “letter e?”
5. How do you estimate field of view?
6. How do you scale diagrams made from microscope observations?
7. How are wet mounts prepared?
8. Compare and contrast two ways plant and animals cells are similar and two ways
they differ.
9. Compare and contrast two ways prokaryotic and eukaryotic cells are similar and
two ways they differ.
10. Draw and describe three organisms from a pond water sample or prepared slides.
11. Describe several points regarding microscope safety.

Vocabulary:

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Amyloplasts are starch granules that can be observed in a potato tuber slide.
A Binocular compound microscope has two ocular (eyepiece) and several objective
(lower) lenses and can magnify objects up to 1000x.
A Dissecting or Stereoscope magnifies to lower limits (about 30x) and gives three
dimensional views of objects.
Eukaryotic cells are more complex and have a distinct nucleus and membrane-bound
organelles such as chloroplasts and mitochondria. Examples are plant, animal, fungus,
and protist cells.
Field of view is the size of the diameter observed under a given objective lens.
Magnification refers to the number of times an object viewed under the microscope has
been enlarged as compared to its original size.
Microscope parts are listed and described in the Compound Microscope section.
A Nanometer (nm) is one billionth of a meter. This small unit of measurement is used in
referring to distances observed under a microscope.
Numerical aperature (n.a.) refers to the ability of microscope lenses to gather light and
resolve fine specimen detail.
Prokaryotic cells are less complex and lack a separate nucleus and membrane-bound
organelles; examples are bacterial cells.
Resolution refers to how clearly objects on a slide can be distinguished as being separate.
Scaling is used to determine what the distance shown in a drawing or map represents in
real life.
Stratified squamous epithelium is a type of tissue that consists of flattened, layered
cells. Vertebrate skin or the mucous membrane lining the inside of the mouth are
examples.

Introduction and Background:

Biologists spend a lot of time looking at organisms. Many are clearly visible to
the naked eye, but others are not, therefore, to see them their images must be magnified.
Sometimes a magnifying glass will do, but frequently, a microscope is needed.
There are several different types of microscopes. You will become familiar with
two kinds: a compound microscope and a dissecting or stereoscope. These two
microscopes differ in three respects: 1) the compound microscope can magnify objects up
to 1000-fold, whereas the dissecting or stereo-microscope’s potential is only about 30-
fold. 2) The dissecting microscope gives you a three-dimensional view of the outer
surface of the object, whereas the compound microscope’s image is largely two-
dimensional. 3) To observe an object under a compound microscope, the object must be
extremely thin and placed on a glass slide. Thus, a thick structure must be sliced into thin
sections using a razor blade. Sectioning and slide preparation is not necessary for the
dissecting microscope.
Compound and dissecting microscopes are similar in that they use visible light
(radiation of wavelengths 400 — 700 nm) to illuminate the object. The problem with

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using visible light is that it has a limit of resolution (the smallest structure visible) of 200
µm (.2 mm), regardless of the quality of the microscope’s lenses. Unfortunately, many
sub-cellular features, and even some organisms, are smaller than 200 µm. Thus, they are
too small to be seen with light microscopy, and to see such minute features they must be
illuminated with another type of radiation, such as electron microscopy. This is beyond
the scope of our lab. Complete #1, Table 1 on Page 1 of your Data Sheet.

Materials:
Compound microscope Small beaker tap water with dropper
Stereoscope (1 per class) Single-edged razor blade
Letter ‘e” slide Thin potato slice
1 mm grid slide Dropper bottle of iodine
Thin sheet onion epidermal cells Vertebrate cheek cell (stratified squamous epithelium)
Dropper bottle of methylene blue Toothpick
Blank slides Yogurt
Coverslips Pond water with dropper or prepared slides
Kim wipes Paper towels
Forceps 3 shapes bacteria slide
Immersion oil Lens paper

Safety:

1. Handle the microscope with care and hold with two hands by the base and arm when
moving as it is a very expensive, delicate observation tool.
2. Electricity can be dangerous, be careful when plugging the microscope into the outlet.
If bare wires are seen on the cord do not use and notify your teacher. When
unplugging, always pull by the plug, not the cord.
3. Only use lens paper (never paper towels) to clean microscope eyepiece lens.
4. Be careful handling glass slides and cover slips as they could break and cause cuts to
the skin. Carefully dispose of broken glass following instructor’s directions.
5. Always begin with the lowest objective- 4x to avoid damaging the objective lenses or
breaking slides; then move to the higher powers being very careful with the highest
powers.
6. Be careful with dyes such as methylene blue or iodine as they can stain skin or cause
eye irritation.
7. Take care when slicing onion and potato with a single-edged razor blade to avoid
cuts.
8. Avoid squirting pond water which might have living bacteria or protists into anyone’s
eyes and wash hands carefully after this section of the lab.
9. When using oil for 100x objective when observing bacteria, take care to not use too
much oil and carefully wipe microscope with lens paper when finished. Also clean oil
from stage.
10. Remove slides from microscope when finished and follow instructor’s directions for
disposal. Wipe stage and protect microscope with dust cover.

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11. Clean your lab area by disposing of all paper and lab debris in the appropriate
container, wipe table, and always wash hands before leaving the lab.

Procedure:
COMPOUND MICROSCOPE

There are binocular compound microscopes on each of the lab tables. Go to the
table as directed by your instructor and remove the dust cover from the microscope.
Notice that there are a variety of controls and other parts. These will be discussed in turn.
Figure 1 is a compound microscope.
Starting at the top, you will be looking through the twin eyepieces or ocular
lens. Notice that the rim has “l0X” engraved into it. That means that the lens magnifies
all images 10-fold. In looking through one of the ocular lenses you will notice a dark line.

That is a hairline, and is useful for pointing to objects viewed under the microscope.
Next, look at the nosepiece and objective lens assembly. Your microscope has four
objective lenses, and the whole assembly can be rotated. Carefully rotate the assembly
and notice that each lens clicks into place. The lens in front of you is the one that is
engaged. Observe that the four objective lenses each have a different color band. The
short one with the red band has a 4x power of magnification. The lens with the yellow

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band is 10x. The lens with the blue band is 40x. The lens with the white band is 100x.
The total magnification is calculated by multiplying the ocular lens (l0x) times the
objective lens. Complete #2, Table 2 on Page 1 of your Data Sheet. Another important
feature which will not be discussed in detail in this lab is numerical aperature, which
refers to the quality of the lens. This is the decimal number that can be seen on the
objectives on college level microscopes, such as the one you are using today.

TO PREVENT DAMAGE TO THE LONGER LENSES, ALWAYS

ENGAGE THE 4X (RED) LENS WHEN PLACING OR REMOVING A
SLIDE FROM THE MICROSCOPE!

Next, notice the stage, which is the black, flat surface beneath the objective
lenses. The slide is to be positioned on the stage, secured by the stage clip and bracket
assembly. Move the stage clip to the right, and notice that it will return to its original
position by spring action. The horizontal and vertical adjustment knobs are located to
the lower right of the stage. Turning the upper knob will cause the stage to move toward
or away from you. Turning the lower knob will cause the clip and bracket assembly to
move to the left or right.
Beneath the stage you will find the condenser assembly. The condenser controls
and focuses the light passing to the slide. The single round knob under the left side of the
stage controls the height of the condenser. Lowering the condenser decreases the amount
of light reaching the slide. Notice the diaphragm lever at the front of the condenser.
Moving the lever to the left and right adjusts the size of the iris diaphragm, which
further regulates the amount of light passing to the slide.
The two large knobs at both sides of the base of the arm are the focus adjustment
knobs. The inner (large) knob on either side is the coarse focus. Turn that knob and
notice the stage moves up and down slightly. The outer (smaller) knob is the fine focus,
and allows for more precise focusing. By focusing through a thick object, it is possible to
determine its three-dimensional nature.
Finally, the light source is located beneath the condenser. The on-off switch is
located on the rear of the scope base. At the left of the microscope base is the rheostat
wheel, which controls the brightness of the light source. Always start with it at “0”,
which would be as far to the left as the red disk will turn, as the stronger light might
be uncomfortable to those persons with sensitive eyes. You may then want to
increase this depending on what you are observing. The body tube is the region
between the eyepiece and nosepiece through which light passes. The part used to hold the
microscope between the body tube and the focus adjustments knobs is the arm, and the
solid bottom of the microscope is called the base. Always hold the microscope with two
hands on the arm and base when moving. Complete # 3, Page 2 of your Data Sheet; list
12 important parts of the compound microscope from top to bottom. Carefully plug
your microscope into the closest outlet and turn on the switch.
The only maintenance you will need to perform on the microscope is periodically
cleaning the objective and ocular lenses, with lens paper provided. Should the light bulb
burn out, or any other malfunction occur, notify the instructor immediately so it can be
corrected.

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VIEWING A PREPARED SLIDE

Obtain a slide of the letter “e.” Use the coarse focus adjustment knob to raise the
nosepiece as far as possible, and then click the 4x (red) lens into place. Place the slide on
the stage, and then carefully bring the letter into focus as best you can using the coarse
focus knob. Next, bring it into best focus using the fine focus adjusting knob.

Note the following and write your observations for #4, Page 2 of your Data Sheet.

1) How does the position of the “e” you see under the microscope differ from
how the “e” appears when you view the unmagnified slide?
2) What happens to the image when you move the slide from left to right?
3) What happens to the image when you move the slide from front to back?
4) When an object is in focus under 4x, it is safe to switch to l0. How does the
“e” differ using the 10x objective?
5) After refocusing, it is safe to then switch to 40x. DO NOT switch to 40x until
you have focused the object under l0x. Failure to do so may damage the 40x
lens because it rides ‘very close to the slide, and the l00x lens rides even closer.
Do not hesitate to ask for help. Describe the “e” under the 40x objective.

ESTIMATING THE FIELD OF VIEW

The field of view is the size of the diameter observed under a given
objective lens. The field of view decreases in size as you switch from low power lenses to
higher power lenses.
You can observe the field of view directly when the 4x lens is in place by
obtaining a grid slide with 1 mm squares and placing it on your stage.
Examine the grid slide with the 4x lens in place and after determining the
diameter of the field of view in millimeters (1 square = 1 mm), enter that value in Table
11 on page 1 of your data sheet. It is not possible to directly measure the field of view
under l0x, 40x, or l00x.
Instead, please estimate them using the formula A x B = C x D, where:
A equals the low power magnification (40x)
B equals the diameter of the field of view at 40x measured by using the grid
slide (100x, 400x or 1000x)
C equals the higher power (l0x, 40x, or l00x)
D equals the field of view of the higher power

Example: With the objective lens at 4x (a total magnification of 40x), a grid slide placed
on the stage shows the diameter of the field of view 5mm. A X B is 200.

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If A x B = C x D, A x B =200=C x D

Answer the question if A x B = 200 and C x D = 200; if C = 100, what is the value of D
(the new field of view)? Solve for D by dividing A x B by C or 200/100.

The value of D is then 2.0, a 2.0mm field of view at l00x.

Work out all the values of field of view for your scope, beginning with the 4x lens. Enter
the values in #5, Table 3 on Page 2 of your Data Sheet. If you do not have time to do
the actual observations and calculations using the grid slide, do this using the numbers
listed in the Example on Page 4.

SCALING

Today you will be making diagrams of objects viewed under the

microscope. It is important in scientific observation and research to know how large the
various structures really are. This topic is more advanced and might be beyond your
instructor’s scope and time allotment for this lab, but this section explains how it would
be done.
The idea behind scaling should be familiar to anyone who has used a map. You
can estimate the distance between two cities by measuring the distance between them on
a map, then consulting the bar scale to determine the distance that it represents in real
life. For example, if two cities are 5cm apart on a map, and 1cm represents 50 miles, then
you can estimate the two cities are 250 miles apart. For each diagram that you make, you
will provide a scale. Often for small measurements such as for a bacterial cell
micrometers (µm) are used; a micrometer = one thousandth of a millimeter (.001). In
doing so you must decide: l) how long the scale should be (in micrometers, µm) and 2)
how long the line that you draw on your data sheet to represent that scale should be (in
mm). To decide how long the scale should be you need to know: 1) which objective lens
you have in place, and 2) the field of view (µm). The scale should be a round number
between 1/10 to 1/3 of the field of view. For example, assume that you were observing
something under low power and that the field of view is 5000µm (5 mm). The range of
possible scale lengths would be 500µm (l/l0 x 5000) to 1700µm(1/3 x 5000).
Within that range, round numbers such as 750µm and 1000µm would all be
acceptable. Let’s assume that you select l000µm. To decide how long the line you draw
on your paper to represent l000µm should be, you use the equation:
a=c
b d
where: “a” is the length of the line (in mm) that you want to draw
“b” is the diameter of the circle (in mm) that you drew on your data sheet
( representing the circle that you observe under the microscope)
“c” is the length of the desired scale (in µm)(in this case it would be 1000 µm)
“d” is the field of view (in µm)(in this case it would be 5000µm)

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Notice that b, c, and d are all known. Therefore, you merely need to solve for a, by
dividing b x c by d and that will give you the length of the line that you need to draw on
your data sheet to represent 1000µm.

MAKING A WET MOUNT

Obviously, not everything that you want to observe under a microscope will be
prepared for you. You will often need to make your own slides from fresh material.
In this lab, you will be making four wet mounts and observing five slides. The
first is of onion epidermis in order to clearly see cell structure. The second will be potato
tuber, to allow you to see how stains make certain features more evident. The third will
be a prepared slide of vertebrate cheek cells, so that you can compare animal cells with
plant cells. The fourth will be yogurt, in order to compare prokaryotic and eukaryotic
cells. The final slide will be of pond water or prepared slides if pond water is not
available, in order to gain some appreciation for the diversity of microorganisms.

ONION EPIDERMIS

Break or slice with a razor blade a piece of a single layer of onion. Don’t use the
outermost layer because most of the cells there will be dead. Snap the piece in half; then
use forceps to peel off a bit of tissue-like transparent epidermis from the inner layer.
Mount the epidermis in a drop of methylene blue on a slide so that it is flat and not
doubled over on itself. Add a coverslip and use absorbant paper to draw out
any excess dye.
View the cells under low power. The layer of cells is just one cell thick. Place the
slide on the stage and focus under the 4x objective. Notice the brick-shaped cells, then
switch to l0x and refocus. If you desire, switch to 40x, however if a single cell cannot fit
within the field of view, return to the l0x.
Using a pencil, draw the cells of the onion epidermis in the 10 cm circle on # 6
Page 3 of your Data Sheet so that they are the same shape and proportion as the
cells appearing within the microscope. Draw just a few cells, but draw them carefully
to show that you were really looking at them. Draw only 4 or 5 cells, do not fill the
circle.
Next, scale your diagram as outlined above, and label the visible structures by
drawing a line to them with a ruler and labeling them outside the circle. Write the
magnification and provide a discussion in which you describe the shape, size, and
color of the cells using at least one or more complete sentences. Cell structures you
might include in your drawing are the cell wall, cytoplasm, nucleus, nucleolus,
nuclear membrane.

POTATO TUBER

Using a razor blade, obtain an extremely (paper) thin section (ca. 5mm x 5mm)

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of the white part of a potato tuber. Add a drop of water and a coverslip. Examine under
your microscope using the 4x objective and notice the whitish spherical structures. Note
though, that the structures are not as distinct as they were for the onion epidermis. Do not
make a diagram at this time.
To make the structures in the potato tuber more visible you will stain them with
iodine. Since potatoes contain lots of starch, a noticeable blue color should
develop. Add Lugol’s iodine to your original potato slide by slowly inserting a drop under
one corner of the coverslip. You can place a bit of paper towel or kim wipe along the
opposite edge of the coverslip to absorb the water already there and to draw the dye under
the cover slip. Wait about two minutes for the color to develop and observe under your
microscope switching to the 10x objective. Notice the spherical, dark blue structures
(these are starch granules or amyloplasts) and the larger, ghost-like cell walls.
Draw the cellular portion (and a few of the loose amyloplasts) in the circle on
# 7, Page 3 of your Data Sheet in the same manner as you did for the onion
epidermis and write the magnification. In your discussion, comment on the size, shape,
and color of the potato cells. Describe the difference in appearance of the amyloplasts
caused by adding iodine.

CHEEK CELLS (Stratified squamous epithelium, smear)

Due to health concerns and to avoid the spread of disease, you will observe
prepared slides of vertebrate cheek cells. Observe first using the 4x objective, you may
then switch to 10x . Some cell structures you might observe include the nucleus,
cytoplasm, and location of cell membrane. Draw and label a few cheek cells in the
circle on #8, Page 4 of your Data Sheet, write the magnification, and in your
discussion describe the size, shape, and color of the cells.

PROKARYOTIC CELLS

Prokaryotic cells are structurally much simpler than eukaryotic cells. They lack
distinct internal structures such as a nucleus. So far the cells observed have been
eukaryotic because they have had a nucleus and other membrane enclosed organelles. In
this lab, you will examine prokaryotic cells from two sources: yogurt and prepared slides.
You will note their size and shape. Examination of their internal contents will be
exceedingly difficult, due to the small size of the cells.
A container of yogurt containing an active bacterial culture is available. Using
a toothpick, place a tiny dab of yogurt on your slide. Add a
small drop of water and stir the toothpick around to spread out the cells. Add a
coverslip.
Focus on the slide under low power and then switch to the 10x and then the 40x
objective, which will be useful to most successfully observe the bacterial cells.
The swarms of organisms that you see are cells of two different species of yogurt
bacteria: Lactobacillus bulgaricus and Streptococcus thermophilus. In order for a product
to be called “yogurt” in the U.S., it must be made using these two cultures, referred
commonly as “live and active cultures.” Observation hints: the suffix bacillus means
“rod-shaped” bacteria, and streptococcus means “chains of circular bacterial cells.”

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Write the magnification, diagram and label a few cells of each species in opposite
sides of the circle in #9, Page 4 of your Data Sheet. Show proper shape and size of the
cells.
Next, observe prepared slides of bacterial cells. First focus on the 4x objective,
then switch to 10x or better 40x since bacterial cells are much smaller in comparison to
other specimens observed in this lab. If time permits, you may try to use the oil
immersion lens, or 100x objective. This lens is very fragile and easily scratched, so take
care and carefully remove the oil with lens paper when you are finished. After focusing
on lower powers, add 1 drop of oil to the top of the slide in the correct location. Slowly
and carefully while watching from the side lower the 100x objective into the oil, then
slowly raise very slightly until the cells come into view. This procedure takes practice,
and you might not meet with success on the first attempt. Write the magnification
and make a separate diagram for of each of the 3 main categories of bacterial cells:
coccus (round), bacillus (rod), and spirilla (spiral) from the slide in one of the three
sectors of the circle in # 10, Page 5 of your Data Sheet. Be sure the organisms are
drawn in the correct proportion.
Compare the cell structure of prokaryotes (yogurt/bacteria) with those of
eukaryotes (onion/potato/cheek). Complete #11, Table 4 on Page 5 of your Data Sheet
in which you list two characteristics that differ between prokaryotic and eukaryotic
cells, and two that are similar. Use your textbook or the Internet if you need help to
complete this table.

POND LIFE

Prepare a wet mount of the pond water available, or observe prepared slides. Try
to place the dropper into the part of the pond water with debris to have the best chance of
observing organisms. Observe at least three different species. Examine the slide using 4x,
l0x, and 40x objectives. Write the magnification, and in each third of the circle in #
12, Page 6 of your Data Sheet draw a different type of organism. In your discussion
comment on the number of different kinds of organisms which you observed, their
relative sizes, and whether the majority appeared to be unicellular or multicellular.

SUMMARY

In #13 on Page 6 of your Data Sheet write a paragraph using

complete sentences with correct grammar and spelling
describing at least five new procedures, facts, or concepts you
have discovered during this laboratory activity.

References:
Baranoski, K. 2010. Revision of existing lab sheet from primary
sources and Data Sheet. Wilkes-Barre (PA):Wilkes University.

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Eberhard, C. 1996. Saunders general biology laboratory lab

manual. Philadelphia (PA): Harcourt College Publishers. p.
15-24.

Terzaghi,W. 2010. Introduction to Microscopy.

http://staffweb.wilkes.edu/willterzaghi/BIO-121/labs/lab3.html.

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