MICROSCOPIC EXAMINATION OF CELLS

Introduction
Cells are the basic units of all living organisms. Cells determine the organization of
all plants and animals. Some organisms that have only one cell in their structural
design, yet they are capable of performing the tasks of food procurement, digestion,
internal transport, excretion, and reproduction. Other organisms require the
operation of billions of cells to perform similar tasks.
There are different kinds of cells. Two basic types are prokaryotic and eukaryotic.
Prokaryotic cells are found in bacteria and blue-green algae. These are simple cells
with no nuclear membrane or membranous organelles. Protistans, fungi, animals, and
plants all have eukaryotic cells. Eukaryotic cells have a nuclear membrane that
clearly separates the nucleus from the rest of the cell as well as membranous
organelles such as mitochondria. While they are both eukaryotic, animal and plant
cells differ in many aspects of structure and function.
Cells are the simplest components of living things. When grouped together to perform
a similar function, they form tissues. Organs are composed of various tissues
grouped together and functioning as one unit. Organs acting together to perform a
similar function are termed organ systems. Organ systems comprise multicellular
organisms like animals.
The microscope is an instrument used to examine cells which are simply too small to
be seen with the human eye. There are three basic types of microscopes used in
science today including the compound microscope, the stereoscopic dissecting
microscope, and the electron microscope. A compound microscope has two lens
systems called the ocular and the objective. The lens systems provide the
microscope with the capacity for magnification and resolving power.
Resolving power is the ability to see separate images of objects that fall very close
together on the eye. In essence, resolving power gives the microscope objectives the
ability to reveal fine detail. It could be stated as the least distance between two points at
which they are seen as two, rather than a single blurred object. This power is a function of
the wavelength of light used and the greatest cone of light which can enter the objective.
When you look through the ocular lens at a slide the portion of the slide that is visible
is called the field of view. This is the part of the slide that is illuminated by the circle
of light. The higher the magnification the smaller the field of view.
The most common objectives are listed below with their technical names:
4x = Scanning Objective
10x = Low Power Objective
43x = High Dry Objective
100x = Oil Immersion Objective

The magnification capacity is marked on the side of the objective lens. The
magnification capacity may be expressed as 4x, 10x, 43x, 97x, or other depending
upon its capacity. The "10x" simply means that this lens can multiply the image of the
specimen ten times the normal size. When the low dry objective is used you can
see more of the slide at one glance. In other words the field of view is larger. The low
and high dry objectives are the two objectives you will use the most.
The oil immersion lens allows you the greatest magnification but you can only see a
small part of the slide at one time since the field of view is much smaller. The oil
immersion objective is so called because it requires a drop of oil on top of the slide.
The oil acts as an additional magnifying lens. While the oil immersion lens must be in
contact with the oil to work properly the other objectives must never be immersed in
anything on the slide.
The working distance is that distance between the bottom of the objective and the
top of the stage. The higher the magnification the shorter the working distance is. The
oil immersion lens has the shortest working distance.
During this exercise you will be introduced to the compound microscope. You will use
it to examine both plant and animal cells and observe their basic characteristics. You
will also be introduced to basic aspects of staining which entails a process which allows
you to see the otherwise transparent cell.

Methods
Safety Information
1. Put anything (toothpicks, slides, coverslips, etc.) that comes into contact with
your saliva into the bleach bath.
2. Notify your instuctor of any broken glass.
Microscope Parts
Get a compound microscope from the cabinet and plug it in. Be sure the light is
working. Use lens paper to clean all of the lenses. Your instructor will go over the
parts of the microscope with you. Identify the following parts:
1.
Lens:
a. Ocular
b. Objective
2.
Nosepiece holds the objective lenses
3.
Ocular tube holds the ocular lens
4.
Arm holds the ocular tube and nosepiece
5.
Stage holds the slide
6.
Slide clips holds the slide in place on the stage
7.
Adjustment knobs focuses light
a. Coarse adjustment used to find specimen on slide
b. Fine adjustment used to adjust focus once specimen is found
8.
Light source of illumination for specimen
9.
Condenser focuses light on specimen

10. Diaphragm Lever adjusts the amount of light coming through the
condenser
11. Base holds the light and rest of microscope.
Care and Use of the Microscope
1.
Carry the microscope with one hand under the base.
2.
Clean the lenses with lens paper only.
3.
Clean all the lenses before using the microscope.
4.
Clean the slides with lens paper both before and after use.
The working distance is one of the features that you must understand to
prevent damaging the microscope. Rotate each objective into position and
look at the distance between the lens and the stage. When using the higher
powered objectives the working distance is so short you can actually force the
lens into the slide damaging the lens and breaking the slide. Use only the
fine focus with the high dry and oil immersion lenses. We will not use
the oil immersion lenses in this class.
5.
Put your slide on the stage using the slide clips to hold it in place over the light.
6.
Use the position adjustment knobs, if present, to move the slide side-to-side and
forward-and-back. Remember the microscope inverts the image. What seems to
be on the right side is really on the left and what seems to be on the top is really
on the bottom.
7.
Start with the scanning or low dry lens when looking for your slide specimen.
At this magnification less light is needed. Use the diaphragm lever or the light
intensity lever to regulate the amount of light coming through the field of vision.
Remember the field of view is the area you see as a circle of light when you look
through the ocular lens.
8.
The focusing knobs move your view up and down through different layers of the
slide and specimen. The layers you can look through include the top of the
coverslip, the bottom of the coverslip, the various layers of the specimen itself, the
top of the underlying slide and the bottom of the slide. Use the coarse focusing
knob to find the level your specimen is on. A slide is like a layer cake you have to
figure out which layer you specimen is on before you can use the fine focus
adjustment knob.
9.
Use the position adjustment knobs or your fingers to move the object you are
seeking to the center of your field of vision.
10. Use the fine adjustment to focus on the specimen as clearly as possible.
11. Without moving the slide, gently change to the high dry objective and use only
the fine focus knob to bring the object into clear focus.
12. Use the oil immersion only when instructed to do so. Clean the oil immersion
lens well after use.
13. Remove the slide from the microscope before storing the microscope.
14. Always turn the light off before storing.
15. Wrap the cord around the arm between the stage and the base for storage.
16. Do not set the microscope on its cord.
17. Cover the microscope before storage.
18. Replace the microscope in the cabinet it came from.

Determining Working Distance

1.
Put a plain glass slide on the stage of your microscope but do not try to
focus.
2.
Using your scanning objective or low dry objective and a ruler, measure the
working distance (distance between the bottom of the objective and the top of
the slide) in millimeters.
3.
Without touching the focusing knobs, turn to the high dry objective.
4.
Measure the working distance of the high dry objective in millimeters.
Determining Magnification
Magnification is not just supplied by the objective lenses but also by the ocular lens.
Total magnification is determined by multiplying the magnifying capacity of the ocular
lens times the capacity of the objective lens.
Ocular x Objective = Total Magnification
Determining the Diameter of the Field of View
While the total magnification tells you how much larger your specimen looks than it really
is, it does not tell you the actual size of your specimen. We are going to estimate the size
of your specimen by comparing it to an object of known size. After all, you can estimate
the height of another person by comparing their height to your own. In this instance we
are going to compare the length of your specimen to the diameter of your field of view. To
do this we are going to have to measure the diameter. While there is special
instrumentation that can do this it is not always available so this exercise will teach you
how to approximate the size by measuring the diameter manually.
1.
2.
3.
4.

Use the low dry objective only.

Pick out a ruler that has millimeters and is transparent or translucent.
Place the ruler so that the millimeter edge cuts across the center of your
field of view.
Move the ruler so that one of the millimeter lines touches the outer edge of
the field of view. Remember the lines are highly magnified so they will not
look as black or as sharply defined.

Objective lens

Slide clip
Ruler (in position)
Opening for
light source
Stage

5.

As you are looking through the microscope, estimate how many millimeters
it is straight across the center of your field of view.
Field of view showing ruler (remember the view is inverted):

Edge of ruler

Markings
on ruler

6.

7.

Compare the size of your specimen to this distance. If you estimated the
diameter is 4 mm across and your specimen is only 1/10 that long how long
is your specimen? 4 X 1/10 = 0.4 mm
If your specimen is 1/50 of the diameter then 4 X 1/50 = ?
Convert the diameter from millimeters to microns.

General Information on Preparing Slide

In this lab you are going to prepare several slides of living cells. As a safety
precaution remember that any human material or glassware contaminated with
human material should be put in the bleach bath. Since these cells are mostly water
you need to add a drop of water to the slide to prevent the cells from drying out. Once
the cells are on the slide, cover the sample with a coverslip. A coverslip holds the
specimen in place and protects the specimen and the lens.

Preparing Human Epithelial Cell Slide

1.
Gently scrape the inside of your cheek with a toothpick to obtain epithelial cells.
2.
Add a drop of 0.87% saline solution to a slide and spread the cells on the slide.
You will not be able to see the cells but they will be present. The saline solution
is isotonic to the cells and will prevent them from either losing or gaining too
much water.
3.
Place your used toothpick in the bleach bath.
4.
Place a coverslip over your preparation.
5.
Find a cell using the low dry objective then switch.
6.
Estimate the size of the cell by comparing its size to your previously measured
diameter of your field of view.
7.
Switch to the high dry objective and draw the cell and its visible structures.
8.
Label the parts of the cell visible on your drawing. Be sure to note the nucleus.
9.
You will use this slide in the next phase of you observations. You probably
noted that the cell appears very transparent. This transparency makes detailed
studies impossible. Let's stain the same specimen to make its structures more
visible. Please procede to the next set of instuctions.
Procedure for Staining Epithelial Slide
1.
Add a drop of methylene blue stain to the edge of the coverslip. It will begin to
move under the coverslip as a result of capillary action.

Eye dropper
Drop of
methylene blue
dye

Coverslip

Place a Drop of Methylene Blue Touching the Edge of the Coverslip

2.

To facilitate the movement of the dye under the coverslip you may place a piece
of tissue paper at the opposite edge of the coverslip.

Drop of dye
touching
coverslip

Coverslip

Direction of
movement
of liquids

Tissue paper
touching
coverslip

Place a Piece of Tissue Opposite the

Drop of Dye Touching the Coverslip so
the Tissue Can Absorb the Water
under the Coverslip

3.
4.
5.

Because the Dye Is Attracted to the

Water under the Tissue Due to
Cohesion, the Tissue Will Absorb the
Water and Pull the Dye Under the
Coverslip

Allow the stain to stand for one minute before you proceed with the microscopic
examination.
Using the high dry magnification, draw and label structures visible in the stained
cell.
Place both the toothpick and the slide in the bleach bath when you are finished.
Throw away the used tissue.

Procedure for Preparing Onion Skin Cell

1.
Cut an onion bulb into pieces and separate one of the layers.
2.

Remove a small piece of the thin epidermis (onion skin) found between the thick
fleshy layers.

Remove Single Epidermal Layer

3.

Spread the thin transparent onion skin layer flat on a microscope slide and add a
drop of water then cover with a coverslip.

Remove Wrinkles and Air Bubbles from Specimen

Before Covering with Coverslip
4.
5.
6.

Remember it is necessary that you keep your preparation wet to prevent drying, so
you may have to add water periodically to the specimen by placing water along the
edge of the coverslip.
Using the low dry objective, estimate the length of one of the cells by comparing it
with the diameter of your field of view.
Using either the low or high dry objective, draw one of the cells and label its
structures. Be sure to note the cell walls and determine whether the nucleus is
visible or not.

Procedure for Preparing Elodea Slide

1.
Obtain a leaf from an Elodea plant located in the beaker.

Remove One Leaf

2.
3.
4.
5.
6.

Place the Leaf on a Slide with Water

Place the leaf on a microscope slide with several drops of water and cover the
leaf with a coverslip.
Examine the leaf with the low dry objective. Note that you can see more than
one layer of cells. Focus on one layer.
Estimate the length of one of the cells.
Gently switch to the high dry objective and focus on a single cell.
Draw and label the structures of the cell. Be sure to note the chloroplasts as
well as the cell walls.

During this exercise you can locate the cell membrane which is normally very difficult to
locate due to its tightness to the cell wall.
Procedure for Locating the Cell Membrane during Plasmolysis
1.
Remove the coverslip from the elodea slide.
2.
Place several drops of 50% saline solution on the leaf. You will see that the salt
solution causes the water to diffuse from the cell which causes the cell to
shrink away from the wall. This consequently exposes the cell membrane as it
separates from the cell wall.
3.
Diagram the changes that you witnessed during the application of the salt solution
to the cells
Cleaning Up Before Leaving (REQUIRED)
1. When finished put everything back where it belongs.
2. Place anything that came in contact with your saliva (toothpicks, slides,
coverslips) into the bleach bath.
3. Put used paper in the trash.
4. Wash other used slides and coverslips with soap and water.
5. Wipe down the tables with damp sponges.

Student ____________________ _________

Date ______________________

Instructor Signature ____________________

Results
1. Complete the following chart:
Objective
Low dry objective
High dry objective

Working Distance in Millimeters

2. Complete the following chart based on numbers your instructor gives you:
Ocular Magnification

Objective Magnification

Total Magnification

3. Fill in the chart below with the results of determining the diameter of the field
of view using the clear millimeter ruler:
Objective Lens
Low Dry

Millimeters

Microns

4. Estimate the length of the specimen below using the diameter of your field of
view for the low dry objective:

? m

Show calculations for conversions.

Estimated Length of Cell = _________ mm.
Estimated Length of Cell = _________ um.
Conversion calculations:
5. Draw two human epithelial cells illustrating the difference(s) before and after
staining. Label all visible structures using the high dry objective. At a minimum

you should be able to identify the nucleus and cytoplasm.

Unstained

Stained

Millimeters (mm)

Microns (um)

Estimated size of cell

(low dry objective)
Conversion calculations:

6. Draw and label all structures of an onion epidermal cell. At a minimum you
should be able to identify the cell wall and cytoplasm. The nucleus may be
visible in some cells as well.

Millimeters (mm)

Microns (um)

Estimated length of cell

(low dry objective)
Conversion calculations:

7. Draw and label one Elodean cell before and after salt treatment. At a
minimum you should be able to identify the cell wall, cell membrane, nucleus

and chloroplasts.
Before

After

Millimeters (mm)

Microns (um)

Estimated length of cell

(low dry objective)
Conversion calculations:

Discussion
8. In your own words, explain how you calculate the size of a real object as seen
in the field of view.

9. Explain why a 0.87% saline solution was used with human epithelial cells
instead of tap water.

10. If you are trying to observe a cell under the microscope and all you see is a
circle of light when you focus, what might you do to improve the image?

11. If you are trying to observe a cell under the microscope and all you see is
darkness when you focus, what should you do?

12. Explain in your own words what happened to the Elodea cell after the salt
treatment. What biological term describes the events just witnessed?

13. Describe four differences between prokaryotic and eukaryotic cells.

Prokaryotic

Eukaryotic

a.
b.
c.
d.
14. Name three differences between plant and animal cells.
Plants
a.
b.
c.
15. State three postulates of the cell theory:
a.
b.
c.

16. Name three structures common to all cells.

Animals

a.
b.
c.
17. Do all living plant cells contain chloroplasts?
18. Label the following:

Typical animal cell (eukaryote)

18. Label following:

Typical plant cell (eukaryote)

19. Give a brief function for each of the following components of a microscope:
a. Condenser b. Nosepiece c. Stage d. Diaphragm lever e. Coverslip -

20. Examine the microscope diagram shown below. Label the components.