Lab 5 - THE MICROSCOPE and CELL STRUCTURE

OBJECTIVES

By the end of this exercise you should be able to:

1. Demonstrate the proper use and care of the microscope.
2. Identify the parts of a digital compound microscope and state the function of each.
3. Define these terms: total magnification, paracentric, parfocal, field of view, depth of
focus, and working distance.
4. Prepare a wet mount.
5. Distinguish between plant and animal cells.

INTRODUCTION

One of the fundamental principles of biology is Cell Theory, which states that all living things
are composed of cells. The cell is the basic unit of life; it is the smallest structure that exhibits
the characteristics of life. There are two distinct types of cells: prokayotes and eukaryotes. The
simple prokaryotic cells (bacteria) lack the nucleus and complex, membrane bound organelles
found in the eukarotic cells. In multicellular eukaryotic organisms (fungi, plants, and animals),
individual cells tend to lose some functions such as motility or protection, and to specialize in
other functions, such as absorption, secretion or contraction. In unicellular eukaryotic
organisms (protozoa, algae, and yeast), all functions are handled by a single, complex cell. Most
eukaryotic cells are between 10 and 30 micrometers (µm) (1 mm = 1000 µm) in diameter, so
they are not visible unless magnified under the microscope. The compound light microscope is
a precision instrument and should be treated accordingly.

Use and Care of the Digital Compound Microscope

Your instructor will demonstrate to you the proper way to carry the microscope. It should
always be carried using two hands, one grasping the arm and the other supporting the base. The
instrument should be carried in a vertical or near vertical position. This type of microscope is
called a compound microscope because it consists of a series of lenses that magnify objects.
These lenses are located in the ocular or eyepiece and in the objectives. The microscope works
by passing light upward through the specimen and the magnifying lenses to your eye.

Parts of the Compound Microscope (see figure on page 44):

1. Ocular (eyepiece) –uppermost lens; magnifies objects ten times their size (10X)
2. Head – holds the oculars
3. Arm - supports the head, stage and adjustment knobs; used in carrying
4. Prism Slider Bar –directs the beam of light between the ocular and computer
5. Nosepiece – revolving part to which the objectives are attached

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6. Objectives – set of four lenses attached to the revolving nosepiece. Objective lenses are
always used in order: scanning, low, high-dry, oil immersion. The magnifying power of
each objective is labeled on its side. Total Magnification is equal to the magnification of
the ocular lens times the magnification of the objective lens. For each of your objectives,
fill in the magnification and the total magnification.

Total Working Diameter

Objective Size Magnification
magnification Distance of Field

Scanning Small _______ X _______ X 25 mm 3.3 mm

Low Medium _______ X _______ X 8.3 mm 1.5 mm

High Large _______ X _______ X 0.5 mm 0.3 mm

Oil immersion
not used in Largest _______ X _______ X 0.1 mm 0.02 mm
BI-201

Note that the working distance (space between objective lens and slide) and the diameter
of the field of view (circle of light seen through the scope) decrease as magnification
increases.

7. Stage - supports the slide over a hole that admits light from the light source. The
stage clips hold the slide firmly in the desired position.
8. Mechanical Stage Knobs – two knobs located below the stage; one controls
forward/backward movement of the slide, and the other controls right/left movement
9. Rheostat – knob located on the right side of the base that regulates voltage and light
intensity. Use a medium rheostat setting (around 4-5). Too high a setting generates
excessive heat and reduces the life of the $50 bulb.
10. Condenser – lens system located beneath the stage, it focuses light onto the specimen; the
condenser should always be in its highest position.
11. Condenser Adjustment Knob – located below the stage; the condenser should always be
in its highest position.
12. Iris Diaphragm – regulates the amount of light passing through the specimen; located
beneath the condenser, controlled by a lever.
13. Coarse Adjustment Knob – used for preliminary focusing by lowering the stage; used
only with the 4X scanning and 10X low power objectives.
14. Fine Adjustment Knob – used for final or fine focusing by raising or lowering the stage

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 Eyepiece

Prism Slider
Bar
Revolving
Nosepiece

Objectives Arm

Specimen
Holder Stage

Condenser X&Y movement

of the stage
Iris Diaphragm Fine Focus
Control Knob for Knob
condenser Coarse Focus
Knob
Illuminator

Base Rheostat

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Use of the Microscope: Letter “e” slide
1. Clean the microscope lenses with lens paper. Do not use Kimwipes, tissues, paper towels,
cloth or anything else.

2. Plug the power cord into the round hole in the back of the microscope and into the outlet.

3. Plug the square end of the USB cable into the back of the microscope and put the small
flat end into the USB port on the back of the iMAC.

4. Check to be sure the rheostat is turned all the way down to 1 before turning on the
power switch in the back of the microscope.

5. Make sure the prism sliding rod is pushed all the way in.

6. Turn on the light and adjust the light intensity to medium (4 or 5).
Caution: Higher settings/voltage generate too much heat and reduce bulb life.

7. Rotate the nosepiece so that the 4X scanning objective clicks into place.

8. You will probably need to adjust the eyepieces to fit the distance between your eyes.
Look into the ocular lenses and adjust them to match the distance between your eyes. As
you look through the lenses, pull the lenses apart or push them together until you see a
single round field of view. In your lab notebook, make a note of the interpupillary distance
on the scale between the oculars. Before you begin the use the microscope in future labs,
set this distance.

9. Place the slide of the letter “e” on the stage (oriented so that you can read it properly), and
use the mechanical stage knobs to center it over the hole in the stage.

10. While watching from the side, gently raise the stage until it stops moving by turning the
coarse adjustment knob away from you.
Caution: Always watch the stage from the side while raising it to be sure that it
doesn’t hit the objective.

11. Looking through the ocular, bring the letter into rough focus by slowly turning the coarse
adjustment knob toward you so that the stage is lowered until the object comes into focus.
Turn the fine adjustment knob away from you to bring the specimen into sharp focus.
Caution: Never turn the fine adjustment knob more than half a turn.

12. While looking through the ocular, adjust the iris diaphragm to increase image contrast.

13. Notice that the image is inverted. This is a characteristic of compound microscopes that
should be kept in mind when studying other specimens. Move the slide to the left while
looking through the ocular. Which way does the image move ? __________

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 Move the slide way from you while looking through the ocular. Which way does the
image move ? _________________

Draw the letter “e” as it appears when observed with each of the following:

Unaided Eye 4X objective 10X objective

Lenses are parfocal – objectives are aligned so that rotation to another lens can be
done with only minor fine focusing.

14. Center the letter and rotate the low-power 10x objective into position. Look through the
ocular and slowly turn the coarse and then fine adjustment knobs away from you until the
image is sharp.
Caution: After the initial focus at 4x and 10X magnification, you should only use the
fine adjustment knob to sharpen the focus. Using the coarse adjustment at 40X can result
in damage to the lens, slide or both.

15. The nosepiece can now be rotated so that the high-dry power 40X objective clicks into
position. As you do this, watch from the side, with your eyes at the level of the stage, to be
sure that the high power objective does not contact and break the slide. Thick preparations
cannot be observed under high power due to the small working distance (the space
between the objective lens and the slide).
Caution: The microscope is paracentric; the object must be centered before rotating
the objective.

16. Once the object is visible under high power, only the fine adjustment knob should be used.
Do not use the coarse adjustment knob while using the high-power objective.

Threads slide
Depth of focus refers to the ability to see through the thickness of a specimen. At higher
magnification, it becomes more apparent.

Place the slide with colored, overlapping threads on the stage of your microscope. With the
scanning objective in place, focus on the threads. Once the threads are in focus, change the
magnification to 10X and then to 40X.

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At high power, focus on the location where the threads cross. Using the fine adjustment only,
determine which color thread is at the bottom, middle and top.

Top thread color ____________________

Middle thread color ____________________

Bottom thread color ____________________

Preparing a Wet Mount

A wet mount is a temporary preparation used to study fresh, living material. Specimens are
mounted in a drop of water and covered with a cover slip.

1. Place a drop of water in the center of a clean microscope slide.

2. Place the specimen in the drop.

3. Touch the coverslip to the edge of the drop, and gently lower it as shown below. If you are
careful, the slide will not have air bubbles (visible as circles of various sizes with very
dark edges).

Why is a coverslip necessary? __________________________________________

Cover slip Lower

slowly

Plant and Animal Cells

Plant Cells:
A. Elodea Leaf

1. Prepare a wet mount with a leaf from a sprig of Elodea, a common pond plant.

2. Examine the leaf first with 4X, then 10X and finally 40X magnification. Each of the
small, box-like units is a cell.

3. As the leaf warms, you may see the green chloroplasts moving due to cytoplasmic
streaming. This circular flow of cytoplasm speeds up the distribution of materials
within the cell.

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 4. Draw a diagram of an Elodea cell on page 49.

B. Onion Epidermis

1. Place a drop of water on a clean slide.

2. Cut an onion into quarters and remove a leaf. Snap the leaf backward and remove the
thin piece of epidermis (transparent membrane that can be peeled from the layer of
onion) at the break point.

3. Place the transparent onion epidermis in the drop of water and add a cover slip.

4. Staining will make the transparent onion cells easier to observe. Place a small amount
of iodine next to the edge of the coverslip. Then draw the stain under the coverslip by
touching a piece of blotting paper or paper towel to the opposite edge of the coverslip.

5. Examine the epidermis first with 4X, then 10X and finally 40X power. Carefully focus
to distinguish the vacuole from the cytoplasm. A nucleolus can sometimes be seen
inside the nucleus.

6. Draw a diagram of an onion epidermis cell on page 49.

C. Potato Cells

1. Place a drop of water on a clean slide.

2. Use a razor blade to make a thin section of potato.

3. Place the potato section into the water drop and add a coverslip.

4. Stain the potato cells with iodine. Place a small amount of iodine next to the edge of
the coverslip. Then draw the stain under the coverslip by touching a piece of blotting
paper or paper towel to the opposite edge of the coverslip.

5. Examine the potato with first with 4X, then 10X and finally 40X power.

6. Draw a diagram of a potato cell on page 49.

7. It is difficult to observe the nucleus and vacuole because the cells are filled with dark
staining organelles called amyloplasts.

What color are the amyloplasts ? ________________

Based on this staining reaction, what do amyloplasts contain? ___________________

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Draw diagrams of an Elodea, onion and potato cell in the spaces below. Make them large
and clear. Label the following structures: cell wall, cell membrane, large central vacuole,
chloroplasts, cytoplasm, nucleus, amyloplasts, and cytoplasmic streaming (indicate by
arrows).

Elodea Cell Onion Cell Potato Cell

Animal Cells:
A. Human Cheek Cells – work independently

Note that these cells are thin and flat and somewhat irregular in shape. The edges of the
cells often become folded over during preparation and the cells tend to clump together.
Some cells may be covered with very small, dark blue rods or spheres. These are bacteria.

1. Place a drop of water on a clean slide.

2. Use a clean toothpick to gently scrape the inside of your cheek.

3. Stir the scrapings into the drop of water on the slide and add a coverslip.

4. Staining will make the transparent cells easier to observe. Place a small amount of
methylene blue next to the edge of the coverslip. Then draw the stain under the
coverslip by touching a piece of blotting paper or paper towel to the opposite edge of
the coverslip.

5. Observe the cheek cells first with 4X, then 10X and finally 40X power.

6. Draw a cheek cell in the space on the next page. Make it large and clear. Label cell
membrane, cytoplasm, and nucleus. If bacteria are present, indicate them also.

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 Human Cheek Cell

UNKNOWNS (optional)

Examine the pond/aquarium water or unknown culture provided by the instructor; it

may contain a mixture of protozoa, algae, and microscopic animals. Protozoa (single-
cell organisms) are especially interesting to observe because of their unique shapes,
modes of locomotion, and large organelles.

1. Make a wet mount with some of the culture debris from the bottom of the
container.

2. Locate the organisms under 4X, then 10X and finally 40X power.

3. Use the wall charts of pond organisms to identify as many organisms as possible.

Are there any ciliates ? ______

Are there any flagellates ? ______

You may draw the organisms here.

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 You are responsible for your assigned microscope!

Microscope Storage

Proper preparation of the microscope for storage reduces the chances of damage, and ensures
that it will be ready for use by the next student. You should know and follow the proper steps
for storage.

Date ___________ Microscope # ______

Prism sliding bar all the way in?
No slide on stage?
Stage lowered fully?
Mechanical stage retracted?
4X objective returned to viewing position?
Rheostat returned to lowest setting?
Power switch off?

Questions for Further Thought and Review

1. Why must you center and focus the object in the field of view under low power before
switching to high power?

2. Why is only the fine adjustment used for high power ?

3. Describe how each of the following is affected by switching from the low to high power
objective.
total magnification - _________________ brightness of field - _________________

diameter of field - ___________________ working distance - _________________

depth of focus - ____________________

4. Why must a specimen viewed with a compound microscope be very thin? Why are
specimens sometimes stained with dyes?

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5. Human cheek cells are epithelial cells, which are highly specialized for protection. List
some of the functions that these cells cannot carry out.

6. Complete the following chart. You may need to look up some of these in your notebook or
textbook.

Cell Found in
Function
organelle or structure plant, animal or both

Nucleus

Cell wall

Chloroplast

Amyloplast

Cell membrane

Cytoplasm

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