LABORATORY 2

INTRODUCTION TO MICROSCOPY PART B

LEARNING OBJECTIVES
When you have completed this lab, you will be expected to:
1. Prepare specimens of cells on a slide;
2. Use a compound light microscope to magnify a specimen and bring the image into
fine focus;
3. Identify some cellular structures, such as the plasma membrane, the nucleus, the
cytoplasm, and the cell wall (in plant cells);
4. Estimate the average size of a cell or sub-cellular structure in a specimen;
5. Identify the various components of a stereoscopic microscope and associate each
component with a function in microscopy;
6. Use a stereoscopic light microscope to magnify a specimen and bring the image into
fine focus;
7. Discuss the advantages and disadvantages of a compound light microscope and a
stereoscopic light microscope.

INTRODUCTION
Cells
Although some cells are very large (e.g., human egg cells, some unicellular protists),
most cells are too small to see with the naked eye. Most eukaryotic cells range in size
between 10 and 100 micrometers (m). In contrast, most prokaryotic cells range in size
between 1 and 10 m. Therefore, in order to view cells, biologists use microscopes.
The cell is the unit of structure, function, and reproduction of all organisms. All cells are
surrounded by a fatty boundary made up of phospholipids and proteins called a plasma
membrane. In addition, all cells contain deoxyribonucleic acid (DNA) as their genetic
material. In the case of eukaryotic cells, DNA is enclosed in membrane-bound
compartment called the nucleus. In the case of prokaryotic cell, the DNA is in a
specialized region but not in a membrane-bound compartment. So, prokaryotic cells do
not contain a nucleus. In addition to the nucleus, eukaryotic cells also have many other
internal membrane-bound compartments specialized for various functions. These
specialized compartments are called organelles (“little organs”). Prokaryotic cells lack
these organelles. The region of a eukaryotic cell outside of the nucleus contains a gel-
like substance called the cytoplasm. The cytoplasm contains organelles and dissolved
chemicals.

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Fig. 1 illustrates some structures seen in a typical animal (a) and plant cell (b).

Figure 1. Structures observed in stained cell specimens under a compound light

microscope. a) Animal cheek epithelial cells stained with methylene blue. b) Onion
epidermal cells stained with iodine.

In addition to their plasma membranes, plant cells, and many other cells, also contain a
more rigid cell wall that surrounds the plasma membrane (see Fig. 1 b). The cell wall is
a tough layer of structural material (e.g., cellulose) external to the plasma membrane
whose function is to protect the cell. In some multicellular organisms, such as plants,
cells in tissues are attached together through their cell walls. Animal cells, in contrast, do
not have a cell wall, but many animal cells in tissues (e.g., muscle, heart, etc.) are also
attached together via cellular junctions.

Lab Exercise 1: Using your Compound Light Microscope to View

a Specimen of Plant Cells

In this exercise, you will use your microscopy skills developed in Part A to view onion
epidermal (skin) cells as an example of a plant cell. The bulb of the onion is actually
made up of modified leaves that are “chubby” and specialized for food (energy in the
form of starch, for e.g.) storage. You will identify sub-cellular structures and estimate the
average size of these cells.

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Exercise 1-1
Preparing and viewing a specimen of onion tissue on a slide

1. Take a piece of onion tissue. Using tweezers, slowly peel off a THIN layer of the
inner of the tissue. You want a section that is about 1 cm2.
N.B. You want only one layer, which is as thick as a layer of our skin. Having
multiple layers or an uneven sample will not allow you to view individual cells
very well (if at all).

2. Place the specimen on a clean glass slide. Make sure that the specimen is laid out
flat without any folds.

3. Add a drop of distilled water (dH2O) next to (NOT ON) your specimen and then a
drop of methylene blue. Place the cover slip at a 45o angle next to the drop of water.
Carefully drop the coverslip to push the solution over your specimen. See Fig. 2.
N.B. You can use your fingers or forceps to carry out this step. If you are
using your hands, only handle the sides of the cover slip to avoid transferring
oil and other materials to the area where the light of the microscope will pass.

Figure 2. Preparing your onion cell specimen.

4. View your specimen under the microscope to see the cells when unstained. View at
40X total magnification and raise the magnification stepwise until you reach 400X
total magnification. Use the condenser and light intensity controls to optimize
contrast and reduce glare.
N.B. You should see rectangular cells attached to each other in a brick
formation.

5. Position your slide under the microscope and observe your stained specimen at the
various magnification levels specified in Step 4. At 400X total magnification, try to
identify the following sub-cellular structures: Plasma Membrane, Nucleus,
Cytoplasm, and Cell Wall.
N.B. Controlling the condenser height and fine focus effectively is essential
for observing sub-cellular structures. What does the condenser system allow
you to optimize?

6. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

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Exercise 1-2
Estimating the average size of an onion epidermal cell

These plant cells are rectangular. You will measure the average length (long side) and
width (short side) of these cells. To do so, you will take advantage of the brick formation
of these cells in the tissue and the fact that the cells extend across your field of view at
any magnification. Size estimation can be done at any magnification because we know
the field diameter at all magnifications from lab1A. The key to getting a good estimate of
length or width is to include a reasonable number of cells in your assessment and take
an average.

1. To measure the average length of these cells, view the specimen at 100X total
magnification. Count the number of cells in three (3) independent series of cells that
extends lengthwise. A series is a chain of cells that extends from one end of your
field of view to the other. Enter your results in the Lab Report section. Calculate the
average length of the cells for each series then calculate the mean length for the
three series.

2. To measure the average width of these cells, view the specimen at 400X total
magnification. Count the number of cells in three (3) independent series of cells that
extends widthwise. Enter your results in the Lab Report section. Calculate the
average width of the cells for each series then calculate the mean width for the three
series.

3. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Lab Exercise 2: Using your Compound Light Microscope to View

a Specimen of Animal Cells

In this exercise, you will use your microscopy skills developed in Part A to view human
epithelial (skin) cells as an example of an animal cell. You will identify sub-cellular
structures and estimate the average size of these cells and their nuclei.

Exercise 2-1
Preparing and viewing a specimen of cheek epithelial cells on a slide
1. Take a clean glass slide and place it in front of you on the bench.
2. Add a drop of saline to the middle of the slide. Add a drop of methylene blue stain to
the drop of saline (NaCl solution). Mix using a toothpick and discard the toothpick in
the designated container.
3. Take a clean toothpick and gently scrape the side of your mouth to gather cells. Mix
the cells into the solution on the slide. Mix well.
N.B. You want to disperse the cells and break their attachments (cell
junctions) so that you can look at cells individually.
4. Take a clean plastic cover slip. Gently cover the solution with the cover slip by
placing one end near (NOT IN) the solution and carefully dropping the other end
over the solution (see Fig. 3).
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N.B. This technique will minimize air bubbles. If too much liquid is present and
the cover slip is floating (not a tight seal), you will have to remove the excess
liquid by putting a tissue at the edge of the coverslip. The tissue will draw
liquid out. Draw liquid out until the coverslip is tightly attached to the slide.
Clean the slide with tissue and clean up any liquid spilled on the sample stage
with tissue before you proceed. Discard all used tissues into the garbage can.
If air bubbles are visible, use the forceps provided or a toothpick to tap on the
coverslip and push them out.

Figure 3. Preparing your cheek cell specimen.

5. View your specimen under the microscope at 40X total magnification. Try to find an
isolated cell.
N.B. You should see structures that look like light blue fried eggs each with a
small, dark blue yolk.

6. Once you find an isolated cell that you want to magnify further, use the stage control
knobs to center this cell in your field of view. Increase the magnification to 100X
total, focus, re-center, and increase the magnification to 400X total.

7. One you have your isolated cell at this magnification, use the condenser to optimize
contrast and reduce glare.

8. Try to identify the following structures: Nucleus, Plasma Membrane, Bacteria (inside
and/or outside of cell) if present. Bacteria will look like dark blue circles or rods in
and/or around your cells.
N.B. Again, controlling the condenser and fine focus effectively is essential
for observing sub-cellular structures.

9. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

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Exercise 2-2
Estimating the average diameter of a cheek cells and its nucleus

These animal cells are circular. You will measure the average diameter of the cells. In
this case, the cells are isolated from one another, so we will have to view a few isolated
cells and estimate their size separately. We will also estimate the average size of the
nucleus of these cells.

1. To estimate the diameter of a cell, view an isolated cell at 400X total magnification.
Estimate the fraction of the field diameter taken up by the cell. Use this information
to calculate an estimate of the diameter of the cell. Record your data in the Lab
Report section.
2. To estimate the diameter of the nucleus, estimate the fraction of the cell’s
diameter taken up by the nucleus. Use this information to calculate an estimate of
the diameter of the nucleus. Record your data in the Lab Report section.
3. Repeat this procedure for two other isolated cells in your specimen.
4. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.
5. Complete the following checklist to correctly put away your compound light
microscope and tidy up your bench before moving on to the next exercise.
Task Done (ü)
Light intensity knob turned all the way down and light switched off
Rotate the nosepiece to the 4X objective lens
Sample stage brought down to lowest position (use coarse adjustment
knob)
Slide removed and placed in appropriate disposal bin
Sample and mechanical stages centered
Microscope observed for spilled liquid or other mess and cleaned if
necessary
Wound-up region of the power cord around one of the oculars, oculars
together to hold the power cord in place
Plastic bag replaced

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Exercise 3: Using a Stereoscopic Light Microscope to View a

Specimen

The Stereoscopic Light Microscope

Compound light microscopes are very useful for viewing thin specimens when
magnification and resolution power are important. However, in many instances in
biology, you will need to magnify thicker specimens or visualize live organisms. For
instance, you may want to magnify a small organ during a dissection or examine an
insect. In this situation, a stereoscopic (dissecting) microscope is the best instrument.
Dissecting microscopes can handle thicker specimens because of a larger working
distance. They also offer a 3D image of a specimen that is ideal for dissections and
observation of a specimen as a whole. However, one disadvantage of a dissecting
microscope is that the overall magnification possible is low when compared to a
compound light microscope. Fig. 4 illustrates some of the various components of a
stereoscopic microscope.

Figure 4. Components of a stereoscopic light microscope.

You will use ONE (1) microscope in teams of 2 to complete the following
exercises. Please ensure that each team member takes part.

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Exercise 3-1
Getting to know the components of a stereoscopic light microscope

1. Familiarize yourself with the components of the stereoscopic light microscope and
their functions in light microscopy. Compare these to the various components of a
compound light microscope.

2. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 3-2
Observing a biological specimen with a stereoscopic microscope

In this exercise, you can observe several large biological specimens that are not moving
using a stereoscopic light microscope.

1. Choose one or several of the specimens available on the side bench.

2. Put the specimen on the sample stage.

3. Use the zoom control to select the lowest setting then use the focus adjustment
knob to bring a region of the specimen into fine focus.

4. Increase the magnification using the zoom control knob in steps. As you do so you
will have to use the focus adjustment knob to focus the image. Continue until you
are able to view a sharp, magnified image of your specimen at the highest setting.

5. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

Exercise 3-3
Identifying flatworms with a stereoscopic microscope

In this exercise, you will view a moving animal specimen. The specimen is a particular
species of flatworm. All flatworms belong to the animal phylum of Platyhelminthes. You
will use the stereoscopic microscope to observe the specimen and identify which genus
the particular species of flatworm belongs to using a key that outlines the body form of a
representative species of 5 different genera of flatworms.

Viewing a moving specimen under the stereoscopic microscope will take practice. In this
case, you will need to constantly move the petri dish on the sample stage as you are
controlling the microscope.

1. Place the Petri dish containing the selected specimens on the sample stage and
remove the lid.

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2. Set the microscope to the lowest possible magnification using the zoom control
knob. Try to follow the specimen and focus the image using the focus adjustment
knob. To follow your specimen, you will have to move the Petri dish, as there are no
controls on this microscope to move the specimen. Try to keep the specimen in your
field of view to observe it.
N.B. Do not go straight to the highest magnification! You must practice
manipulating the specimen while adjusting the magnification and focusing.
This is much easier to do at the lower magnification levels.

3. Once you are accustomed to keeping the specimen in your field of view, increase
the magnification in steps to observe the specimen in more detail. Remember to
focus the specimen every time you change the magnification level!

4. The species in each flatworm genus share certain morphological (structural) traits
(e.g., body shape, position of eyespots) which differ from species in the other
genera. Use the key in Fig. 5 to identify the genus of the flatworm in the specimen
under observation based on its key morphological traits.

Figure 5. Representatives of various genera of flatworms

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5. COMPLETE THE LAB REPORT SECTION FOR THIS EXERCISE.

6. At the end of this exercise put away the stereoscopic microscope the same way as
you did for the compound microscope. If you need help let your instructor know.

7. Make sure you clean up your bench area and station. Put all materials back in the
appropriate location and clean your lab bench with water from the sinks on the side
benches and paper toweling.

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NAME: ______________________________ LAB SECTION: ____

LABORATORY I
INTRODUCTION TO MICROSCOPY
LAB REPORT
Exercise 1-1
Preparing and viewing a specimen of onion tissue on a slide

1. Use the circle on the right as your field of view. Draw

and label a diagram of ONE (1) onion cell to
scale as viewed at 400X total magnification.
Label the following sub-cellular structures
using the acronyms provided: Plasma
Membrane (PM), Nucleus (N), Cytoplasm (C),
and Cell Wall (CW).

2. What is the importance of staining a cell

specimen?

3. Which cell components were stained by methylene blue in the plant cell?

Exercise 1-2
Estimating the average size of an onion epidermal cell

1. Complete the following tables.

Table 1-2 a) Average LENGTH of an onion epidermal cell.

Number of Cells Field Diameter (m) Estimated

Series (lengthwise across field) at 100X average Length (m)

Mean

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Table 1-2 b) Average WIDTH of an onion epidermal cell.

Number of Cells (widthwise Field Diameter (m) Estimated

Series across field) at 400X average Width (m)

Mean

Exercise 2-1
Preparing and viewing a specimen of cheek epithelial cells on a slide

1. Use the circle on the right as your field of view. Draw

and label a diagram of ONE (1) cheek cell to scale as
viewed at 400X total magnification. Label the following
sub-cellular structures using the acronyms provided:
Label the Plasma Membrane (PM), Nucleus (N),
Cytoplasm (C), and Bacteria (B) if present.

2. Which cell components were stained by methylene blue in

the animal cell?

Exercise 2-2
Estimating the average DIAMETER of a cheek cells and its NUCLEUS

1. Complete the following table.

Table 2-2. Average diameter of a cheek epithelial cell and its nucleus.
Fraction of Fraction of
Field
field occupied Diameter of cell occupied Diameter of
Cell Diameter (m)
by cell cell (m) by nucleus nucleus (m)
at 400X
diameter diameter
1

Mean: Mean:

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Exercise 3-1
Getting to know the components of a stereoscopic light microscope

1. Which component can be used to control the magnification level?

2. What is the range of magnification possible for the stereoscopic light microscope?

3. Both compound and stereoscopic light microscopes have a focus adjustment knob
to control focus. How do the mechanisms for focusing differ between the two types
of microscopes?

Exercise 3-2
Observing a biological specimen with a stereoscopic microscope

1. How does the image of a specimen viewed by this microscope differ from the image
viewed by the compound light microscope? Describe some different
characteristics.

2. Describe ONE (1) specific task in a lab where a stereoscopic light microscope would
be appropriate and not a compound light microscope?

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Exercise 3-3
Identifying flatworms with a stereoscopic microscope

1. Identify the genus of the species in the specimen: _______ [use letter label from the
key in the procedure].

2. Describe one or two key morphological (structural) features that allowed you to
identify its genus from the comparative anatomy key.

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