LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.

2021

LAB 1. UNDERSTANDING THE WORK OF

MICROSCOPES

(Referred to Principles of Biology Lab Manual, Bishop et al., 2009;

quoted with adjusment from Visualising Human Biology Lab Manual, Ellie, 2011,
Campbell Biology, Reece et al, 2016))

INTRODUCTION
Imagine you work in a clinical laboratory that specializes in diagnosing cancer.
Every day, you examine biopsies (tissue specimens) from patients and look for any
signs of disease. The biopsies shown in Figure 2.1 have just been shipped to your lab
for analysis; how can you tell whether these specimens appear normal or abnormal?
Since cells are too small to be seen with the naked eye, you rely on a microscope to
magnify these specimens for analysis. (The cells in Figure 2.1 have already been
magnified with a microscope.) Once the specimens are in focus, you draw on your
knowledge of typical and atypical cell structure to detect any signs of disease. Within
a matter of minutes, you conclude that the first patient’s biopsy appears normal.
However, the second patient’s biopsy contains cells that appear to be cancerous.
Throughout the course of this semester, you will use microscopes to view cells,
and other biological matter in greater detail. During this lab, you will learn the proper
way to handle and focus a compound light microscope, in theory.

There are two types of light microscopes. Dissecting microscopes and

compound microscopes differ from one another based on structure and function.
Nevertheless, they do share several features in common:

- The specimen of interest (a microscope slide, an organism, etc.) is set on the

stage of the microscope for viewing.
- The light source focuses visible light onto the specimen.
- Glass lenses magnify the specimen of interest. Ocular lenses are located in the
eyepieces; the prefix ocul- actually means eye in Latin. Objective lenses point
toward the object (specimen) of interest.
- Adjustment knobs bring the specimen into sharp focus.
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

A typical dissecting microscope is shown in Figure 2.2, and a typical compound

microscope is shown in Figure 2.3. Microscope models differ from one another based
on their parts and capabilities, so the microscopes owned by your university may
differ slightly from these examples. Ask your instructor if you have questions
regarding the unique features of your microscope model.

DISSECTING MICROSCOPES
A dissecting microscope shows the three-dimensional surface of a specimen in
greater detail. The objects viewed through a dissecting microscope—organs, insects,
leaves, etc.—are generally too large or too thick to be viewed through a compound
microscope. As the name implies, dissections can also be performed under this
microscope, when desired. The magnification ranges of a dissecting microscope
(typically 5x-50x) is much lower than the magnification range of a compound
microscope. For this reason, cells cannot be distinguished in most cases. The basic
parts of a dissecting microscope are labeled on Figure 2.2, and the steps for proper
usage are summarized next to the figure.

1. Using two hands, carry a dissecting microscope to your workstation. One

hand grips the arm of the microscope, while the other hand holds the base of
the microscope.
2. Use lens paper to clean the microscope lenses prior to use. Note: Only use
lens paper to clean the microscope lenses. Other materials (paper towels,
Kleenex, shirt sleeves, etc.) can scratch the lenses.
3. Place the specimen of interest (a microscope slide, a leaf, a bug, etc.) onto the
stage.
4. Plug the microscope into the power outlet. Turn on the light source and point
it toward the specimen.
5. Rotate the zoom control knob and bring the low-power objective lens
(example: 0.5x) into the viewing field.
6. View the specimen by looking through the oculars.
7. Slowly turn the focus knob to bring the specimen into sharp focus.
8. To view the specimen at a higher level of magnification, rotate the zoom
control knob and bring the high-power objective into the viewing field. Bring
the specimen into sharp focus, as needed, using the focus knob.
9. When you are finished viewing the specimen, turn off the light source and
clean off the stage. Use lens paper to clean off the microscope lenses.
10. Using two hands, carry the dissecting microscope back to its storage cabinet.
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

COMPOUND LIGHT MICROSCOPES

Biologists and medical professionals regularly use compound microscopes to
observe thin, two-dimensional tissue sections. The basic parts of a compound
microscope are labeled on Figure 2.3, and the steps for proper usage are summarized
under the figure. The magnification power of a compound microscope (typically 40x-
1000x) far exceeds the magnification power of a dissecting microscope. For this reason,
individual cells can easily be distinguished.
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

1. Using two hands, carry a compound microscope to your workstation. One

hand grips the arm of the microscope, while the other hand holds the base of
the microscope.
2. Use lens paper to clean the microscope lenses prior to use. Note: Only use lens
paper to clean the microscope lenses. Other materials (paper towels, Kleenex,
shirt sleeves, etc.) can scratch the lenses.
3. Plug the microscope cord into the power outlet, and turn on the light source.
While viewing microscope slides, you can adjust light intensity (and therefore
contrast) with the iris diaphragm.
4. Make sure the lowest power objective (example: 4x objective) is located directly
above the hole in the stage. If needed, turn the revolving nosepiece to bring this
objective into position.
5. Secure the microscope slide on the stage. If your microscope has a manual
stage, then the stage clips are placed on top of the slide. If your microscope has
a mechanical stage, then the stage clips grip the sides of the slide.
6. Center the microscope slide on the stage. If you are using a mechanical stage,
use the stage control knobs to center the microscope slide.
7. Carefully watch this step from the side of the microscope. Turn the course focus knob
to lift the stage toward the objective. Move the slide as close as possible to the
objective, but do not allow the slide to collide with the objective.
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

8. View your specimen by looking through the oculars from this point forward. Using the
course focus knob, move the slide away from the objective until the image
comes into rough focus.
9. Slowly turn the fine focus knob and bring the image into sharp focus. Only use
the fi ne focus knob to sharpen images from this point forward.
10. Scan the microscope slide in a pattern similar to the one shown below. If your
microscope has a mechanical stage, use the stage control knobs to move the
slide.

11. Center the object of interest before moving to a higher level of magnification.
12. Turn the nosepiece and position the next objective (example: 10x objective)
directly above the light beam traveling through the slide. Since your
microscope is parfocal, the image should still be in focus and require little (if
any) adjustment. Bring the image into sharp focus using the fi ne focus knob, if
needed.
13. Center the object of interest before moving to a higher level of magnification.
14. Turn the nosepiece and position the next objective (example: 40x objective)
directly above the light beam traveling through the slide. Bring the image into
sharp focus using the fine focus knob, if needed. The 100x objective lens (if
present) will not be used during Lab 2.
15. When you are finished observing the microscope slide, turn the nosepiece and
bring the lowest power objective (example: 4x objective) back into the viewing
field.
16. Remove the microscope slide from the stage. If you are using a mechanical
stage, move the slide away from the objectives prior to removal.
17. Turn off the light source, unplug the microscope, and wrap its cord around the
cord holder (if present) or base. Use lens paper to clean the microscope lenses.
18. Using two hands, carry the microscope back to its storage cabinet.

FIELD OF VIEW AND TOTAL MAGNIFICATION POWER

You may wonder why a wide range of magnification levels are available on
microscopes. Why isn’t the highest level of magnification always desired? One answer
to this question deals with the relationship between magnification power and the field
of view. Figure 2.4 shows the relationship between magnification power and field of
view.
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

The magnification power of the ocular lens is written on the eyepiece, while the
magnification power of each objective lens is written on the side of the objective. The
total magnification of a specimen can be calculated using the following equation:

Magnification of Ocular Lens × Magnification of Objective Lens =

Total Magnification Power

For instance, a specimen is magnified one hundred times (100x) when the 10x ocular
lens is used in conjunction with the 10x objective lens.

DEPTH OF FIELD
Since compound microscopes create two-dimensional images, only one plane (or
layer) of a specimen is in sharp focus at any given time. The thickness of this plane—
the depth of field—is dependent on the magnification power being used.

ELECTRON MICROSCOPE
Until recently, the resolution barrier prevented cell biologists from using
standard light microscopy when studying organelles, the membrane-enclosed
structures within eukaryotic cells. To see these structures in any detail required the
development of a new instrument. In the 1950s, the electron microscope was
introduced to biology. Rather than focusing light, the electron microscope (EM)
focuses a beam of electrons through the specimen or onto its surface. Resolution is
inversely related to the wavelength of the light (or electrons) a microscope uses for
imaging, and electron beams have much shorter wavelengths than visible light.
Modern electron microscopes can theoretically achieve a resolution of about 0.002 nm,
though in practice they usually cannot resolve structures smaller than about 2 nm
across. Still, this is a 100-fold improvement over the standard light microscope.
The scanning electron microscope (SEM) is especially useful for detailed study
of the topography of a specimen. The electron beam scans the surface of the sample,
usually coated with a thin film of gold. The beam excites electrons on the surface, and
these secondary electrons are detected by a device that translates the pattern of
 LAB MANUAL | PRINCIPLES OF BIOLOGY I | SEMESTER 1 | 2020.2021

electrons into an electronic signal sent to a video screen. The result is an image of the
specimen’s surface that appears three-dimensional.
The transmission electron microscope (TEM) is used to study the internal
structure of cells. The TEM aims an electron beam through a very thin section of the
specimen, much as a light microscope aims light through a sample on a slide. For the
TEM, the specimen has been stained with atoms of heavy metals, which attach to
certain cellular structures, thus enhancing the electron density of some parts of the cell
more than others. The electrons passing through the specimen are scattered more in
the denser regions, so fewer are transmitted. The image displays the pattern of
transmitted electrons. Instead of using glass lenses, both the SEM and TEM use
electromagnets as lenses to bend the paths of the electrons, ultimately focusing the
image onto a monitor for viewing.
Electron microscopes have revealed many subcellular structures that were
impossible to resolve with the light microscope. But the light microscope offers
advantages, especially in studying living cells. A disadvantage of electron microscopy
is that the methods used to prepare the specimen kill the cells. Specimen preparation
for any type of microscopy can introduce artifacts, structural features seen in
micrographs that do not exist in the living cell.