ANATOMY AND PHYSIOLOGY LABORATORY EXERCISE 2

MICROSCOPY

OBJECTIVES:
At the end of this exercise, each student must be able to:

 Identify each major part of a compound light microscope and describe its function
 Describe the correct use and care of the microscope
 Determine the total magnification at different settings
 Learn the basic procedures on how to use a microscope to observe prepared specimens
( LPO, HPO )
 Accurately record microscopic observations

INSTRUCTIONS:
 Study the principles of microscopy and the correct procedure of carrying, focusing and
storage of the microscope
 Watch the video tutorial on how to use the compound microscope.
 https://youtu.be/lo2aC_m2vyo
 Accomplish Lab Report #1

 PRINCIPLES OF MICROSCOPY

The microscope is an essential tool in microbiology laboratory, thus it is the

responsibility of the student to take proper care of the microscope and to learn to use it
correctly. The microscope most commonly used in the laboratory is the compound light
microscope wherein the specimen is observed using visible light and two sources of
magnification are used. Magnification is a result of 2 lenses: the objective and the
ocular. The total magnification is computed by multiplying the power of the ocular and
the power of the objective lens.
The iris diaphragm controls the amount of light that enters the condenser which
will allow for appropriate observation of contrast and depth of field.
As two small objects are moved closer to each other, a point is reached where
the eye is unable distinguish the objects as separate entities, and only a single object is
observed. The smallest distance at which two points can be seen separately is called the
resolving power of the lens. The resolving power of the human eye at ten inches is 0.1
mm. This resolution increases if the microscope is used to aid the eye, and increases as
magnification is increased. However, there is a limit to the resolution of even the highest
magnification lens on a light microscope, and other types of microscopes, like electron
microscopes, must be used to resolve smaller structures of bacteria and viruses.
As the light from the light source passes through the slide from below, and then
enters air again, the light is bent, and goes off to one side or the other, rather than
continuing on through the lens and to the eye. This bending is called refraction, which
occurs any time light passes from material of one density to material of another density.
When refraction occurs, the refracted light is lost, and it is harder to see object.
Refraction can be partially overcome if material of the same density is placed between
the slide and the glass lens of the objective. Modern light microscopes use a particular
oil for this purpose, and a particular kind of objective. These objectives are referred to as
oil-immersion objectives, and are meant to be used with immersion oil.
 USING THE MICROSCOPE:
Basic rules
 Always carry the microscope with 2 hands: one under the base and the
other grasping the arm
 Gently place the microscope on the lab table with the arm facing you.
The microscope's base should be resting evenly on the table,
approximately 10 cm from the table's edge.
 Make sure the stage and objective are at their farthest distance apart and
that the lowest power objective is in position. Start each new observation
at low power.

Focusing the Microscope using LPO and HPO

1. Obtain some prepared microscope slide.

2. Carefully place the slide on the stage and secure it with stage clips. Move the slide
so that the specimen is centered in the stage’s hole. Click the scanning power (4X)
or the low power objective (if the scanning power objective is not present) into place
on the nosepiece
3. While looking from the side, use the coarse-focus knob to move the objective as
close as possible to the slide.
4. Look through the ocular and use the coarse – focus knob to slowly move the
objective and slide apart. When the image becomes clear, switch to the fine focus
knob
5. Adjust the light intensity by using the diaphragm until the detail of the image is at its
clearest.
6. Sketch the entire field of view
7. Center the part of the specimen that you wish to see more clearly and switch to high
power objective. Most scopes are parfocal which means that the image remains
focused when you change objectives. However, some minor adjustment with the
fine focus is usually necessary.
8. Sketch the entire field seen using the high-power objective.
9. When the slide is removed, make sure that the low power objective is in viewing
position and that the objective and stage are as far apart as possible.
. (Note: When focusing, always start at low power and work your way up. This not only
helps find and focus on specimens quickly but also alleviates the potential of ramming a
long, high-powered objective through a slide when trying to focus with the course
adjustment knob under high power. Pay attention to the working distance of the lens.
This is the distance between the lens and the slide when the specimen is seen in sharp
focus. The higher the magnification, the smaller the working distance. To avoid ramming
a long objective into a slide, observe the Working Distance Rule: Use the coarse
adjustment knob on low power only .

RULES FOR PROPERLY RECORDING MICROSCOPIC OBSERVATIONS

 Label each sketch with the name of specimen and what type of section it is.
 Label each sketch with the total magnification
 Label as many parts of the sketch as you can.
  Sketches should be done with care and not with haste

Proper Storage of the Microscope

To practice proper care of the microscope, make certain to clean it and put it away
properly at the end of this exercise.
1. Make certain the slide is removed from the stage.
2. Clean all lens with Lens Paper if there is excess oil.
a. Obtain a clean sheet of lens paper.
b. Rub oculars to clean as demonstrated.
c. Rub objectives to clean, starting with the scanning power objective through
the high power objective. Clean the oil immersion objective last.
3. Put the scanning power objective in place.
4. Pull the body tube away from the stage (i.e. lower the stage as far as possible)
5. Wrap the cord.
6. Return the proper storage location in the cabinet.

Reference:
How to use a compound light microscope laboratory exercise materials from the virtual microbiology classroom. (n.d.). Science
Prof Online Free Science Education Website. https://www.scienceprofonline.com/vmc/vmc-
lab/vmc-laboratory-microscopy.html

Mc Laughin. (2016). Laboratory exercises in MIcrobiology: Discovering the unseen world

through hands-on investigation. CUNY Academic
Works. https://academicworks.cuny.edu/cgi/viewcontent.cgi?article=1015&context=qb_oers

Microscopes – General microbiology. (n.d.). Open Textbooks – Oregon State University, Open
Educational Resources
Unit. https://open.oregonstate.education/generalmicrobiology/chapter/microscopes/

How to use a compound light microscope laboratory exercise materials from the virtual
microbiology classroom. (n.d.). Science Prof Online Free Science Education
Website. https://www.scienceprofonline.com/vmc/vmc-lab/vmc-laboratory-microscopy.html

.
Lab Report # 1 ( MICROSCOPY)

NAME: Verdote, Charles Denver A. Date: 09/23/21

Year and Section: 1-YA-17

I. Identify the parts of the compound microscope. Choose from the word bank below.

Ocular/eyepiece

Body tube

Revolving
nosepiece
Arm
LPO
Oil immersion
objective
Stage
HPO

Stage clips Coarse focus knob

Iris diaphragm Fine focus knob

adjustment

Mirror/light
source
Base

Microscope labeling. (n.d.). The Biology Corner. https://www.biologycorner.com/worksheets/microscope_labeling.html

Word Bank:

-Arm - Eye piece - Iris Diaphragm Adjustment

-Base - Fine Focus Knob -Mirror (or light source)
-Coarse Focus Knob -High-power objective -Revolving Nosepiece
-Condenser -Low-power objective -Stage
-Condenser Focus Knob - oil immersion objective
II. Give the functions of each part of the microscope listed in the word bank.

Main Parts of Microscope

1. Head - This is also known as the body, because it houses the optical components of the
microscope's top section.
2. Base - It serves as a support for microscope. It also has tiny illuminators on board.
3. Arm - This is the component of the microscope that connects the base to the head and
the eyepiece tube to the base. It serves to support the microscope's head and is also
used to transport the microscope. Some high-end microscopes include an articulated
arm with several joints that allows the tiny head to move more freely for improved
viewing.

Sub parts of the Microscope

1. Eyepiece – also known as the ocular. this is the part used to look through the
microscope. Its found at the top of the microscope. Its standard magnification is 10x with
an optional eyepiece having magnifications from 5X – 30X.

2. Eyepiece tube – it’s the eyepiece holder. It carries the eyepiece just above the objective
lens. In some microscopes such as the binoculars, the eyepiece tube is flexible and can
be rotated for maximum visualization, for variance in distance. For monocular
microscopes, they are none flexible.

3. Objective lenses – These are the major lenses used for specimen visualization. They
have a magnification power of 40x-100X. There are about 1- 4 objective lenses placed
on one microscope, in that some are rare facing and others face forward. Each lens has
its own magnification power.

4. Nose piece – also known as the revolving turret. It holds the objective lenses. It is
movable hence it cal revolve the objective lenses depending on the magnification power
of the lens.

5. The Adjustment knobs – These are knobs that are used to focus the microscope. There
are two types of adjustment knobs i.e fine adjustment knobs and coarse adjustment
knobs.

6. Stage – This is the section on which the specimen is placed for viewing. They have
stage clips that hold the specimen slides in place. The most common stage is a
mechanical stage, which allows the control of the slides by moving the slides using the
mechanical knobs on the stage instead of moving it manually.

7. Aperture – This is a hole on the microscope stage, through which the transmitted light
from the source reaches the stage.

8. Microscopic illuminator – This is the microscopes light source, located at the base. It is
used instead of a mirror. it captures light from an external source of a low voltage of
about 100v.

9. Condenser – These are lenses that are used to collect and focus light from the
illuminator into the specimen. They are found under the stage next to the diaphragm of
the microscope. They play a major role in ensuring clear sharp images are produced
 with a high magnification of 400X and above. The higher the magnification of the
condenser, the more the image clarity. More sophisticated microscopes come with an
Abbe condenser that has a high magnification of about 1000X.

10. Diaphragm – it’s also known as the iris. Its found under the stage of the microscope and
its primary role is to control the amount of light that reaches the specimen. It’s an
adjustable apparatus, hence controlling the light intensity and the size of the beam of
light that gets to the specimen. For high-quality microscopes, the diaphragm comes
attached with an Abbe condenser, and combined they are able to control the light focus
and light intensity that reaches the specimen.

11. Condenser focus knob – this is a knob that moves the condenser up or down thus
controlling the focus of light on the specimen.

12. Abbe Condenser – this is a condenser specially designed on high-quality microscopes,

which makes the condenser to be movable and allows very high magnification of above
400X. High-quality microscopes normally have a high numerical aperture than objective
lenses.

13. The rack stop – It controls how far the stages should go preventing the objective lens
from getting too close to the specimen slide which may damage the specimen. It is
responsible for preventing the specimen slide from coming too far up and hit the
objective lens.

III. Research online photomicrographs of specimen observed using LPO, HPO

( Give 2 examples for each )

Low Power Objective

High Power Objective

IV. Answer the following questions.

1. Explain the meaning of parfocal.
Parfocal - having all of the matching focus points in the same plane: having a set of
objectives or eyepieces mounted in such a way that they may be swapped out without
changing the focus of the instrument (such as a microscope).

2. Describe the difference between coarse focus knob and fine focus knob.

The coarse adjustment knob sharpens the image when the power is low (usually the
bigger knob)
Fine adjustment knob—sharpens the image at all magnifications (usually the smaller
knob).
3. What does the term field-of-view mean with respect to your microscope? How does the
scopes field-of-view relate to the importance of centering your specimen using the
mechanical stage, before switching to the next higher objective magnification?

The diameter of the field of an optical light microscope is the field number, which is the
diameter of the field view in mm measured at the intermediate image plane. The field number
is typically listed on the microscope eyepiece. The microscope field of view is found with the
following formula:
Field of View = Field Number (FN) ÷ Objective Magnification

The higher your magnification, the smaller the microscope field of view will be. If you think of
looking at the above aphid through the microscope, if you were to zoom in to view only the leg
of the aphid, your field of view would definitely be smaller, while the magnification is increased.

You must have the object centered before you change objectives to increase the magnification,
because the field of view becomes smaller; if the object is off to the side, it may disappear when
you go to higher magnification. The higher the power of the objective lens, the less will be the
depth of field.