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: ___________________________________ Date: ____________

Year and Section:___________________________

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

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.
III. Research online photomicrographs of specimen observed using LPO, HPO
 ( Give 2 examples for each )
IV. Answer the following questions.
1. Explain the meaning of parfocal.
2. Describe the difference between coarse focus knob and fine focus 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?