BACHELOR OF SCIENCE IN BIOLOGY:

MICROBIOLOGY LABORATORY
LABORATORY EXERCISE WEEK
2 MICROSCOPY 3

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, Oil immersion )
● Accurately record microscopic observations

MATERIALS
● Laboratory Report
● Power Point Presentation
● Videos about using the microscope
● Additional required lab readings
●
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://www.youtube.com/watch?v=f7KlFSgdUGU
● https://youtu.be/lo2aC_m2vyo
● https://www.youtube.com/watch?v=m4XuYWhvDdU
● Read the following:
 o Smith, Molly and Selby, Sara, "Microbiology for Allied Health Students: Lab
Manual" (2017). Biological Sciences Open Textbooks. 16.
https://oer.galileo.usg.edu/biology-textbooks/16 2.3 Instruments of microscopy

● Accomplish Lab Report #2

INTRODUCTION

● PRINCIPLES OF MICROSCOPY

The microscope is one of the most essential tools in microbiology. The compound light
microscope is the most commonly used microscope in the laboratory. It uses light to observe a
specimen. It also has at least two sources of magnification, the eye piece or ocular lens and the
objective (low power or high power). When viewing objects under the microscope, it is important
to calculate for the magnification which is the product of the ocular and the power of the
objective lens.

When viewing a specimen under the microscope, there comes a point wherein two
objects cannot be seen as separate objects. The resolving power of a microscope is the
smallest distance at which two objects can be seen as separate. Increasing the magnification of
the light microscope does not necessarily increases its resolution. Other types of microscopes
such as the electron microscope have greater resolving power, thus are used to view more
detailed structures of bacteria and viruses.

As light passes from one medium to another such as when it passes from the light
source, then through the air and then through the slide, its direction changes. This is called
refraction. When refraction occurs, it becomes harder to view an object using the lower and high
power objectives. To remedy this, a few drops of oil is placed between the glass slide and the
lens of the objective. The oil immersion objective has a higher refractive index and allows the
user to view finer details of a certain specimen

● USING THE MICROSCOPE:

Basic rules
● The microscope should always be carried with two hands: one hand under the
base and the other hand holding the arm of the microscope.
● The microscope should be carefully placed on the table. The base of the
microscope should be about 10 cm away from the edge of the table.
● The stage and the objectives should be positioned farthest from each other.
● Always start with the lowest power objective, such as the scanning objective or
the low power objective when observing specimen.
PROCEDURE
MATERIALS
● Prepared slides
● Compound microscope with oil immersion objective
● Cedarwood oil

Focusing the Microscope using LPO and HPO

1. Obtain prepared microscope slides.

2. Look through the eyepiece and adjust the light by moving the mirror.
3. Move the nosepiece upward and carefully place a prepared slide at the center of the stage
of the microscope. Use the stage clips to secure the slide. Move the nosepiece so that the
scanning power or the low power objective is in the middle.
4. Look through the ocular and move the low power objective as close as possible to the
slide by using the coarse focus knob
5. Use the coarse focus knob to slowly move the low power objective upward until the image
becomes clear.
6. Move the slide and center the part of the specimen which you want to view more clearly.
Then move the nosepiece to high power objective. Use the fine focus knob to view the
object clearly. Do not move the coarse adjustment knob since most compound light
microscopes are parfocal.
7. Sketch the entire field.
8. After observing and sketching the specimen, move the objectives so that they are far away
from the stage. At this point, you can remove the slide.

Focusing the Microscope Using the Oil Immersion Objective

1. Repeat steps 1-6

2. Move the nosepiece so that the slide is between the high power and oil immersion objective.
3. Place a 1-2 drops of cedarwood oil on the center of the slide.
4. Move the nosepiece so that the oil immersion objective touches the oil on the glass slide.
5. Use the fine adjustment knob to focus the specimen.
6. Record your observations
7. Move the objective and stage as far apart as possible.
8. Wipe the 100x objective and glass slide gently with lens paper xylol or toluene to remove the oil.

Note: The low power and high power objectives cannot be viewed once oil is placed on the
slide, since the oil will blur the image.
 .

RULES FOR PROPERLY RECORDING MICROSCOPIC OBSERVATIONS:

● Always write the name of the specimen and section used.
● Indicate the total magnification.
● Label the parts of the specimen.
● Sketch observations carefully and neatly.

Proper Storage of the Microscope

1. Clean the microscope before storing it.

2. Be sure that there is no slide on the stage.
3. Wipe all ocular and objective lenses with lens paper or tissue.
4. Move the objectives and stage as far away from each other.
5. Wrap the cord around the base of the microscope.
6. Store the microscope in the assigned storage cabinet.

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
 MICROBIOLOGY LABORATORY
Lab Report # 2 ( MICROSCOPY)

NAME___________________________________ Date: ____________

Year and Section:___________________________

I. Identify the parts of the compound microscope.

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. Answer the following questions
1. What is parfocal microscope?
2. Explain the principle behind oil immersion objective.
3. When is it recommended to use the oil immersion objective?
4. How have microscopes improved our understanding of microbes?
5. What are the limitations of microscopes and the information that we get from them?

IV. Draw or attach pictures of specimen observed using LPO, HPO, Oil Immersion objective.
Objective. ( Give 2 examples for each )