Activity 1

BASIC MICROSCOPY
The Word of God in the book of Hebrew 4:13 says, “Neither is there any creature that is not manifest in
his sight: but all things are naked and opened unto the eyes of him with whom we have to do.” KJV

New International Version says, “Nothing in all creation is hidden from God’s sight. Everything is
uncovered and laid bare before the eyes of him to whom we must give account. Hebrews 4:13”

Microbiology usually is concerned with organisms so small they cannot be seen distinctly with unaided
eye. Because of the nature of this discipline, the microscope is of crucial importance; much of what is known about
microorganisms has been discovered with microscopes. Thus, it is important to understand how the microscope
works and the way in which specimens are prepared for examinations

With the invention of the microscope, microbiologists gained valuable tool to observe and study
structures that are too small to be seen by the unaided eye. As a result, many of the theories basic to the
understanding of biologic sciences have been established. This exercise will familiarize you with the workhorse of
microscope—the compound microscope and provides you with the necessary instructions for its proper use.

Learning outcomes:

After this activity, the student should be able to:

1. For the students to know more about God, the Creator of the heavens and the earth as
the Searcher of every person’s heart in relation to the primary function of the microscope.
2. Locate and name the parts of a microscope and explain the function of each part.
3. Define and interconvert different metric units.
4. Compare and contrast the simple and compound light microscope.
5. Practice the proper way of handling the microscope to prevent damage.
6. Know how to operate the microscope.

Materials:

• Microscope (monocular or binocular bright field microscope)

• Ruler or measuring tape
• Lens paper
• Prepared blood smear slide
• Prepared paramecium slide

I. Care of the Compound Microscope

The Compound Microscope is a precision instrument and should always be handled with care. At all times
you must observe the following rules for its transport, cleaning, use and storage:
 • When transporting the microscope, hold it in an upright position with one hand on its arm and the
other supporting its base. Avoid jarring the instrument when setting it down.

• Position the microscope on the table’s edge so as to keep your back straight while looking into the
microscope.

• Wipe the eyepieces and objectives with clean lens paper.

• Open the iris diaphragm all the way.

• Look through the eyepieces and adjust the interpupillary distance, then manipulate the concave
mirror until the light evenly distributed throughout the microscope field.

• Raise the condenser until it is level with the stage.

• Place the slide over the stage aperture and fasten with stage clips.

• Raise objective with coarse focus knob while looking through the eyepiece until the image appears.
Use fine focus knob to acquire the best resolution.

• Regulate iris diaphragm to control brightness.

REMINDERS:

• The use of direct sunlight as a light source may damage your eyes.

• Never lower the objective while looking through the ocular.

II. Parts of the Microscope

Basically, the students have to master the parts and functions of the microscope.
Simple microscope (Reading glass)
Compound microscope (Composed of several parts)

A. Mechanical Parts:

• Base – a horseshoe foot, which supports and steadies the rest of the microscope.
• Pillar – serves as a firm support which is the post or vertical extension of the base and to which the arm is
attached.
 • Arm – carries the tube and is connected to the base. The arm of a binocular microscope (which you will be
using) is fixed unlike that of a freshman microscope (monocular microscope) which is inclinable.
• Inclination joint – movable hinge that attaches the frames of the microscope to the base. It is used for tilting
the microscope whenever necessary for the convenience of the user.
• Stage – a square platform with a circular aperture at the center upon which the specimen is placed. It is
provided with a central opening allowing light to pass from below into the object of study.
• Substage condenser – concentrates light rays received from the mirror and sends them to the objective.
• Substage condenser knob – used to move the substage condenser up and down.
• Stage clips – holds the slide in place on the stage.
• Mechanical stage – some microscopes are provided with mechanical stage which moves the specimen by
screw movement either forward, backward or sideward.
• Revolving nosepiece – to which the three-parfocal objectives are attached.
• Binocular tube – a hollow tube through which the light from the objective to the ocular passes.
• Clamping screw – holds the alternative body in the desired position.
• Binocular alternative body – carries paired eyepieces and can be rotated frontally or dorsally.
• Diopter Setting Adjustment Ring/ Diopter Adjustment – find on the left eyepiece and corrects any
difference in strength in both eyes.
• Interpupillary Adjustment Wheel/ Interpupillary Adjustment – located at the center of the two eyepiece
and controls the horizontal distance between them.
• Focusing Knobs – this consist of two pairs of knobs which, when turned clockwise or counter-clockwise,
lower or raise the body tube. These are used to adjust the objective when focusing.

▪ Coarse Focus Knob – brings the object into approximate focus, while the

▪ Fine Focus Knob – gives the object maximum definition.

• Mirror Rack – it holds the mirror and is provided with a hinge to facilitate tipping to any desired angle.

B. Illuminating Parts

• Mirror – collects and reflects light into the condenser. The medical microscope mirror is concave on one side
and flat on the other.

▪ Concave Mirror – it is used when the source of light (natural or artificial) is of low density.

▪ Plane Mirror – this is used when the source of light is very bright.

• Iris diaphragm – controls the numerical aperture by regulating the amount of light entering the condenser.
• Abbe condenser – this is used to condense or concentrate the reflected light from the mirror to the object or
specimen being examined.

C. Magnifying Parts

• Eyepiece (ocular) – this is a detachable cylinder situated on top of the draw tube or alternative body tube. It
is equipped with a lens that magnifies the object several times and enables one to see the specimen by looking
through it. Sometimes it is provided with a pointer which is used in pointing at the specific parts of the
specimen.
 • Objectives – this is the most important part of the microscope and is the primary magnifier of the object.
▪ Low Power Objective (LPO) – this is a short cylinder with a lens of low magnifying power. It is used in
observing the general outline and locating the various parts of the specimen.

▪ High Power Objective (HPO) – this is long cylinder with a lens of high magnifying power. It is used to
study the detailed part of the specimen.

▪ Oil Immersion Objective (OIO) – this is long cylinder with a lens of a very high magnifying power. It
focuses very close to the slide and needs a small drop a special oil (cedar oil).

III. Characteristics of Objective Lens

1. Resolution. Property of an objective to distinguish the distance between two points as separate and distinct.

2. Numerical Aperture. A measure of the resolution of an objective. An objective with a 0.15 N.A. permits you to
distinguish 15,000 lines per inch. If you view 16,000 lines per inch specimen through a 0.15 N.A. objective, you
will never see the lines as separate no matter how powerful the magnification.

3. Focal length. The distance from the center of the lens to a point where the parallel light rays is brought to a focus.
It is a constant of the lens system.

4. Working Distance. The distance between the front lens of an objective and the surface of the specimen when it
is in focus.

5. Parfocal. The optical and mechanical design where only slight refocusing with the fine focus knob is sufficient to
obtain clarity when changing from one lens to another.

IV. Care of the Microscope

1. Never drop the microscope heavily on the table. Place it gently without jarring.

2. When transporting the microscope, grasp the arm firmly with one hand and support it at the base with the other.

3. Do not touch the lenses with your finger – use only lens paper for cleaning.

4. Before returning the microscope, remove the slide and make sure the 10X objective is fully lowered. Turn the
concave mirror upside down.

V. Procedure

A. Determination of Microscope Field Area

1. Adjust the microscope for viewing under 10X objectives. Place a metric ruler or tape on the stage with
its millimeter scale over the stage aperture.

2. Move one of the millimeter markings to one edge of the field. The distance between the borders of the
two consecutive millimeter markings is 1,000 micrometers.

3. Count the number of millimeters across the field diameter and add the additional fraction that may
exist.

4. Calculate mathematically the high dry and oil immersion objective microscope field diameters using the
formula:
Given:
Low Power Magnification = 4X
Medium Power Magnification = 10X
High Power Magnification = 40X
Oil Immersion Magnification = 100X

OI/High/medium Power Magnification = Low Power Field Diameter (LPFD)

Low Power Magnification OI/High/Medium Power Field Diameter (HPFD)

5. Cross multiply and solve for X.

6. Record the diameter, area and radius of the 3 objectives. Area is obtained by multiplying the square of
the radius by 3.1416. Answers should be in micrometers.

7. Given: Low Power Field Diameter (PDFD) = 4 mm

LPO [Low Power Obj.] MPO [Medium Power Obj.] HDO [High Dry Obj.] OIO [Oil Immersion Obj.]
DIAMETER 4 mm
RADIUS
AREA

Formula for Area: A = πr2 π = 3.1416

Formula for Radius: r = d/2, d = diameter

B. Measurement of Specimen

1. Secure prepared Paramecium slide and measure the length of one the Paramecia that can be seen in the
field view.

2. Calculate the approximate length of the Paramecium based on the field diameter of your objective.
 3. Draw the image observed and label its visible parts.

PARAMECIUM

C. Use of Oil Immersion Objective

1. Place the stained blood smear on the stage and examine under the 10X objective.

2. Center the white blood cells on the microscope field view.

3. Use the fine focus knob to sharpen the image.

4. Shift the objective to 45X and manipulate the fine focus knob to obtain the clear image.

5. Shift the nosepiece and apply a small drop of immersion oil at the center of the slide.

6. Rotate the Oil Immersion Objective into place. Make sure the Oil Immersion lens is immersed in oil.

7. Use the fine focus knob for maximum resolution.

8. Draw and label.

 BLOOD SMEAR
(Note: Remove oil from oil immersion lens with lens paper after the exercise)

QUESTIONS

1. What is the purpose of oil used in the Oil Immersion Objective (OIO)?

2. When is the OIO used?

3. Is the relationship between objective magnification and field diameter directly or indirectly proportional?
Why?

4. Why does one never lower the objective with the Coarse Focus Knob while looking through the eyepiece?

5. What kind of microscope uses the following?

a. Reflected Light

b. Transmitted Light

6. What is Ramsden’s disk?

 7. What is meant by Binocular Compound Microscope?

8. Why there is a need for microscopy in Microbiology?

9. What can be clearly focused to you under the microscope may not be in focus to somebody? Why?

10. Describe Electron microscopy. Compare the processes in Electron microscopy with the Word of God
in Hebrew 4:12

VI. Basic Apparatus used in Microbiology

Learning Outcomes:

• To identify the different apparatus/equipment used in the laboratory

• Describe the function of each apparatus/equipment

Procedure

• Know the different laboratory apparatus/equipment you see inside the laboratory.

• Discuss with you group the functions of each equipment.

• Draw the different apparatus you see inside the laboratory.

Integration of Faith, Values and Learning (IFVL):