MICROBIOLOGY AND PARASITOLOGY Laboratory

Lab Lesson6
Bright Field Microscopy
Background Information
A microscope is a precision instrument use to magnify objects too small to be seen by the eye. It is
the window to the unseen world. Human visual acuity is limited to 4 µm (micrometer) and some
specimens and organisms are smaller than this dimension. Thus, the microscope is an indispensible
instrument in biology.
At present there are different kinds of microscopes available. Modern technology has provided to
biologists a wide range of instruments to observe the specimens too small to be seen by the eyes. These
instruments range from the most common bright field microscope to several specialized instruments and
the highly sophisticated electron microscope. All these operate on a common principle: a type of energy is
projected toward the object; the energy bounces off the object and creates an impression on a sensing
device. This may be television screen, a photographic plate, or the human eye. The image reveals the
shape, size and arrangement of the microorganism.

Bright Field Light Microscope

This is the most familiar microscope. The light microscope is a two lens system in which the lens
nearest the object is known as the objective lens and that nearest the eye, is known as the ocular lens.
Visible light rays are projected through a condenser that focuses them into a sharp cone, then sends
them through the opening in the stage on which the slide rests.
The light passes through the slide, bounces off the object, then passes through the objective lens to
form a magnified image darker than the background around it. This image is a real image that can be
projected onto a screen. The image now becomes an object for the ocular lens and the light rays are
magnified a second time creating a virtual image in space visible only to the observer.
The dual lens system is referred to as a compound microscope to distinguish it from a single lens
system such as used by van Leeuwenhoek. In addition, a basic laboratory microscope usually has three
objective lenses called the very low power (scanner), low power, high power lenses.
The knowledge on the care and use of this microscope is vital to the success of its operation
especially for beginners. Extreme care must be practiced in handling this versatile precision instrument.
In this laboratory you will be learning the basics of student microscope, the bright field microscope.

Components
The major parts and functions of a compound microscope can be categorized as mechanical part and
optical parts (table6-1).

Lens System
The oculars, the objectives and the condenser constitute the three lens system of all microscopes.
The ocular lens located on the upper end of the drawtube. It is also known as the eyepiece and has a
magnification of 10X. There are usually three objective lenses: low power objective (LPO) has a
magnification of 10X, high power objective (HPO) has a magnification of 40-45X and oil immersion
objective (OIO) the longest objective with a magnification of 60-100X. The structure below the stage
that concentrates light on the slides, the condenser, comprised the third lens. The condenser moves up
and down through the condenser control knob. A diaphragm, within the condenser, regulates the
amount of light that will reach the slide. There are two types: a) disc diaphragm- circular plate with
holes of various diameters and b) iris diaphragm- with opening regulated by a lever.

Focusing knobs
These knobs found at the side of the microscope bring the specimen in the slide into focus. The
larger coarse adjustment knob allows rough focus while the smaller fine adjustment knob allows sharper
focus.

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Table 6-1 Mechanical parts of the microscope.

Part Description/Functions
Base The bottom portion which hinged a rigid support. It carries a mirror or a
built-in light source and light switch.
Pillar The vertical extension of the base.
Arm The structure that arises from the base that supports the stage, lenses
and control mechanism. It is where you grasp the microscope when
carrying.
Body Tube It bears the barrel that has the ocular lens at the upper end and the
revolving nosepiece at the lower end.
Draw Tube The upper part of the body tube that houses the ocular lens.
Revolving Nosepiece The circular structure at the lower end of the body tube to which the
objectives are attached.
Stage The platform on which a microscope slide is placed for viewing.
Stage Aperture The opening at the center of the stage. It allows the passage of light into
the specimen.
Mechanical Slide Holder The device that holds the slide in the stage. It fastens the slide into the
stage. It allows smooth movement of slide in the stage.

Magnification
This refers to the number of times the object is enlarged. It is determined by multiplying the
magnification indices of the ocular and the objective lenses.

Working distance
The working distance of an objective is the actual distance between the front surface of the lens and
the cover slip. It is dependent on the numerical aperture (N.A.) of the lens and decreases as actual image
magnification increases. It is a very important factor the selection of objectives.

Resolution
The ability of the microscope to reveal the clearest pattern is known as resolution. The best image is
not the largest; it is the clearest. The two major factors that affect resolution, the wavelength of light used
and the angular aperture of the lens system involved.For microscopes with substage condenser,
calculation of the resolving power can be approximated by applying the following formula:

wavelength of light
Resolution = ----------------------------- N.A. = numerical aperture
2 x N. A.

Numerical Aperture of Objective lenses

Objective Lens Numerical Aperture

Low power 0.25
High power about 0.65
Oil immersion about 1.25

Depth of Field
Depth of field refers to the thickness of the plane of focus. With a large depth of field, all of the
threads can be in focus at the same time. With a narrow depth of field, only one thread can be focused
while everything else will be out of focus.

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Diameter of the Microscopic Field

The microscopic field is the circular lighted view field when one looks in the ocular lens. It is
measured simply by determining the diameter of the widest area of the field express in mm or µm (0.001
mm).

Setting up an Elementary Microscope

1. Tilt the body back to a convenient angle if using microscope without built-in lighting system. Turn the
10X lens into place and adjust the concave mirror in relation to the source of light. The objective is
aligned with the eyepiece when you hear a “click” sound. Peep into the eyepiece. You will see a
bright circular field- the microscopic field.
2. Place a slide carrying a mounted specimen on
the stage fastened to the mechanical stage clip
(Figure 6-1).
3. While looking through the side (not through the
eyepiece) use the coarse adjustment to bring
the objective to the mounted slide about 3mm.
If the light is too bright, adjust by constricting the
iris diaphragm (electric microscope) or tilting the
concave mirror a little from the source of light.

Figure 6-1.The correct way of putting a slide on the stage.

Focusing and Shifting Objectives

a) Turn the objective lens marked 4X until it is secured in place.
b) Rotate the coarse adjustment knob to move the stage upward until it stops.
c) View through the ocular lens and slowly turn the coarse adjustment knob to lower the stage until
the specimen is in focus.
d) To set the specimen in sharp focus, turn the fine adjustment knob gradually. Maximize contrast
by turning the disk diaphragm or iris diaphragm level to adjust light intensity.
e) In necessity of higher magnifications, just rotate the 10X or 40X objectives in place. Most
microscopes are parfocal (specimen is still in focus) and parcentric (specimen is still at the
center) though objectives are changed. Minor adjustments in lighting and contrast may be called
for.
f) In using the oil immersion objective, an oil immersion is placed in the slide before place the
objective marked 100X in viewing position. Specimen is still in focus and minor adjustments still
may be needed each time your change objective.

Illumination
a) Check the light source of the microscope. If a box lamp is used, make sure that it is position exactly
under the center of the condenser. If a mirror is used, make sure that it is free of dust and used the
concave side at lower magnification and convex side at higher magnification.
b) Adjust the amount of light coming through the condenser using the diaphragm located under the
stage in the front of the microscope. Light adjustment using the diaphragm is critical to obtaining
proper contrast. For oil immersion microscopy (1000X), the diaphragm should be set almost all the
way open (to your left for maximum light). For low powers such as 100X the diaphragm should be
set mostly closed (to your right for minimum light).

Care of the microscope

Transport and Placement

1. The microscope should be held firmly with both hands while carrying it. One hand is grasping the
arm of the microscope while the other supports the base of the microscope (Figure 6-2).
2. Always place the microscope in the center of a firm stable disk. Extreme care is needed to prevent
costly accidents. An ordinary microscope costs P16,000.00 pesos.
3. Avoid overcrowded working conditions.

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4. Do not remove any part of the microscope. If there are

mechanical problems, notify your instructor.

Cleaning the microscope

Clean the exterior lenses of the eyepiece and objective
before and after each lab using lens paper only. Remove
any immersion oil from the oil immersion lens before
putting the microscope away.

Reason for using immersion oil

Normally, when light waves travel from one medium
into another, they bend. Therefore, as the light travels from
the glass slide to the air, the light

Figure 6- 2. Correct way of handling a

microscope.

waves bend and are scattered similar to the "bent pencil" effect when a pencil is placed in a glass of
water. The microscope magnifies this distortion effect. Also, if high magnification is to be used, more light
is needed.
Immersion oil has the same refractive index as
glass and, therefore, provides an optically
homogeneous path between the slide and the lens
of the objective. Light waves thus travel from the
glass slide, into glass-like oil, into the glass lens
without being scattered or distorting the image (Fig.
6-3). In other words, the immersion oil "traps" the
light and prevents the distortion effect that is seen as
a result of the bending of the light waves.

The immersion oil has the same refractive index

as the glass lens and the glass slide. Because the
light waves follow a homogeneous path, there is no
distortion.

Figure 6-3.The oil immersion lens.

Returning the Microscope to the Cabinet

1. If you are using a microscope in an inclined position, return it to an erect position.
2. Remove the slide from the stage.
3. Wipe all oil from the lens and stage.
4. Rotate the scanner or LPO into position.
5. If the microscope has a movable body tube, lower it to its lowest position
6. If the microscope has a built-in movable lamp, raise the lamp to its highest position and wrap the
electrical cord around the base.
7. Adjust the mechanical stage so that it does not project too far on either side.

Objectives
At the end of the exercise the student must be able to:

1. Identify the parts of a compound microscope and state their functions.

2. Demonstrate the correct way to:
a. carry and set-up a microscope

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b. adjust the light using the disc or iris diaphragm lever for optimum contrast after focusing.

c. mount a slide in the slide holder of a mechanical stage correctly.

d. focus on a specimen using 10X, 40X, and 100X objectives.
e. calculate the total magnification of a lens system when using a 10X, 40X, or 100X objective in
conjunction with a 10X eyepiece.
f. calculate the resolving power of each objectives.

Materials
 compound microscope Puccinia graminis, Entamoeba coli
 prepared slides (letter “e”, or E. histolytica)
Hydrodictyon or Draparnaldia,  celluloid ruler

Procedure
A. Setting up the Microscope
Get a microscope and study the various parts. Identify the parts and familiarize with their
functions. Carry the equipment properly. Place the microscope at the center of the working
table. DO NOT PLACE THE EQUIPMENT ON THE EDGE OF THE TABLE! Set the microscope
as outline above. Draw the microscope and label its parts.

B. Magnification and Resolution

Determine the magnification index and total magnification of each objective. On the basis of
the numerical aperture (N.A.) values for each objective, calculate the resolving power or
resolution of each. Wavelength of yellow light is 0.53 µm.

C. Working Distance
Examine a letter “e” slide under scanning lens. Bring the specimen is focus. Measure the
distance between the tip of the objective lens and the surface of the slide or coverslip. Use mm
units. Repeat the process using LPO. Complete the table the table in the report sheet.

D. Initial Observation on Letter “e”

Obtain a prepared slide of letter “e”, handling it by the edge. Following the procedure
described earlier examine the slide. Move the slide back and forth, side to side. Note the direction
of movement and the position of the object. Shift from one objective to another.

E. Observing Prepared Slides of Microorganisms

1. Examine the demonstration slides of the following microorganisms:

a. Hydrodictyon, alga
b. Aspergillus niger, a fungus
c. Entamoebahistolytica, a protozoon
d. Smear of bacteria showing basic shapes

2. Compare the relative sizes of the specimens by noting the magnification where the
specimens were observed.
3. Draw the specimens.

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Name: _____________________________ Score: ____________________

Lab Schedule: _______________________ Term/Date: ___________________

Lab Report 6 Bright Field Microscopy

Attendance Stamp
Results and Observation

A) 1. List all the parts and functions of the microscope.

Parts of the Microscope Function(s)

1) Arm
2) Base
3) Body tube
4) Condenser
5) Draw tube
6) Illuminator
7) Objective lens
8) Ocular lens
9) Pillar
10) Revolving nosepiece
11) Slide holder
12) Stage
13) Stage aperture

2. Label the parts of the microscope.

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B) Fill-in the table below.

Lenses Magnification Index Total Magnification Resolution

Very low power
Low power
High power
Oil immersion

C) Complete the table with working distance measured.

Objective Lenses Working Distance(mm)

Very low power (scanner)
Low power
High power

What happen to the working distance as you shift to higher magnification? _____________
_________________________________________________________________________

What does this tell you about breaking the slide or running into the stage? _____________
_________________________________________________________________________

D) What is the position of letter “e” as seen under the microscope compared to unaided eye?
________________________

Indicate the direction when you move the slide:

i. to the left _____________________ ii. forward _____________________

Draw the letter as it appears when observed with:

a) Unaided eye b) Scanner (VLPO)

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LPO HPO

E) Observing Prepared Slides of Microorganisms

______________________x____ _________________________ x ____

______________________ x ____ _________________________ x ____

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Lab Lesson 6: Bright Field Microscopy