The Microscope
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Introduction to Brightfield Microscope
The simplest type of
compound microscope is
the Brightfield compound
microscope.
The word “brightfield”
refers to the fact that
magnified objects
appear as dark objects
against a bright
background. A sufficient
contrast must exist
between the magnified
object and the
brightfield background
for the objects to be
visible.
The word “compound”
from “Brightfield
Compound
Microscope” means
that a specimen
positioned properly
on the stage of a
microscope and
illuminated by a light
source will be
magnified by two-lens
system.
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The Path of LIght
04
Then the image of the specimen
is magnified again by the ocular
lens or eyepiece.
03
Light rays then pass into the
objective lenses, the lenses
closest to the specimen.
02
Has lenses that direct the light
rays through the specimen.
Condenser
01
Also known as the light source.
Iluminator
Ocular lens
Objective lenses
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Importance of using Immersion Oil
The immersion oil has the same refractive
index as the glass slide, so the oil becomes
part of the optics of the glass.
When immersion oil is used, the
light rays do not refract when as
they enter the air from the slide.
The use of immersion oil
improves the resolving power
of the lenses.
If oil is not used with an oil immersion
objective lens, the image becomes fuzzy,
with poor resolution.
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How to apply?
The oil used in oil immersion
is CEDAR WOOD OIL. The
refractive index is: 1.516.
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Parts of the Microscope
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Structural Components
01
02
03
HEAD/BODY
Houses the optical parts
in the upper part of the
microscope.
ARM
Connects to the base and supports the
microscope head. It is also used to
carry the microscope.
BASE
Supports the microscope
and houses the illuminator.
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Optical Components
01
Eyepiece/Ocular
Remagnifies the image
formed by the objective
lens; standard magnifying
power is 10x.
02
Eyepiece Tube
Holds the eyepieces in
place above the objective
lenses.
03
Objective lenses
Primary lenses that
magnifies the specimen.
0
4
Nosepiece
A rotating turret that
houses the objectives.
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Objective Lenses
1. Scanner: Magnification: 4x
2. Low Power Objective (LPO):
○ Magnification: 10x
○ It usually forms the general outline or
wider portion of the object.
3. High Power Objective (HPO):
○ Magnification: 45x
○ It is longer than the LPO and it forms a
bigger image if the object in focus. In
most cases, it is used to enlarge
specimens that are so small under LPO.
4. Oil Immersion Objective (OIO):
○ Magnification: 100x
○ HIGHEST DEGREE OF MAGNIFICATION
○ It is used to examine stained smear
preparations of microorganisms using
immerion oil as their medium.
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Optical Components
05
Coarse Adjustment Knob
A bigger wheel used to
adjust the LPO in focusing;
used also for initial
focusing.
06
Fine Adjustment Knob
A smaller wheel for final
focusing of the specimen
using HPO and OIO; used
also to make the specimen
more vivid.
07
Stage
Where the slide/specimen
is placed for focusing.
08
Stage Clips
It is used to hold the slide
in place.
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Optical Components
0
9
Iluminator
The light source of the
microscope.
10
Condenser
It is used to collect and
focus the light from the
illuminator on the specimen.
11
Iris Diaphragm
It controls the amount of
light reaching the
specimen.
12
Condenser Focus Knob
Moves the condenser up or
down to control the lighting
focus on the specimen.
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Correct Manipulation of Microscope
1
Place the specimen slide on the stage and secure it with
the stage clips. Arrange the portion of the slide to be
examined over the central opening stage.
2
Rotate the low power objective into place under the
body tube. You will feel a “click” when it is correctly
place.
3
Raise the condenser as high as it will go.
4
Rotate the coarse adjustment knob clockwise to
bring down the LPO close but not touching the slide,
until the specimen is seen through the eyepiece.
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Correct Manipulation of Microscope
5
Regulate the intensity of light by
opening or closing the iris diaphragm.
6
Sharpen the focus by turning the fine
adjustment knob.
7
Turn the LPO to HPO without elevating the body tube.
Notice that it will be almost in focus because most
microscopes are parfocal. Little adjustments with fine
adjustment knob will only be needed to clearly view the
object in focus. NOTE: The HPO has a smaller aperture
than the LPO, hence it will be necessary to open the iris
diaphragm furher in order to fill the objective aperture
with light.
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Correct Manipulation of Microscope
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9
10
To focus the oil immersion lens, swing the oil immersion
objective halfway towards the specimen in focus. Place a
drop og immerion oil on the smear. Swing the oil immersion
lens in place. Using the coarse adjustment, bring the
objective down until it touches the oil. Find the object by
gently turning the coarse adjustment knob upwards. Sharpen
the focus with the fine adjustment knob.
Compare the relative sizes from those
seen under the LPO, HPO, & OIO.
Clean the stage and the lenses after each use,
before returning the microscope to the
stockroom.
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Care of the Brightfield Compound Microscope
Get the microscope from the
stockroom by taking the arm with
one hand and supporting the
instrument at the base with the
other hand.
Wipe all lenses with lens paper
before and after use. If the oil
immersion lens is sticky, moisten a
piece of lens paper with 95% alcohol
to wipe it clean. If it is still sticky,
wipe the lens clean with xylol, and
immediately remove the xylol with
tissue moistened with 95% alcohol
then wipe dry the lens with lens
paper. Consistent use of xylol may
loosen the lens.
Keep the stage clean and dry.
1
3
5
2
4
6
Place the microscope at
least six inches from the
edge of the laboratory
table with the
microscope arm facing
you.
NEVER USE TISSUE OR CLOTH
when cleaning the objective
lenses or oculars. Failure to
clean the lens will result in
formation of gummy residue
which reduces efficiency of
lenses.
Do not tilt the microscope;
instead, adjust your stool so
you can comfortably use the
instrument. Tilting tends to
distort wet mounts or objects
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covered with oil.
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Calculating the Total Magnification
TOTAL MAGNIFICATION = Magnifying power of the
eyepiece x Magnifying power of the objective used
EXAMPLE:
Magnifying power of the eyepiece = 10
Magnifying power of the objective used = 100 (OIO)
Therefore: 10 x 100 = 1000x (The specimen in focus is
magnified 1000 times its actual size using oil immersion
objective)
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