INTRODUCTION TO MICROSCOPE

Molecular Biology and Diagnostics Laboratory

 HISTORY
Hans and Zacharias Janssen of Holland in the
1590’s created the rst compound
microscope.
Antonie van Leeuwenhoek and Robert Hooke
made improvements by working on the lenses
Required for the morphological study of
microorganisms.
USES - to magnify the image.
To achieve maximum resolution
To provide su cient contrast for
observation.

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THE
MICROSCOPE
WHAT IS THE PURPOSE OF A MICROSCOPE?

To magnify or enlarge the image of

tiny objects.
 EYE PIECE
•Also known as the ocular
•Contains the rst lens you look
through - usually a magni cation of
10x
•Located on the top of the body tube
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OBJECTIVE LENSES
•Used in combination with the eyepiece to
provide a range of magni cation
•Magni cation ranges from 4x to 00x
•Located on the nosepiece at the bottom of
the body tube
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 NOSE PIECE
•Holds the objective lenses
•Rotates to enable magni cation
•Located at the bottom of the body
tube
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 ARM
•Supports the upper parts of the
microscope
•Used to carry the microscope
•When carrying a microscope, always
have one hand on the arm and one
hand on the base. 

Use two hands!!

 BASE
•Supports the whole microscope
•Used to carry the microscope
•When carrying a microscope, always
have one hand on the arm and one
hand on the base. 

Use two hands!!

STAGE
•Supports the slide
•The slide contains the specimen or
object that you are viewing with the
microscope.
STAGE CLIP
•Helps to hold the slide in place
•Usually one on each side of the hole
(stage opening) = 2 stage clips
•The stage opening allows light to pass
from the light source to the lenses.
LIGHT SOURCE
•Provides light necessary for viewing
the specimen
•Usually either a mirror or illuminator
•Sends light through the stage opening
to the diaphragm
CONDENSER
The condenser is used to capture and
focus the light up through the stage.
The condenser lenses have the most
utility at higher magni cation powers
like 400X and above and microscopes
that have condenser lenses will be able
to render a sharper image than those
without and the 400X and above range.
In the example microscope the
condenser is actually built into the
stage of the microscope and achieves a
numerical aperture of 0.65.
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CONDENSER IRIS DIAPHRAGM

•Wheel or lever located below the
stage opening
•Regulates the amount of light that
can enter the lenses
•May need to be adjusted based on the
thickness of the specimen being
studied
COARSE ADJUSTMENT KNOB
•Raises and lowers the stage or
objective lenses
•Used only when focusing the low
power (4x) objective lens
FINE ADJUSTMENT KNOB
•Raises and lowers the stage or
objective lenses a small distance for
exact focusing
•Used when focusing the low power
(10x), high power (40x), and oil
immersion (100x) objective lenses
TYPES OF MICROSCOPE
TYPES OF MICROSCOPES
Microscopes are broadly categorized into light (optical) &
electron depending upon magni cation.

In light microscopy the magni cation is obtained by optical

lenses using light waves. This includes:
Bright-Field microscopy
Phase-Contrast microscopy
Fluorescent microscopy
Dark Field microscopy

Electron microscope – Uses beam of electrons to produce

the image. The specimen samples can be examined by
either transmission or scanning electron microscopy



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 BRIGHT-FIELD MICROSCOPY
The microscopic eld is brightly lightened and the
microorganisms appear dark because they absorb
some of the light.
Microorganisms do not absorb much light, but
when they are stained with dye, it increases their
light absorbing ability.
This results in greater contrast & colour
di erentiation.
This type of microscopes produce about 1,000 to
2,000 magni cation.
If magni es greater than 2,000 the image becomes
fuzzy.
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 DARK FIELD MICROSCOPY

The objects are brightly illuminated against
dark background.
The light microscope is equipped with a
special kind of condenser that transmits a
hallow cone of light from the source of
illumination.
Most of the light directed through
condenser does not enter the objective, but
some of the light rays will be scattered if the
transparent medium contains objects such as
microbial cells.

 DARK FIELD MICROSCOPY

This di racted light will enter the
objective and the reach the eye; thus the
object (microbial cell) will appear bright
in dark microscopic eld.
Dark eld microscopy is valuable for the
examination of unstained
microorganisms suspended in uid wet
mount and hang drop preparations.
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 Treponema pallidum; a spirochete that causes
Syphilis

Dark Field Microscopy

Saccharomyces cerevisiae observed under dark field
microscopy
 USES OF DARK-FIELD MICROSCOPY
Dark- eld microscopes are used in the
microbiology laboratory for the
following purposes;
Visualization of spirochetes such as
Treponema pallidum (syphilis),
Borrelia burgdorferi (lyme
borreliosis) and Leptospira
interrogans (leptospirosis) in clinical
samples.
Spirochetes can not be seen by light
microscopy because of their thin
dimensions.
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 FLUORESCENCE MICROSCOPY
Chemical substances absorb light but
only some substances will emit light of a
longer wavelength and a lesser energy
content.
Such substances are called uorescent
and the phenomenon is termed as
uorescence.
The microorganisms are stained with a
uorescent dye and then illuminated with
blue light
The blue light is absorbed and green light
emitted by the dye.
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FLUORESCENCE MICROSCOPY
FLUOROCHROMES
• Acridine orange : Orange
• Auramine-Rhodamine : Yellow
• Calco uor white: White
• Fluorescein Isothiocyanate (FITC) : Green
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 FLUORESCENCE MICROSCOPY
The uorescent dyes are combined with
antibodies which are speci c to
microorganisms.
Antibodies to which uorescent dye is
attached are known as labelled antibodies.
The labelled antibodies are then mixed with
a suspension of bacteria & examined by
uorescent microscopy.
The bacterial cell combined with labelled
antibody will be visible in microscope,
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CARING FOR A
MICROSCOPE
CARING FOR A MICROSCOPE
Clean only with a soft cloth/tissue
Make sure it’s on a at surface.
Don’t bang it.
Carry it with 2 HANDS...one on the arm and
the other on the base.

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 HOW TO USE A MICROSCOPE

Start on the lowest magni cation
Don’t use the coarse adjustment
know on high magni cation…you’ll
break the slide!!!
Place slide on stage and lock clips
Adjust light source (if it’s a mirror…
don’t stand in front of it!)
Use ne adjustment knob to focus
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LET’S REVIEW…