MICROSCOPE
The parts of a brightfield microscope are divided into three groups:
[Link] Parts
Base: It holds various parts of the microscope, such as the light source, the fine
and coarse adjustment knobs
C-shaped arm: It holds the microscope, and it connects the ocular lens to the
objective lens
Mechanical stage: The arm bears a stage with stage clips to hold the slides and
the stage control knobs to move the slide during viewing. It has an aperture at the
center that permits light to reach the object from the bottom.
[Link] Parts
Ocular lens: The arm contains an eyepiece that bears an ocular lens of 10x
magnification power.
Micro scopes with two eye pieces are called as binocular microscopes
Objective lens: The arm also contains a revolving nose piece that bears three to
four objectives with lenses of differing magnifying power (4x, 10x, 40x and 100x).
[Link] Parts
Condenser: It is mounted beneath the stage which focuses a cone of light on the
slide
Iris diaphragm: It controls the light that passes through the condenser
Light source: It may be a mirror or an electric bulb
Fine and coarse adjustment knobs: They sharpen the image
MICROSCOPE
Properties of a Microscope
A good microscope should have at least three properties:
1. Good resolution: Resolution power refers to the ability to produce separate images of closely placed objects so that they can be
distinguished as two separate entities.
The resolution power of:
Unaided human eye is about 0.2 mm (200 µm)
Light microscope is about 0.2 µm
Electron microscope is about 0.5 nm.
Resolution depends on refractive index of the medium.
Oil has a higher refractive index than air; hence, use of oil enhances the resolution power of a microscope.
2. Good contrast: This can further be improved by staining
the specimen. When the stains bind to the cells, the
contrast is increased
3. Good magnification: This is achieved by use of lenses.
There are two types of convex lenses used:
Ocular lens with a magnification power of 10x
Objective lenses—scanning (4x), low power (10x), high power (40x) and oil immersion (100x).
The total magnification of a field is the product of the magnification of the objective lens and ocular lens:
Scanning field (40x)
Low power field (100x)
High power field (400x) and
Oil immersion field (1000x).
BRIGHT-FIELD OR LIGHT MICROSCOPE
Working Principle
The rays emitted from the light source pass through the iris diaphragm
and fall on the specimen. The light rays passing through the specimen
are gathered by the objective and a magnified image is formed. This
image is further magnified by the ocular lens to produce the final
magnified virtual image.
Applications
1. Gram staining of clinical specimen
• Helps in rapid presumptive identification of the organism for
starting empiric antimicrobial treatment
• Aids in detecting the presence of inflammatory cells
2. Stool microscopy: To detect ova/cysts, RBCs, pus cells etc.
3. KOH mount: To identify fungal elements from clinical material
4. Peripheral blood smears: To detect parasites like Plasmodium,
Leishmania and microfilariae
5. Identification of an organism from a culture medium by culture
smears
DARK FIELD MICROSCOPE
Working Principle
In dark field (or dark ground) microscope, the object appears
bright against a dark background. This is made possible by use
of a special dark field condenser.
The dark field condenser has a central opaque area that
blocks light from entering the objective lens directly and has a
peripheral annular hollow area which allows the light to pass
through and focus on the specimen obliquely
Only the light which is reflected by the specimen enters
the objective lens whereas the unreflected light does not enter
the objective. As a result, the specimen is brightly illuminated;
but the background appears dark.
Applications
Dark field microscope is used to identify the living, unstained
cells and thin bacteria like spirochetes which cannot be
visualized by light microscopy.
PHASE CONTRAST MICROSCOPE
Working Principle
The condenser is similar to that of dark field microscope, consists of an
opaque central area with a thin transparent ring, which produces a
hollow cone of light.
As this cone of light passes through a cell, some light rays are
bent due to variations in density and refractive index within the
specimen and are retarded by about onefourth of a wavelength
The undeviated light rays strike a phase ring in the phase plate, (a
special optical disk located in the objective), while the deviated
rays miss the ring and pass through the rest of the plate
The phase ring is constructed in such a way that the undeviated
light passing through it is advanced by one fourth of a
wavelength, the deviated and undeviated waves will be about half
wavelength out of the phase and will cancel each other when they
come together to form an image
The background, formed by undeviated light, is bright, while the
unstained object appears dark and well defined.
Applications: Phase contrast microscopy is especially useful for studying:
Microbial motility
Determining the shape of living cells, and
Detecting microbial internal cellular components, such as the cell membrane, nuclei, mitochondria, spindles, chromosomes,
Golgi apparatus, endospores and inclusion bodies; which become clearly visible because they have refractive indices markedly
different from that of water.
FLUORESCENCE MICROSCOPE
Working Principle
When fluorescent dyes are exposed to ultraviolet (UV) rays, they become excited and
are said to fluoresce, i.e. they convert this invisible, short wavelength rays into light of
longer wavelengths (i.e. visible light).
• The source of light may be a mercury lamp which emits rays that pass through
an excitation filter
• The excitation filter is so designed that it allows only short wavelength UV light
(about 400 nm, called as the exciting wavelength of light) to pass through;
blocking all other long wavelength rays
• The exciting rays then get reflected by a dichromatic mirror in such a way that
they fall on the specimen which is previously stained by fluorescent dye and
focused under the microscope
• The fluorescent dye absorbs the exciting rays of short wavelength, gets
activated and in turn emits rays of higher wavelength
• A barrier filter positioned after the objective lenses removes any remaining
ultraviolet light, which could otherwise damage the viewer’s eyes, or blue and
violet light, which would reduce the image’s contrast.
Applications: Fluorescence microscopy is especially useful for studying:
• Auto fluorescence –eg: cyclospora
• Microbes coated with fluorescent dye-
✓ Acridine orange –For detection of malarial parasite by QBC examination.
✓ Auramine phenol- For tubercle bacilli
• Direct and indirect immunofluorescence to detect antigen and antibody respectively.
ELECTRON MICROSCOPE
Working Principle
Electrons are generated by electron gun, which travel in high speed.
The medium of travel in EM should be a fully vacuum path because in
air path, electrons can get deflected by collisions with air molecules.
Electrons pass through a magnetic condenser and then bombard on the
thin sliced specimen mounted on the copper slide
The specimen scatters electrons passing through it, and then the
electron beam is focused by magnetic lenses (objective lens followed
by projector lenses) to form an enlarged, visible image of the
specimen on a fluorescent screen
A denser region in the specimen scatters more electrons and therefore
appears darker in the image since fewer electrons strike that area of
the screen. In contrast, electrontransparent regions are brighter.
Applications
1. Detection of viruses.
It can detect viruses- Directly from the clinical specimens (e.g. rotavirus from stool) or
From tissue cultures or
After adding specific antiviral antibody to the specimen (known as immuneEM)
2. Study of ultra structural details of various microbes.