PAPER CCEN-103

UNIT-I
Topic: MICROSCOPY
CONTENT
• Introduction of Microscope
• Light Microscopy
• Phase Contrast Microscopy
• Fluorescence Microscopy
• SEM & TEM
• Confocal Microscope
• Fixation and Staining
Introduction of Microscope
• First Microscope was invented by Antoni Van
Leeuwenhoek
• Microscope is a simple as well as important
instrument in the Laboratory
• There are many small objects or details of
objects which cannot be seen by the naked
human eye.
• Microscopes are used to observe the shape of
bacteria, fungi, parasites and host cells in
various stained and unstained preparations.
Components of Compound Microscope
• Eye-pieces
• It has a lens which magnifies the image formed by the
objective. The magnifying power of the eye-piece is in the
range 5x20x. A movable pointer may be attached to the
inside of the eye-piece.

• Objectives
• Objectives with magnifying powers 4x, 10x, 40x and 100x
are commonly used. The magnifying power is marked on
the lens and is usually color-coded for easy identification.
• The 100x objective is for oil immersion.
• Mechanical stage
• The mechanical stage holds the slide and allows it to be
moved to the left, right, forward or backward by rotating
the knobs.
• It is fitted with fine Vernier graduations as on a ruler.
This helps in relocating a specific field of examination.

• Two-sided mirror
• The two-sided mirror provides necessary illumination
through reflection of natural or artificial light. It has two
surfaces, one plain for artificial light and other concave
for natural light.
• Coarse and fine focusing knobs
• The coarse and fine focusing knobs are used to change the
distance between the specimen slide and the objective.
• The coarse focusing knob changes the distance rapidly and
the fine focusing knob changes the distance very slowly.
• One revolution of the fine focusing knob should generally
move the mechanical stage by 100 µm. The movement
should be smooth and free from jerks.
• Numerical Aperture (NA)
• It is a dimensionless number that characterizes the range of
angles over which the system can accept or emit light.
• The angle is denoted by θ and is expressed as a sine value.
•  Numerical aperture (NA) is Sine
value of the half-aperture angle
multiplied by the refractive index (η)
of the medium.

• Refractive index is a measure of the

bending of a ray of light when
passing from one medium into
another.
Light Microscopy
• It have 2 types i.e.
1. Darkfield Microscopy
2. Brightfield Microscopy

• Darkfield Microscopy
• In which the field became darker where as the
object will illuminate. It requires special type of
condenser and used to observe bacteria like
leptospires.
• Brightfield Microscopy
• It is commonly used microscopy in which the field
is bright and the object or specimen would be
colored with stain so, one can easily visualize the
shape and structure of object.
• Differential staining may be used depending on
the properties of different structures and
organisms.
1. Gram’s Staining
2. Acid-Fast staining
3. Negative Staining
4. Cell wall Staining etc.
Phase Contrast Microscopy
• It is a special type of light microscopy
• Phase-contrast microscopy allows the examination
of live unstained organisms.
• In this method the light passing through the object
and that passing around it.
• It requires special objective lens known as phase-
contrast lens
Fluorescence Microscopy
• Object is stained with fluorochromes
(Fluorochromes absorb light energy of a specific
wavelength and re-emit it at a longer wavelength)
and are exposed with the light of higher energy or
shorter wavelength.
• These fluorochrome stained object absorbs the light
and emits the light with brilliant colors (specific
and bright color).
• Auramine differential staining
• It requires specific microscope
Scanning and Transmitting electron
Microscope
• SEM and TEM are the types of Electron
Microscope.
• Electron microscopes are scientific instruments that
use a beam of energetic to examine objects on a very
fine scale.
• The image produced bye electron microscopes is
perceived by CRT or X-ray plates.
• It produces 10,000x plus magnification with use of
Electron beam.
SEM
• It provides a valuable combination of high resolution
imaging, elemental analysis, and recently,
crystallographic analysis.
• It is used for inspecting topographies of specimens
at very high magnifications using a piece of
equipment called the scanning electron microscope.
• SEM magnifications can go to more than 3,00,000X.
• Electrons from the beam hit the surface of the object
and bounce off(back scattered) it. A detector
registers these scattered electrons and turns them
into a picture.
TEM
• It can be used only for the thin sample as much as
<200nm.
• In a conventional transmission electron microscope, a
thin specimen is irradiated with an electron beam of
uniform current density.
• Principle of TEM is same as light microscope that is
energy source passes through specimen and provides the
idea of the internal and outer structure of the object. But
the difference is in TEM the energy source is electron
beam rather then visible light.
• All deflected electrons are captured by detector and
produced the image of object.
• Maximum resolution of TEM can go up to 10,00,000x.
Confocal Microscope
• Similar to the widefield microscope, the confocal
microscope uses fluorescence optics.
• Instead of illuminating the whole sample at once, laser
light is focused onto a defined spot at a specific depth
within the sample. This leads to the emission of
fluorescent light at exactly this point.
• A pinhole inside the optical pathway cuts off signals
that are out of focus, thus allowing only the
fluorescence signals from the illuminated spot to enter
the light detector.
• It is broadly used to resolve the details structure of
specific objects within the cell.
Fixation
• To study the microorganism we have to observe it carefully,
and to observe under microscopy they have to be fix.
• Fixation is the process which will immobilize the organism
and its structure, in order to prevent washing away of smear.
• Smear is a thin and even distribution of organisms on glass
slide using wire loop to fix it properly.
• It can be achieved by 2 ways.
1. Use of Physical agents
2. Use of Chemical agents
• Physical agent
• The most common agent is heat. By passing the slide from flam of
burner can fix the smear on glass slide.

• Chemical agent
• Chemicals maybe used where heat proves deleterious to cell.
• Chemical usually includes acids, heavy metal and oxidizing agent. e.g.
glacial acetic acid, formalin, picric acid, chloroform etc.

• After successful fixation one can perform staining procedure.

Staining
• It is extremely difficult to 1. Neutral dye
visualize 2. Leuco dye
microorganisms in living state
because they are tiny and 3. Metachromatic dye
transparent. Hence, it is 4. Synthetic dye
necessary to stain them for 5. Natural dye
proper visualization.
• There are large number of
colored compounds use for the
staining of bacteria.
• There are 7 class of dyes.
1. Basic dye they are cationic in nature, chromogen have positive charge
which have higher affinity for the negative constituents of the cell. e.g.
methylene blue, safranin etc.
2. Acidic dye they are anionic in nature, chromogen have portion of negative
charge which have higher affinity towards positive constitution of cell. e.g.
eosin, Congo red, rose Bengal etc.
3. Neutral dye mixing positive and negative compound neutral dyes are
formed. e.g. Romanowasky’s stain, Giemsa’s stain.
4. Leuco dyes Reduced form of dye will colorless dur to unsaturation in cyclic
ring. e.g. methylene blue, used as indicators of oxidation-reduction potential.
5. Metachromatic dye dye appears to be of different color in contrast to its
original color due to change in concentration.
6. Synthetic dye at modern time synthetic dyes are used, also known as aniline
as well as coal tar dye.
7. Natural dye naturally occurring dyes was used at early time e.g. saffron,
indigo etc.
Gram’s Staining
• Working of gram’s staining is based on
physicochemical nature of the cell wall of
bacteria.
• Cell wall of gram -ve bacteria are generally
thinner than gram +ve.
• Gram positive bacteria have higher amount of
peptidoglycan and low amount of lipids where
gram negative bacteria have high amount of lipids.
• It involves the use of two different stains.
1. Crystal Violet
2. Safranin

• Crystal violet and Gram’s iodine forms the CI complex in the cell wall and
fix the stain.
• When decolorizer applied, at that time thin layered microbes release the
color complex and thick layered retain the CI complex.
• After decolorization counter stain safranin is applied, therefore safranin
can deposited in to thin layered microorganisms.
• Hence, we can differentiate the gram positive and negative bacteria.
• Gram positives are violet or purple in color where gram negative are pink
in color.
Acid-Fast Staining
• Some bacteria have high lipoidal content in the cell wall compared to
gram positive and negative.
• Basic fuchsin or carbolfuchsin can penetrate the waxy cell wall with
the help of phenol and heat.
• Then acid-alcohol is used as a decolorizer. As fuchsin primary stain is
more soluble in lipoidal content than alcohol so acid-fast organism
remains colored and non acid fast organisms faces decolorization.
• Then counter stain Malecite green or methyl blue is used to recolorize
non acid fast organism.
• There 3 methods of acid-fast
staining
1. Ziehl-Neelson Method
2. Gross’s Cold method
3. Kinyoun Method