BIOANALYTICAL TECHNIQUES

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Microscope is the most characteristic instrument of the
Microbiology Lab. It provides magnification which
enables us to visualize microorganisms and their
structures, which could not be resolved with the help of
an unaided eye.
Magnification Range: X100 – X400,000

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Light (Optical) Electron

Bright-field Microscope Transmission Electron Microscope

Dark-field Microscope Scanning Electron Microscope

Fluorescence Microscope 2

Phase-contrast Microscope
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Physical properties of Light
Light is an electromagnetic (EM) radiation composed of 2 electric
and magnetic fields propagating together through space and a
variety of media.
Common interactions of Light

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Reflection
When light strikes an object & bounces back to the original
medium
Transmission
It refers to the passing of light through an object. Transparent
objects have 100% transmission. M/o are visible to us as
transmission is allowed through them.

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Absorption
It occurs if light is absorbed by an object. Light rays neither pass
through nor bounce back, but are rather taken up by the object.
Refraction
When light passes from one medium to another, it changes its
direction. This bending of light is known as refraction and angle
through which it bends is called angle of refraction.
Diffraction 4
When the light waves pass through an opening or around a
barrier in a path, then there is change in direction of waves
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History of Microscopes
The term microscope was coined by Faber in 1623.
First well-known users of microscopes were Antony van Leeuwenhoek & Robert
Hooke.
Leeuwenhoek (1632 to 1723) developed a first simple microscope with a
magnification of 200x– 300x. It was a single lens microscope with powerful
magnifying glass. He was the first person to observe bacteria & protozoa in water,
as well as, maiden person to detect bacteria under microscope. He is also known

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as “Father of microscopy”.

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The Leeuwenhoek Microscope
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History of Microscopes
Robert Hooke (1665) was the first person to build a microscope with multiple
lenses, i.e., laboratory compound microscope.

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In 1830, Joseph Jackson Lister made the first improved version of compound
microscope. Later on, series of improvements resulted in the final shape of a
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modern compound microscope.
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Parts of microscope

• Illuminator - This is the light source located below the specimen.

• Condenser - Focuses the ray of light through the specimen.
• Stage - The fixed stage is a horizontal platform that holds the specimen.
• Objective - The lens that is directly above the stage.
• Nosepiece - The portion of the body that holds the objectives over the stage.

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• Iris diaphragm - Regulates the amount of light into the condenser.
• Base – Base supports the microscope which is horseshoe shaped.
• Coarse focusing knob - Used to make relatively wide focusing adjustments to the
microscope.
• Fine focusing knob - Used to make relatively small adjustments to the
microscope.
• Body - The microscope body.
• Ocular eyepiece - Lens on the top of the body tube. It has a magnification of 10×
normal vision. 8
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Principle
In bright-field microscopy, the microscopy field is brightly lighted
and the m/o appear dark because they absorb some of the light.
Generally, m/o don’t absorb much light, however, staining them
with a dye greatly increases their light-absorbing ability, resulting
in greater contrast and colour differentiation.

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Baccili and cocci under light microscope 9

Magnifications: X1,000 to X2,000 (>2,000, image becomes fuzzy)

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Important concepts in Microscopy
Resolution
The ability to resolve small details of an object and to reveal closely
adjacent structural details as separate and distinct identity.

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Resolving power
• It is the ability to differentiate two close points as separate
• The resolving power of human eye is 0.25 mm
• The light microscope can separate dots that are 0.25 µm apart 10
• The electron microscope can separate dots that are 0.5 nm apart
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Important concepts in Microscopy

Types of microscope Resolving power

Compound Microscope 200 nanometers

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Scanning Electron 10 nanometers
Microscope
Transmission Electron 0.2 nanometers
Microscope

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Important concepts in Light Microscopy
Limit of resolution (D)
The minimum distance between two distinguishable objects is
known as limit of resolution.
D depends on:
1. Angular aperture α,
half angle of cone of

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light entering
objective lens from
specimen
2. Refractive index (n)
of medium between
specimen and
objective lens
3. Wavelength (λ) of
incident light
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Important concepts in Light Microscopy
Limit of resolution or D = 0.61 λ/n sinα
The sine value of angular aperture (sinα) multiplied by refractive
index (n) of medium filling the space between objective lens and
cover slip gives the Numerical aperture (NA), which is an index of
resolving power of microscope.

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Moving the object lens closer to
specimen increases angular
aperture, thus value of
n sinα increases, and value
of D decreases, resulting
in better resolution.
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Important concepts in Light Microscopy
Eg. Air R.I. = 1; Immersion Oil R.I. = 1.5
When we replace air by immersion oil between specimen &
objective lens, bending of direction of light coming out from the
specimen will be more, angular aperture, α will increase,
resulting in better resolution.
Shorter the wavelength of light of incident ray, lower will be D,

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better resolution.

Max α with best objective lens - 70° (sin 70° = 0.94)

Shortest λ = 450 nm (blue); immersion oil (n) = 1.5
D = 0.61 X 450 nm/1.5 X 0.94 = 194 nm ~ 0.2 µm

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Max. resolution of Light microscope using visible light is 0.2 µm
(200 nm)
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Magnification
It is the ratio of the size of an object seen under microscope to the actual size
observed with unaided eye.
Total magnification of light microscope is determined by X magnification power
of objective lens by that of ocular lens (eye piece). Compound microscope can
have up to 6 interchangeable objective lenses, with different powers of
magnification. Ocular lenses generally have a magnification of X10.
Resolving power is determined solely by objective lens

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Scanning 4X X 10 = 40 X magnification
Low-power 10X X 10 = 100 X
High-dry 40X X 10 = 400 X
Oil-immersion 100X X 10 = 1000 X
The basic limitation of bright-field microscope is one not of magnification, but
of resolving power. Mere increase in size (greater magnification) without the
ability to distinguish structural details (greater resolution) is not beneficial.
Magnification beyond resolving power is of no value since the larger image will
be less distinct in detail and fuzzy in appearance. 15
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Objective
PROPERTY LOW POWER HIGH POWER OIL IMMERSION
Magnification of 10x 40-45x 90-100x
objective
Magnification of 10x 10x 10x
eyepiece

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Total magnification 100x 450 – 450x 900 – 1000x

Numerical aperture 0.25 – 0.30 0.55 – 0.65 1.25 – 1.4

Mirror used Concave Concave Plane
Focal length (Approx) 16 mm 4 mm 1.8 – 2 mm
Working distance 4 – 8 mm 0.5 – 0.7 mm 0.1 mm
Iris diaphragm Partially closed Partially opened Fully opened
Position of condenser Lowest Slightly raised Fully raised 16
Maximum 0.9 µm 0.35µm 0.18µm
resolution(Approx)
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Figure shows the sizes visible to unaided human eye and by various types of
microscopy
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Dark-Field Microscopy: In a light microscope, the light coming through a
condenser is directly transmitted through a specimen. Under a bright field of
illumination, the contrast relative to the background decreases, and most of the
live samples can’t be stained as they get distorted by staining. Then, where
contrast is needed to visualize a specimen, dark field illumination is used.
Principle: A bright-field
microscope can be adapted
as a dark-field microscope by
adding a special disc called a

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stop to the condenser. Most
of light directed through
condenser doesn’t enter the
objective; the field becomes
dark. However, some of the
light rays gets diffracted
through microbial cells. The
diffracted light will enter
objective, thus
object/microbes will appear 18
bright in an otherwise dark
microscopic field.
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Applications of Dark-Field Microscopy:
• It provides little details of the specimen, the contrast is exceptional
• It is particularly valuable for examination of unstained microorganisms
suspended in fluid – wet mount & hanging drop preparations
• It is used to examine spirochetes (Leptospira & Treponema pallidum) in the
exudates from leptospiral and syphilitic Infections
• Useful in examining Campylobacter jejuni and endospores

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Treponema pallidum Leptospira
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Images seen under Dark-Field Microscopy

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Paramecium

Treponema vincenti

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Volvox and Spirogyra

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Dark-field vs Bright-field
Microscopy: The appearance of a
white blood cell (eosinophil)
surrounded by red blood cells, as

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viewed by
(A) dark-field
(B) Bright-field microscopy

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Phase Contrast Microscopy:
In 1935 Frits Zernike, a Dutch physicist produced the phase contrast microscope
(Zernike microscope)
It uses a conventional light microscope fitted with a phase-contrast objective and
a phase-contrast condenser. This special optical system helps to distinguish
unstained structures within a cell which differ only slightly in their refractive
indices or thicknesses.
Principle: This technique is based on the fact that light passing through one
material and into another material of a slightly different refractive index and/or

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thickness will undergo a change in phase. These differences in phase are
translated into variations in brightness of the structures and hence are detectable
by the eye.

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Phase Contrast Microscopy

Applications:
Valuable for studying microbial motility, organelles of
living cells, eukaryotic cells, shape of bacterial cells,
detecting bacterial components such as endospores,

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inclusion bodies
Diagnosis of heamatologic diseases: identification and
characterization of cells from blood & bone marrow. It is
able to detect the minute alterations in cellular
morphology between normal & pathologic cell forms.
Diagnosis of tumor cells
Examination of growth, dynamics, and behavior of a wide 25
variety of living cells in cell culture
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Phase Contrast Microscopy

Macronucleus

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Micronucleus
Paramecium

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Phase Contrast Microscopy

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Rhodospirillum rubrum