Microbes and Microscopy: Chapter - 1 Lecture - 2

Microbes and Microscopy
Chapter – 1
Lecture – 2
Noshin Azra Rahman (Sr. Lecturer - SLS) 1

Microscopy
• Microscopy is the technical field of using
microscopes to view samples and objects that
cannot be seen with the unaided eye (objects
that are not within the resolution range of the
normal eye).
• There are two well-known branches of
microscopy:
– Optical or light microscopy
– Electron microscopy
Light microscopy
• Light microscopy, uses a system of lens that manipulate the

path of light beam that travels between the object that being
studied includes:
• Bright-field
• Dark-field
• Differential interference contrast (DIC)
• Fluorescence and
• Phase-contrast microscopy

Electron Microscopy
• The Electron Microscopy, as the name suggests, uses a beam

of electrons in place of light waves to produce the image.
• Specimens can be examined by either
• Transmission electron microscopy
• Scanning electron microscopy
• Scanned probe microscopy is a modern day microscopic
technique widely used now-a-days. The technique includes
scanning tunneling microscopy and atomic force microscopy.

Light Microscopy: Bright Field Microscopy
• Under usual operating conditions, the field of vision in a

compound light microscope is brightly illuminated. By focusing
the light, the condenser produces a bright-field illumination.
• The specimen is illuminated by the transmitted white light and
contrast is caused by absorbance of light at the denser areas of
the specimen.
• The typical appearance of a bright-field microscopy image is a
dark sample on a bright background, hence the name.
• Ordinarily, microbes do not absorb much light, but staining
them with a dye greatly increases their light absorbing ability
resulting in greater contrast and color differentiation.

• Light Path
– The light path of a bright-field microscope is
extremely simple, no additional components are
required beyond the normal light microscope setup.
The light path therefore consists of:
– a transillumination light source, commonly a halogen
lamp in the microscope stand;
– a condenser lens which focuses light from the light
source onto the sample; and
– oculars to view the sample image.
• How it Works
A series of finely ground lenses forms a clearly focused image
that is many times larger than the specimen itself. This
magnification is achieved when light rays from an illuminator,
the light source, are passed through a condenser, which has
lenses that direct the light rays through the specimen.
From here, light rays pass into the objective lenses, the lenses
closest to the specimen.
The image of the specimen is magnified again by the ocular lens,
or eyepiece

• To achieve high magnification (100X) with good resolution, the

lens must be small.
• To preserve the direction of light rays at the highest magnification,
immersion oil is placed between the glass slide and the oil
immersion objective lens.
• The immersion oil has the same refractive index as glass, so oil
becomes part of optics of the glass of the microscope.
• Unless immersion oil is used, light rays are refracted as they enter
the air from the slide, and the objective lens would have to be
increased in diameter to capture them.

• Advantages
– Simplicity of setup with only basic equipment
required.
• Limitations
– Very low contrast of most biological samples.
– Low apparent optical resolution due to the blur of
out of focus material.
– The sample often has to be stained before viewing.
Therefore, live cells cannot usually be viewed.
Limitations of Bright Field Microscopy
• Staining, although a widely used procedure in

light microscopy, kills cells and can distort
their features. Two forms of light microscopy
improve image contrast without the use of
stain, and thus do not kill cells. These are:
– Phase-contrast microscopy and
– Dark-field microscopy

Light Microscopy: Dark-Field Microscopy
• The dark-field microscope is a light microscope in which
light reaches the specimen from the sides only. The only
light that reaches the lens is that scattered by the specimen,
and thus the specimen appears light on a dark background
(Figure 2.5c).
• A darkfield microscope is used to examine live

microorganisms that either are invisible in the ordinary
light microscope, cannot be stained by standard methods,
or are so distorted by staining that their characteristics then
cannot be identified.
• Instead of the normal condenser, a darkfield

microscope uses a darkfield condenser that
contains an opaque disk. The disk blocks light
that would enter the objective lens directly.
Only light that is reflected off (turned away
from) the specimen enters the objective lens.
Because there is no direct background light,
the specimen appears light against a black
background-the dark field.
• Principal uses:
– Resolution by dark-field microscopy is somewhat better than by
light microscopy, and objects can often be resolved by dark-
field that cannot be resolved by bright-field or even phase-
contrast microscopes.
– This technique is frequently used to examine unstained
microorganisms suspended in liquid, as bundles of flagella (the
structures responsible for swimming motility) are often
resolvable with this technique.
– One use for darkfield microscopy is the examination of very
thin spirochetes, such as Treponema pallidum, the causative
agent of syphilis.

Light Microscopy: Phase Contrast
Microscopy
• Phase-contrast microscopy is especially useful because it permits
detailed examination of internal structures in living
microorganisms. In addition, it is not necessary to fix (attach the
microbes to the microscope slide) or stain the specimen procedures
that could distort or kill the microorganisms.
• Phase-contrast microscopy is based on the principle that cells

differ in refractive index (a factor by which light is slowed as it
passes through a material) from their surroundings.
• Phase-contrast microscopy depends on the wave nature of light

rays and the fact that light rays can be in phase (their peaks and
valleys match) or out of phase.
Microscopy
• If the wave peak of light rays from one source coincides with
the wave peak of light rays from another source, the rays
interact to produce reinforcement (relative brightness).
• However, if the wave peak from one light source coincides
with the wave trough from another light source, the rays
interact to produce interference (relative darkness).
• In a phase-contrast microscope, one set of light rays comes
directly from the light source. The other set comes from light
that is reflected or diffracted from a particular structure in the
specimen. (Diffraction is the scattering of light rays as they
"touch" a specimen's edge. The diffracted rays are bent away
from the parallel light rays that pass farther from the
specimen.)
Microscopy
• When the two sets of light rays-
direct rays and reflected or
diffracted rays- are brought
together, they form an image of
the specimen on the ocular lens,
containing areas that are
relatively light (in phase),
through shades of gray, to black.
• In phase-contrast microscopy,
the internal structures of a cell
become more sharply defined.

Light Microscopy: Differential Interference
Contrast (DIC) Microscopy
• Differential interference contrast (DIC) microscopy is
similar to phase-contrast microscopy in that it uses
differences in refractive indexes. However, a DlC
microscope uses two beams of light instead of one.
• Differential interference contrast (DIC) microscopy is a
form of light microscopy that employs a polarizer in the
condenser to produce polarized light (light in a single
plane).
• The polarized light then passes through a prism that
generates two distinct beams. These beams traverse the
specimen and enter the objective lens where they are
recombined into one.
• Because the two beams pass through different
substances with slightly different refractive indices,
the combined beams are not totally in phase but
instead create an interference effect.
• This effect visibly enhances subtle differences in
cell structure. Thus, by DIC microscopy, cellular
structures such as the nucleus of eukaryotic cells
(Figure 2.7), or endospores, vacuoles, and granules
of bacterial cells, appear more three-dimensional.

Uses:
• DIC microscopy is typically used for
observing unstained cells because it can reveal
internal cell structures that are nearly invisible
by the bright-field technique.
• https://www.youtube.com/watch?v=TKTGgA
Q2VEs

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Microbes and Microscopy

Noshin Azra Rahman (Sr. Lecturer - SLS) 1

• Light microscopy, uses a system of lens that manipulate the

Noshin Azra Rahman (Sr. Lecturer - SLS) 3

• The Electron Microscopy, as the name suggests, uses a beam

Noshin Azra Rahman (Sr. Lecturer - SLS) 4

• Under usual operating conditions, the field of vision in a

Noshin Azra Rahman (Sr. Lecturer - SLS) 5

Noshin Azra Rahman (Sr. Lecturer - SLS) 7

• To achieve high magnification (100X) with good resolution, the

Noshin Azra Rahman (Sr. Lecturer - SLS) 8

• Staining, although a widely used procedure in

Noshin Azra Rahman (Sr. Lecturer - SLS) 10

• A darkfield microscope is used to examine live

• Instead of the normal condenser, a darkfield

Noshin Azra Rahman (Sr. Lecturer - SLS) 13

• Phase-contrast microscopy is based on the principle that cells

• Phase-contrast microscopy depends on the wave nature of light

Noshin Azra Rahman (Sr. Lecturer - SLS) 16

Noshin Azra Rahman (Sr. Lecturer - SLS) 20

Noshin Azra Rahman (Sr. Lecturer - SLS) 22

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